research paper of genetic engineering

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• Published: 29 October 2020

The genome editing revolution: review

• Ahmad M. Khalil   ORCID: orcid.org/0000-0002-1081-7300 1  

Journal of Genetic Engineering and Biotechnology volume  18 , Article number:  68 ( 2020 ) Cite this article

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Development of efficient strategies has always been one of the great perspectives for biotechnologists. During the last decade, genome editing of different organisms has been a fast advancing field and therefore has received a lot of attention from various researchers comprehensively reviewing latest achievements and offering opinions on future directions. This review presents a brief history, basic principles, advantages and disadvantages, as well as various aspects of each genome editing technology including the modes, applications, and challenges that face delivery of gene editing components.

Genetic modification techniques cover a wide range of studies, including the generation of transgenic animals, functional analysis of genes, model development for diseases, or drug development. The delivery of certain proteins such as monoclonal antibodies, enzymes, and growth hormones has been suffering from several obstacles because of their large size. These difficulties encouraged scientists to explore alternative approaches, leading to the progress in gene editing. The distinguished efforts and enormous experimentation have now been able to introduce methodologies that can change the genetic constitution of the living cell. The genome editing strategies have evolved during the last three decades, and nowadays, four types of “programmable” nucleases are available in this field: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system. Each group has its own characteristics necessary for researchers to select the most suitable method for gene editing tool for a range of applications. Genome engineering/editing technology will revolutionize the creation of precisely manipulated genomes of cells or organisms in order to modify a specific characteristic. Of the potential applications are those in human health and agriculture. Introducing constructs into target cells or organisms is the key step in genome engineering.

Conclusions

Despite the success already achieved, the genome editing techniques are still suffering certain difficulties. Challenges must be overcome before the full potential of genome editing can be realized.

In classical genetics, the gene-modifying activities were carried out selecting genetic sites related to the breeder’s goal. Subsequently, scientists used radiation and chemical mutagens to increase the probability of genetic mutations in experimental organisms. Although these methods were useful, they were time-consuming and expensive. Contrary to this, reverse genetics goes in the opposite direction of the so-called forward genetic screens of classical genetics. Reverse genetics is a method in molecular genetics that is used to help understanding the function of a gene by analyzing the phenotypic effects of specific engineered gene sequences. Robb et al. [ 68 ] defined and compared the three terms: “genome engineering”, “genome editing”, and “gene editing”. Genome engineering is the field in which the sequence of genomic DNA is designed and modified. Genome editing and gene editing are techniques for genome engineering that incorporate site-specific modifications into genomic DNA using DNA repair mechanisms. Gene editing differs from genome editing by dealing with only one gene.

This review briefly presents the evolution of genome editing technology over the past three decades using PubMed searches with each keyword of genome-editing techniques regarding the brief history, basic principles, advantages and disadvantages, as well as various aspects of each genome editing technology including the modes, future perspective, applications, and challenges.

Genome-wide editing is not a new field, and in fact, research in this field has been active since the 1970s. The real history of this technology started with pioneers in genome engineering [ 36 , 59 ]. The first important step in gene editing was achieved when researchers demonstrated that when a segment of DNA including homologous arms at both ends is introduced into the cell, it can be integrated into the host genome through homologous recombination (HR) and can dictate wanted changes in the cell [ 10 ]. Employing HR alone in genetic modification posed many problems and limitations including inefficient integration of external DNA and random incorporation in undesired genomic location. Consequently, the number of cells with modified genome was low and uneasy to locate among millions of cells. Evidently, it was necessary to develop a procedure by which scientists can promote output. Out of these limitations, a breakthrough came when it was figured out that, in eukaryotic cells, more efficient and accurate gene targeting mechanisms could be attained by the induction of a double stranded break (DSB) at a specified genomic target [ 70 ].

Furthermore, scientists found that if an artificial DNA restriction enzyme is inserted into the cell, it cuts the DNA at specific recognition sites of double-stranded DNA (dsDNA) sequences. Thus, both the HR and non-homologous end joining (NHEJ) repair can be enhanced [ 14 ]. Various gene editing techniques have focused on the development and the use of different endonuclease-based mechanisms to create these breaks with high precision procedures [ 53 , 78 ] (Fig. 1 ). The mode of action of what is known as site-directed nucleases is based on the site-specific cleavage of the DNA by means of nuclease and the triggering of the cell’s DNA repair mechanisms: HR and NHEJ.

Genome editing outcomes. Genome editing nucleases induce double-strand breaks (DSBs). The breaks are repaired through two ways: by non-homologous end joining (NHEJ) in the absence of a donor template or via homologous recombination (HR) in the presence of a donor template. The NHEJ creates few base insertions or deletion, resulting in an indel, or in frameshift that causes gene disruption. In the HR pathway, a donor DNA (a plasmid or single-stranded oligonucleotide) can be integrated to the target site to modify the gene, introducing the nucleotides and leading to insertion of cDNA or frameshifts induction. (Adapted from [ 78 ])

One of the limitations in this procedure is that it has to be activated only in proliferating cells, adding that the level of activity depends on cell type and target gene locus [ 72 ]. Tailoring of repair templates for correction or insertion steps will be affected by these differences. Several investigations have determined ideal homology-directed repair (HDR) donor configurations for specific applications in specific models systems [ 67 ]. The differences in the activities of the DNA repair mechanisms will also influence the efficiency of causing indel mutations through NHEJ or the classical microhomology-mediated end joining (c-MMEJ) pathway, and even the survival of the targeted cells. The production of such repair in the cell is a sign of a characteristic that errors may occur during splicing the ends and cause the insertion or deletion of a short chain. Simply speaking, gene editing tools involve programmed insertion, deletion, or replacement of a specific segment of in the genome of a living cell. Potential targets of gene editing include repair of mutated gene, replacement of missing gene, interference with gene expression, or overexpression of a normal gene.

The human genome developments paved the way to more extensive use of the reverse genetic analysis technique. Nowadays, two methods of gene editing exist: one is called “targeted gene replacement” to produce a local change in an existing gene sequence, usually without causing mutations. The other one involves more extensive changes in the natural genome of species in a subtler way.

In the field of targeted nucleases and their potential application to model and non-model organisms, there are four major mechanisms of site-specific genome editing that have paved the way for new medical and agricultural breakthroughs. In particular, meganucleases (MegNs), zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas-9) (Fig. 2 ).

Schematic diagram of the four endonucleases used in gene editing technologies. a Meganuclease (MegN) that generally cleaves its DNA substrate as a homodimer. b Zinc finger nuclease (ZFN) recognizes its target sites which is composed of two zinc finger monomers that flank a short spacer sequence recognized by the FokI cleavage domain. c Transcription activator-like effector nuclease (TALEN) consists of two monomers; TALEN recognizes target sites which flank a fok1 nuclease domain to cut the DNA. d CRISPR/Cas9 system is made of a Cas9 protein with two nuclease domains: human umbilical vein endothelium cells (HuvC) split nuclease and the HNH, an endonuclease domain named for the characteristic histidine and asparagine residue, as well as a single guide RNA (sgRNA). (Adapted from [ 1 , 51 ]; Gaj et al., 2016 [ 53 ];)

Meganucleases (MegNs)

Meganucleases (MegNs) are naturally occurring endodeoxyribonucleases found within all forms of microbial life as well as in eukaryotic mitochondria and chloroplasts. The genes that encode MegNs are often embedded within self-splicing elements. The combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. The high specificity of these enzymes is based on their ability to cleave dsDNA at specific recognition sites comprising 14–40 bp (Fig. 2 a). Unlike restriction enzymes, which provide defenses to bacteria against invading DNA, MegNs facilitate lateral mobility of genetic elements within an organism. This process is referred to as “homing” and gives the name homing endonucleases to these enzymes. The high DNA specificity of MegNs makes them a powerful protein scaffold to engineer enzymes for genome manipulation. A deep understanding of their molecular recognition of DNA is an important prerequisite to generate engineered enzymes able to cleave DNA in specific desired genome sites. Crystallographic analyses of representatives from all known MegNs families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. The functional capabilities of these enzymes in DNA recognition vary widely across the families of MegNs. In each case, these capabilities, however, make a balance between what is called orthogonal requirements of (i) recognizing a target of adequate length to avoid overt toxicity in the host, while (ii) accommodating at least a small amount of sequence drift within that target. Indirect readout in protein-DNA recognition is the mechanism by which the protein achieves partial sequence specificity by detecting structural features on the DNA.

Several homing endonucleases have been used as templates to engineer tools that cleave DNA sequences other than their original wild-type targets.

Meganucleases can be divided into five families based on sequence and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D/E) XK [ 74 ]. I-CreI is a homodimeric member of MegNs family, which recognizes and cleaves a 22-bp pseudo-palindromic target (5′-CAAAACGTCGTGAGACAGTTTG-3′). The important role of indirect readout in the central region of the target DNA of these enzymes I-CreI suggested that indirect readout may play a key role in the redesign of protein-DNA interactions. The sequences of the I-CreI central substrate region, four bp (± 1 and ± 2) called 2NN, along with the adjacent box called 5NNN, are key for substrate cleavage [ 64 ]. Changes in 2NN significantly affect substrate binding and cleavage because this region affects the active site rearrangement, the proper protein-DNA complex binding, and the catalytic ion positioning to lead the cleavage.

An exhaustive review of each MegN can be found in Stoddard [ 75 ] as well as in Petersen and Niemann [ 63 ]. Several MegNs have been used as templates to engineer tools that cleave DNA sequences other than their original wild-type targets. This technology have advantages of high specificity of MegNs to target DNA because of their very long recognition sites, ease in delivery due to relatively small size, and giving rise to more recombinant DNA (i.e., more recombinogenic for HDR) due to production of a 3′ overhang after DNA cleavage. This lowers the potential cytotoxicity [ 53 , 78 ].

Meganucleases have several promising applications; they are more specific than other genetic editing tools for the development of therapies for a wide range of inherited diseases resulting from nonsense codons or frameshift mutations. However, an obvious drawback to the use of natural MegNs lies in the need to first introduce a known cleavage site into the region of interest. Additionally, it is not easy to separate the two domains of MegNs: the DNA-binding and the DNA-cleavage domains, which present a challenge in its engineering. Another drawback of MegNs is that the design of sequence-specific enzymes for all possible sequences is time-consuming and expensive. Therefore, each new genome engineering target requires an initial protein engineering step to produce a custom MegN. Thus, in spite of the so many available MegNs, the probability of finding an enzyme that targets a desired locus is very small and the production of customized MegNs remains really complex and highly inefficient. Therefore, routine applications of MegNs in genome editing is limited and proved technically challenging to work with [ 24 ].

Zinc finger nucleases (ZFNs)

The origin of genome editing technology began with the introduction of zinc finger nucleases (ZFNs). Zinc finger nucleases are artificially engineered restriction enzymes for custom site-specific genome editing. Zinc fingers themselves are transcription factors, where each finger recognizes 3–4 bases. Zinc finger nucleases are hybrid heterodimeric proteins, where each subunit contains several zinc finger domains and a Fok1 endonuclease domain to induce DSB formation. The first is zinc finger, which is one of the DNA binding motifs found in the DNA binding domain of many eukaryotic transcription factors responsible for DNA identification. The second domain is a nuclease (often from the bacterial restriction enzyme FokI) [ 6 ]. When the DNA-binding and the DNA-cleaving domains are fused together, a highly specific pair of “genomic scissors” is created (Fig. 2b ). In principle, any gene in any organism can be targeted with a properly designed pair of ZFNs. Zinc finger recognition depends only on a match to DNA sequence, and mechanisms of DNA repair, both HR and NHEJ, are shared by essentially all species. Several studies have reported that ZFNs with a higher number of zinc fingers (4, 5, and 6 finger pairs) have increased the specificity and efficiency and improved targeting such as using modular assembly of pre-characterized ZFs utilizing standard recombinant DNA technology.

Since they were first reported [ 41 ], ZFN was appealing and showed considerable promise and they were used in several living organisms or cultured cells [ 11 ]. The discovery of ZFNs overcame some of the problems associated with MegNs applications. They facilitated targeted editing of the gene by inducing DSBs in DNA at specific sites. One major advantage of ZFNs is that they are easy to design, using combinatorial assembly of preexisting zinc fingers with known recognition patterns. This approach, however, suffered from drawbacks for routine applications. One of the major disadvantages of the ZFN is what is called “context-dependent specificity” (how well they cleave target sequence). Therefore, these specificities can depend on the context in the adjacent zinc fingers and DNA. In other terms, their specificity does not only depend on the target sequence itself, but also on adjacent sequences in the genome. This issue may cause genome fragmentation and instability when many non-specific cleavages occur. It only targets a single site at a time and as stated above. Although the low number of loci does not usually make a problem for knocking-out editing, it poses limitation for knocking in manipulation [ 32 ]. In addition, ZFNs cause overt toxicity to cells because of the off-target cleavages. The off-target effect is the probability of inaccurate cut of target DNA due to single nucleotide substitutions or inappropriate interaction between domains.

Transcription activator-like effector nucleases (TALENs)

The limitations mentioned in the previous section paved the way for the development of a new series of nucleases: transcription activator-like effector nucleases (TALENs), which were cheaper, safer, more efficient, and capable of targeting a specified region in the genome [ 13 ].

In principle, the TALENs are similar to ZFNs and MegNs in that the proteins must be re-engineered for each targeted DNA sequence. The ZFNs and TALENs are both modular and have natural DNA-binding specificities. The TALEN is similar to ZFN in that it is an artificial chimeric protein that result from fusing a non-specific FokI restriction endonuclease domain to a DNA-binding domain recognizing an arbitrary base sequence (Fig. 2c ). This DNA-binding domain consists of highly conserved repeats derived from transcription activator-like effectors (TALE). When genome editing is planned, a pair of TALEN is used like ZFNs. The TALE protein made of three domains: an amino-terminal domain having a transport signal, a DNA-binding domain which is made of repeating sequences of 34 amino acids arranged in tandem, and a carboxyl-terminal domain having a nuclear localization signal and a transcription activation domain. Of the 34 amino acids, there is a variable region of two amino acid residues located at positions 12 and 13 called repeat variable di-residues (RVD). This region has the ability to confer specificity to one of the any four nucleotide bps [ 15 ].

Unlike ZFNs, TALENs had advantages in that one module recognizes just one nucleotide in its DNA-binding domain, as compared with 3 bps recognized by the first single zinc finger domains [ 39 ]. So, interference of the recognition sequence does not occur even when several modules are joined. In theory, because cleavage of the target sequence is more specific than ZFN, it became possible to target any DNA sequence of any organism genome. This difference facilitates creation of TALEN systems which recognize more target sequences. Another benefit of the TALEN system over ZFN’s for genome editing is that the system is more efficient in producing DSBs in both somatic cells and pluripotent stem cells [ 35 ]. In addition, TALENs exhibit less toxicity in human cell lines due to off-target breaks that result in unwanted changes and toxicity in the genome. Another advantage of TALENs is a higher percentage of success in genome editing through cytoplasmic injection of TALEN mRNA in livestock embryos than observed with ZFN induction [ 39 ]. In addition, TALENs have been more successfully used in plant genome engineering [ 88 ]. It is hoped that TALENs will be applied in the generation of genetically modified laboratory animals, which may be utilized as a model for human disease research [ 24 , 39 ].

The TALEN-like directed development of DNA binding proteins was employed to improve TALEN specificity by phage-assisted continuous evolution (PACE). The improved version was used to create genetically modified organisms [ 34 ]. Nucleases which contain designable DNA-binding sequences can modify the genomes and have the promise for therapeutic applications. DNA-binding PACE is a general strategy for the laboratory evolution of DNA-binding activity and specificity. This system can be used to generate TALEN with highly improved DNA cutting specificity, establishing DB-PACE as a diverse approach for improving the accuracy of genome editing tools. Thus, similar to ZFN, TALEN is used for DSBs as well as for knocking in/knocking out. In comparison with the ZFN, two important advantages for this editing technique have been reported: first, the simple design, and second, the low number of off-target breaks [ 35 ].

In spite of the improvement and simplification of the TALEN method, it is complicated for whom not familiar with molecular biological experiments. Moreover, it is confronted with some limitations, such as their large size (impeding delivery) in comparison to ZFN [ 24 , 39 ]. The superiority of TALEN relative to ZFN could be attributed to the fact that in the TALEN each domain recognizes only one nucleotide, instead of recognizing DNA triplets in the case of ZEF. The design of TALEN is commonly more obvious than ZNF. This results in less intricate interactions between the TALEN-derived DNA-binding domains and their target nucleotides than those among ZNF and their target trinucleotides [ 35 , 39 ].

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)

The CRISPR/Cas system is the most recent platform in the field of genome editing. The system was developed in 2013 and is known as the third generation genomic editing tools. The clustered regularly interspaced short palindromic repeats, which are sometimes named “short regularly spaced repeats” were discovered in the 1980s. Computational analysis of these elements showed they were found in more than 40% of sequenced bacteria and 90% of archaea [ 37 , 56 ]. The acronym CRISPR was suggested, and a group of genes adjacent to the CRISPR locus, which was termed “CRISPR-associated system”, or Cas was established [ 37 ]. Cas proteins coded by these genes carry functional domains similar to endonucleases, helicases, polymerases, and nucleotide-binding proteins. In addition, the role of CRISPRs as bacterial and archaeal adaptive immunity system against invading bacteriophages and other and in DNA repair was realized [ 17 , 77 ].

Unlike the two previous technologies (ZFN and TALEN), in which the recognition of the DNA site was based on the sequence recognition by artificial proteins requiring interaction between protein and DNA, the DNA recognition of the CRISPR/Cas system is based on RNA-DNA interactions. This offers several advantages over ZFNs and TALENs. These include easy design for any genomic targets, easy prediction regarding off-target sites, and the probability of modifying several genomic sites simultaneously (multiplexing). CRISPR-Cas systems are diverse and have been classified thus far into two classes, six types, and over 20 subtypes based on locus arrangement and signature cas genes [ 33 , 44 , 51 ]. Types I, III, and IV, with multiprotein crRNA-effector complexes, are class 1 systems; types II, V, and VI, with a single protein-crRNA effector complex, are class 2. All CRISPR-Cas systems require Cas proteins and crRNAs for function, and CRISPR- cas expression is a prerequisite to acquire new spacers, process pre-crRNA, and assemble ribonucleoprotein crRNA interference complexes for target degradation. Herein, we will focus on the CRISPR-Cas9 technology, the reader should keep in mind other available variants of the system such as CRISPR-Cas6 [ 5 ], CRISPR-Cas12a, -Cas12b [ 42 ], as well as the most recently discovered c2c2 (Cas13a) and c2c6 (Cas13b [ 19 , 69 ]. The CRISPR/Cas9 system is made of Cas9 nuclease and single-guide RNA (sgRNA). The sgRNA is an engineered single RNA molecule containing crispr RNA and tracr RNA parts. The sgRNA recognizes the target sequence by standard Watson-Crick base pairing. It has to be followed by a DNA motif called a protospacer adjacent motif (PAM). The commonly used wild-type Streptococcus pyogenes Cas (SpCas9) protein has a specific PAM sequence, 5’-NGG-3’, where “N” can be any nucleotide base followed by two guanine (“G”) nucleobases. This sequence is located directly downstream of the target sequence in the genomic DNA, on the non-target strand. Targeting is constrained to every 14 bp (12 bp from the seed sequence and 2 bp from PAM) [ 15 ]. SpCas9 variants may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences when used in place of wild-type SpCas9.

DNA cleavage is performed by Cas9 nuclease and can result in DSB in the the case of a wild-type enzyme, or in a SSB when using mutant Cas9 variants called nickases (Fig. 2d ). It should be emphasized that the utilization of this approach in editing eukaryotes’ genome only needs the manipulation of a short sequence of RNA, and there is no need for complicated manipulations in the protein domain. This enables a faster and more cost-effective design of the DNA recognition moiety compared with ZFN and TALEN technologies. Applications of CRISPR-Cas9 systems are variable like those for ZFNs, TALENs, and MegNs. But, because of the relative simplicity of this system, its great efficiency and high tendency for multiple functions and library construction, it can be applied to different species and cell types [ 35 ].

As shown in Fig. 3 , in all CRISPR/Cas systems, immunity occurs in three distinct stages [ 77 , 81 ]: (1) adaptation or new spacer acquisition, (2) CRISPR transcription and processing (crRNA generation), and (3) interference or silencing. The advantages of the CRISPR/Cas system superseded those of both of the TALEN and ZFN tools, the ZFN in particular. This is due to its target design simplicity since the target specificity depends on ribonucleotide complex formation and non-protein/DNA recognition. In addition, the CRISPR/Cas approach is more efficient because changes can be introduced directly by injecting RNAs that encode the Cas protein and gRNA into developing embryos. Moreover, multigene mutations can be induced simultaneously by injecting them with multiple gRNAs. This is an example that explains the rapid spread of CRISPR/Cas 9 application in various fields. Still, the system has certain drawbacks. Although the CRISPR/Cas9 is much less complicated than TALEN, in terms of execution and construction, the off-target effect in CRISPR/Cas9 is higher than TALEN. Since the DSB results only after accurate binding of a pair of TALEN to the target sequence, the off-target effect problem is considered to be low. These two are different in restriction of target sequence. CRISPR/Cas9 is much more efficient than TALEN in multiple simultaneous modification. Table 1 compares the three main systems of site-directed synthetic nuclease employed in genome editing: ZFN, TALEN, and CRISPR/Cas9.

Schematic representation of CRISPR loci and targeting of DNA sequence, which include Cas genes, a leader sequence, and several spacer sequences derived from engineered or foreign DNA that are separated by short direct repeat sequences. The three major steps of CRISPR-Cas immune systems. In the adaptation phase, Cas proteins excise specific fragments from foreign DNA and integrate it into the repeat sequence neighboring the leader at the CRISPR locus. Then, CRISPR arrays are transcribed and processed into multiple crRNAs, each carrying a single spacer sequence and part of the adjoining repeat sequence. Finally, at the interference phase, the crRNAs are assembled into different classes of protein targeting complexes (cascades) that anneal to, and cleave, spacer matching sequences on either invading element or their transcripts and thus destroy them. (Adapted from [ 3 , 53 , 78 ])

The off-target effect is an essential subject for future studies if CRISPR/Cas9 is to achieve its promises as a powerful method for genome editing. Non-specific and unintended genetic modifications (off-target effect) can result from the use of CRISPR/Cas9 system which is one of the drawbacks of this tool. Therefore, this point should be considered for use in researches. One strategy to reduce the off-target activity is to replace the Streptococcus pyogenes Cas9 enzyme (SpyCas9) for a mutant Cas9 nickase (nSpyCas9; ncas9), which cleaves a single strand through the inactivation of a nuclease domain Ruvc or HNH [ 9 ]. Our understanding of off-target effects remains fragmentary. A deeper understanding of this phenomenon is needed. Several approaches that could be followed to characterize the binding domains and consequently Cas9 targeting specificity have been reviewed and summarized [ 83 ].

It has previously been stated that CRISPR/Cas9 system needs both gRNA and PAM to detect its target sequence of interest by integration of a gRNA component that binds to complementary double-stranded DNA sequences. Cell culture studies have shown that off-target effects may be due to the incorrect detection of genomic sequences by sgRNA. This, in turn, affects cleavage when the mismatch is in the vicinity of the PAM (up to 8 bases), but if the PAM is too far apart, these effects will be small [ 4 ], even a slight mismatch between sgRNA and target sequences can lead to a failure. Dependence of this method on specific PAM sequences to act functionally limits the number of target loci, and it can reduce off-target breaks [ 86 ]. For this goal, another type of specific PAM-containing nucleases has been prepared to compensate for this limitation. Genetic engineering and enzyme changing have also been able to overcome the limitation [ 42 ]. For a sgRNA, many similar sequences depending on the genome size of the species may exist [ 86 ]. Interestingly, the initial targeting scrutiny of the CRISPR/Cas9-sgRNA complex showed that not every nucleotide base in the gRNA is necessary to be complementary to the target DNA sequence to effect Cas9 nuclease activity. Regarding that where the similar sequences are found in the genome, their breaks could lead to malignancies or even death [ 86 ]. Various methods have been proposed to prevent off-target breaks, among which the double nicking method, the FokI-dCas9 fusion protein method, and the truncated sgRNA method [ 76 ] (Fig. 4 ).

a Summary of the Cas9 nickases methods in efficient genome editing. Two gRNAs target opposite strands of DNA. These double nicks create a DSB that is repaired using non-homologous end joining (NHEJ) or edits via homology-directed repair (HDR) (adapted from www.addgene.org/crispr/nick ). b FokI-dCas 9 fusion protein method. Two FokI-dCas9 fusion proteins are used to adjacent target sites by two different sgRNAs to facilitate FokI dimerization and DNA cleavage. These fusions would have enhanced specificity compared to the standard monomeric Cas9 nucleases and the paired nickase system because they should require two sgRNAs for activity. c Truncated sgRNA method. Cas9 interacting with either a full-length sgRNA (20 nucleotide sequence complementary to target site) or truncated gRNA (less than 15 nucleotide sequence complementary to target site). (Retrieved from blog.addgene.org )

To overcome these problems, researchers explored another generation of base editing technologies, which combine CRISPR and cytidine deaminase (Fig. 5 ). This is a diverse method called CRISPR-SKIP (Fig. 6 ) which uses cytidine deaminase single-base editors to program exon skipping by mutating target DNA bases within splice acceptor sites [ 25 ]. Given its simplicity and precision, CRISPR-SKIP will be widely applicable in gene therapy. Base editing utilizes Cas9 D10A nickases fused to engineered base deaminase enzymes to make single base changes in the DNA sequence without the need of DNA DSB. Also, base editing does not require an external repair template. The Cas9 nickase part of the base editor protein plays a dual function. The first is to target the deaminase activity to the wanted region and the second is to localize the enzyme to certain regions of double-stranded RNA. The deaminase domains in base editors (BEs) occur in two versions: either adenosine deaminase or cytosine deaminase, which catalyze only base transitions (C to T and A to G) and cannot produce base transversions [ 26 , 68 ]. In these base editing tools, the targeted activity of adenosine deaminase can result in an A:T to G:C sequence alteration in a very similar way [ 26 , 68 ].This approach avoided the requirement of breaking DNA to induce an oligonucleotide. In addition, compared to knocking system, it exerted a higher output with lower off-targets [ 40 , 43 ]. Adenosine is deaminated to inosine (I) that is subsequently utilized to repair the nicked strand with a cytosine, and the I:C base pair is resolved to G:C [ 26 ]. More recently, new genome editing technologies have been developed: glycosylase base editors (GBEs), which consist of a Cas9 nickase, a cytidine deaminase, and a uracil-DNA glycosylase (Ung), are capable of transversion mutations by changing C to A in bacterial cells and from C to G in mammalian cells [ 45 , 89 ]. The new BEs can also be designed to minimize unwanted (“off-target”) mutations that could potentially cause undesirable side effects. The novel BE platform may help researchers understand and correct genetic diseases by selective editing of single DNA “alphabets” across nucleobase classes. However, the technique with this new class of transversion BEs is still at an early stage and requires additional optimization, so it would be premature to say this is ready for the clinic applications.

Base editing uses engineered Cas9 variants to induce base changes in a target sequence. Cas9 nickase is fused to a base deaminase domain. The deaminase domain works on a targeted region within the R-loop after target binding and R-loop formation. Simultaneously, the target strand is nicked. DNA repair is started in response to the nick using the strand which contains the deaminated base as a repair template. Repair leads to a transition mutations: C:G to T:A and A:T to G:C for cytosine and adenosine base editors, respectively [ 68 ]

Essential steps in CRISPR-SKIP targeting approach: a Nearly every intron ends with a guanosine (asterisked G). It is hypothesized that mutations that disrupt this highly conserved G within the splice acceptor of any given exon in genomic DNA would lead to exon skipping by preventing incorporation of the exon into mature transcripts base. b In the presence of an appropriate PAM sequence, this G can be effectively mutated by converting the complementary cytidine to thymidine using CRISPR-Cas9 C>T single-base editors. (From [ 25 ])

Gene delivery

From biotechnology’s point of view, the main obstacle that is facing molecular technology is to select the right method that is simple but effective to transfer the gene to the host cell. The components of gene editing have to be transferred to the cell/nucleus of interest using in vivo, ex vivo, or in vitro route. In this regard, several concerns must be considered including physical barriers (cell membranes, nuclear membranes) as well as digestion by proteases or nucleases of the host. Another important issue is the possible rejection by the immune system of the host if the components are delivered in vivo. In general, the gene delivery routes can be categorized in three classes of physical delivery, viral vectors, and non-viral agents. Although the direct delivery of construct plasmids may sound easy and more efficient and specific than the physical and the chemical methods, it proves to be an inappropriate choice because the successful gene delivery system requires the foreign genetic molecule to remain stable within the host cells [ 52 ]. The other possible procedure is to use viruses. However, because plant cells have thick walls, the gene transfer systems for plants involve transient and stable transformation using protoplast-plasmid in vitro [ 54 ]: agrobacterium-mediated transformation, gene gun and viral vectors (transient expression by protoplast transformation), and agro-infiltration [ 1 ]. Viruses may present a suitable vehicle to transfer genome engineering components to all plant parts because they do not require transformation and/or tissue culture for delivering and mutated seeds could easily recovered. For many years, scientists employed different species of Agrobacterium to systematically infect a large number of plant species and generate transgenic plants. These bacterial species have small genome size and this facilitates cloning and agroinfections, and the virus genome does not integrate into plant genomes [ 1 ].

Of the challenges and approaches of delivering CRISPR, it was pointed out [ 18 , 51 ] that although the present genome engineering is in favor of CRISPR tools, TALENs may still be of a primary choice in certain experimental species. For example, TALENs have been utilized in targeted genomic editing in Xenopus tropicalis by knocking-out Klf4 [ 49 , 50 ] or thyroid hormone receptor α [ 23 ]. In addition, TALENs have been utilized to modify genome of human stem cells [ 47 ]. Also TALEN approach has been applied to create amniotic mesenchymal stem cells overexpressing anti-fibrotic interleukin-10 [ 12 ]. Lately, a geminivirus genome has been prepared to deliver various nucleases platforms (including ZFN, TALENs, and the CRISPR/Cas system) and repair template for HR of DSBs [ 62 ].

To deliver the carrying DNA sequence to target cells, non-viral techniques such as electroporation, lipofection, and microinjection can also be used [ 18 ]. In addition, these techniques also reduce off-target cleavages problems. Gene transfer via microinjection is considered the gold standard procedure since its efficiency is approximately 100% [ 85 ]. The advantage of this approach is its high efficacy and less constrains on the size of the delivery. A disadvantage is that it can be employed only in in vitro or ex vivo cargo. Recently, small RNAs, including small interfering RNA (siRNA) and microRNA (miRNA), have been widely adopted in research to replace laboratory animals and cell lines. Development of innovative nanoparticle-based transfer systems that deliver CRISPR/Cas9 constructs and maximize their effectiveness has been tested in the last few years [ 29 , 58 ].

Applications of gene technology

The ability of the abovementioned gene delivery systems to target and manipulate the genome of living organisms has been attractive to many researchers worldwide. Despite all limitations, the interest in this technology has developed its capabilities and enhanced its scope of applications. Genome/gene engineering technology is relatively applicable and has potential to effectively and rapidly revolutionize genome surgery and will soon transform agriculture, nutrition, and medicine. Some of the most important applications are briefly described below.

Plant-based genome editing

The appearance of genome editing has been appealing especially to agricultural experts. One of the major goals for utilizing genome editing tools in plants is to generate improved crop varieties with higher yields and clear-cut addition of valuable traits such as high nutritional value, extended shelf life, stress tolerance, disease and pest resistance, or removal of undesirable traits [ 1 ]. However, several obstacles related to the precision of the genetic manipulations and the incompatibility of the host species have hampered the development of crop improvements [ 2 ]. The use of site-specific nucleases is one of the important promising techniques of gene editing that helped overcome certain limitations by specifically targeting a suitable site in a gene/genome. The employment of the gene editing technologies, including those discussed in this review, seems to be endless ever since their emergence, and several improvements in original tools have further brought accuracy and precision in these methods [ 78 ].

Animal-based genome editing

Recent genome editing techniques has been extensively applied in many organisms, such as bacteria, yeast, and mouse [ 53 , 73 ]. Genetic manipulation tools cover a wide range of fields, including the generation of transgenic animals using embryonic stem cells (ESC), functional analysis of genes, model development for diseases, or drug development. Genome editing techniques have been used in many various organisms. Among the livestock and aquatic species, ZFN is only used for zebrafish, but two other technologies, TALEN and CRISPR, have been used at the cell level in chicken, sheep, pig, and cattle. Engineered endonucleases or RNA-guided endonucleases (RGENs) mediated gene targeting has been applied directly in a great number of animal organisms including nematodes and zebrafish [ 20 , 57 ], as well as pigs [ 71 , 85 ]. Since the first permission to use CRISPR/Cas9 in human embryos and in vivo genome editing via homology-independent targeted integration (HITI), an increasing number of studies have identified striking differences between mouse and human pre-implantation development and pluripotency [ 66 ], highlighting the need for focused studies in human embryos. Therefore, more specific criteria and widely accepted standards for clinical research have to be met before human germline editing would be deemed permissible [ 31 ]. In this regard, results of some research on the human genome editing have been questioned. The “He Jiankui experiments at the beginning of 2019”, which claimed to have created the world’s first genetically edited babies, is simply the most recent example. He Jiankui said he edited the babies’ genes at conception by selecting CRISPR/cas9 to edit the chemokine receptor type 5 (CCR5) gene in cd4+ cells in hopes of making children resistant to the AIDS virus, as their father was HIV-positive. Researchers said He’s actions exposed the twins to unknown health risks, possibly including a higher susceptibility to viral illnesses. For more information on the scientific reactions around the world, the reader may find helpful several excellent sources of information [ 38 , 49 , 79 , 84 ].

• Gene therapy

The original principles of gene therapy arose during the 1960s and early 1970s when restriction enzymes were utilized to manipulate DNA [ 22 ]. Since then, researchers have done great efforts to treat genetic diseases but treatment for multiple mutations is difficult. Different clinical therapy applications have been attempted to overcome these problems. Much of the interest in CRISPR and other gene editing methods revolves around their potential to cure human diseases. It is hoped that eradication of human diseases is not too far to achieve via the CRISPR system because it was employed in other fields of biological sciences such as genetic improvement and gene therapy. It is important to mention that the therapeutic efficiency of gene editing depends on several factors, such as editing efficacy, which varies widely depending on the cell type, senescence status, and cell cycle status of the target [ 69 ]. Other factors that also influence therapeutic effectiveness include cell aptitude, which refers to the feasibility of accomplishing a therapeutic modification threshold, and the efficient transfer of programmable nuclease system to the target tissue, which is only considered to be effective if the engineered nuclease system reaches safely and efficiently to the nucleus of the target cell. Finally, the precision of the editing procedure is another important aspect, which refers to only editing the target DNA without affecting any other genes [ 80 ].

The genome editing tools have enabled scientists to utilize genetically programmed animals to understand the cause of various diseases and to understand molecular mechanisms that can be explored for better therapeutic strategies (Fig. 7 ). Genome editing gives the basis of the treatment of many kinds of diseases. In preliminary experiments, the knocking-in procedure was used to reach this goal. There are examples of gene editing techniques applied in different genetic diseases in cell lines, disease models, and human [ 48 , 53 , 82 ]. These encouraging results suggest the therapeutic capability of these gene editing strategies to treat human genetic diseases including Duchenne muscular dystrophy [ 8 , 28 , 55 ], cystic fibrosis [ 21 ], sickle cell anemia [ 62 ], and Down syndrome [ 7 ]. In addition, this technology has been employed in curing Fanconi anemia by correcting point mutation in patient-derived fibroblasts [ 60 ], as well as in hemophilia for the restoration of factor VIII deficiency in mice [ 61 , 87 ]. The CRISPR tools have also demonstrated promising results in diagnosis and curing fatal diseases such as AIDS and cancer [ 16 , 30 , 84 ].

Outline of the ex vivo and in vivo genome editing procedures for clinical therapy. Top: In the ex vivo editing therapy, cells are removed from a patient to be treated, corrected by gene editing and then re-engrafted back to the patient. To achieve therapeutic success, the target cells must be capable of surviving in vitro and autologous transplantation of the corrected cells. Below: In the in vivo editing therapy, designed nucleases are administered using viral or non-viral techniques and directly injected locally to the affected tissue, such as the eye, brain, or muscle. (Adapted from [ 48 ])

Other applications

The applications mentioned above were more about knock out or modification of genes Gapinske et al. [ 25 ]. However due to inactivate nuclease activity nature of the dCas9, CRISPR can be used in other applications as well. By selecting the target sequence, gene expression can be controlled by inhibiting the transcription rate of RNA polymerase II (polII) or inhibiting the transcription factor binding [ 65 ]. Additionally, combining gene expression inhibitors such as Krüppel-associated box with the inactivated Cas9 has led to generate a special kind of gene inhibitors, which are called CRISPR interference (CRISPRi), and downregulate gene expression [ 46 ]. It is also possible to control gene expression by fusing transcription-activating molecule, the transcription-repressing molecule, or the genome-modifying molecule to dCas9 [ 27 ].

Genome editing is a fast-growing field. Editing nucleases have revolutionized genomic engineering, allowing easy editing of the mammalian genome. Much progress has been accomplished in the improvement of gene editing technologies since their discovery. Of the four major nucleases used to cut and edit the genome, each has its own advantages and disadvantages, and the choice of which gene editing method depends on the specific situation. The current genome editing techniques are still buckling up with problems, and it is difficult to perform genome editing in cells with low transfection efficiency or in some cultured cells such as primary cultured cells. Genotoxicity is an inherent problem of enzymes that act on nucleic acids, though one can expect that highly specific endonucleases would reduce or abolish this issue. Exceptional efforts are needed in future to complement and offer something novel approaches in addition to the already existing ones. It is anticipated that research in gene editing is going to continue and tremendously advance. With the development of next-generation sequencing technology, new extremely important clinical applications, such as manufacturing engineered medical products, eradication of human genetic diseases, treatment of AIDS and cancers, as well as improvement of crop and food, will be introduced. Combination of genomic modifications induced by targeted nucleases to their own self-degradation, self-inactivating vectors may help overcoming confronting limitations discussed above to improve the specificity of genome editing, especially because the frequency of off-target modifications. Our understanding of off-target effects remains poor. This is a vital area for continued study if CRISPR/Cas9 is to realize its promise. Regarding gene cargo delivery systems, this remains the greatest obstacle for CRISPR/Cas9 use, and an all-purpose delivery method has yet to emerge. The union between genome engineering and regenerative medicine is still in its infancy; realizing the full potential of these technologies in reprograming the fate of stem/progenitor cells requires that their functional landscape be fully explored in these genetic backgrounds. Humankind can only wait to see what the potential of these technologies will be. One major question is whether or not the body’s immune response will accept or reject the foreign genetic elements within the cells. Another important concern is that along with the revolutionary advances of this biotechnology and related sciences, bioethical concerns and legal problems related to this issue are still increasing in view of the possibility of human genetic manipulation and the unsafety of procedures involved [ 49 , 50 , 66 ]. The enforcement of technical and ethical guidelines, and legislations should be considered and need serious attention as soon as possible.

Availability of data and materials

Not applicable

Abbreviations

CRISPR-associated protein 9

Clustered regularly interspaced short palindromic repeats

Double-stranded break

Embryonic stem cells

Homology-directed repair

Homology-independent targeted integration

Homologous recombination

Human umbilical vein endothelium cells

Intron-encoded endonuclease

• Meganucleases

Microhomology-mediated end joining

Non-homologous end joining

Phage-assisted continuous evolution

Protospacer adjacent motifs

RNA-guided endonucleases

Repeat variable di-residues

Single guide RNA

Streptococcus pyogenes Cas9

Single-strand break

Transcription activator-like effector nuclease

Zinc finger nucleases

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Article Contents

Introduction, human enhancement, genetic engineering, conclusions.

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Human enhancement: Genetic engineering and evolution

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Mara Almeida, Rui Diogo, Human enhancement: Genetic engineering and evolution, Evolution, Medicine, and Public Health , Volume 2019, Issue 1, 2019, Pages 183–189, https://doi.org/10.1093/emph/eoz026

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Genetic engineering opens new possibilities for biomedical enhancement requiring ethical, societal and practical considerations to evaluate its implications for human biology, human evolution and our natural environment. In this Commentary, we consider human enhancement, and in particular, we explore genetic enhancement in an evolutionary context. In summarizing key open questions, we highlight the importance of acknowledging multiple effects (pleiotropy) and complex epigenetic interactions among genotype, phenotype and ecology, and the need to consider the unit of impact not only to the human body but also to human populations and their natural environment (systems biology). We also propose that a practicable distinction between ‘therapy’ and ‘enhancement’ may need to be drawn and effectively implemented in future regulations. Overall, we suggest that it is essential for ethical, philosophical and policy discussions on human enhancement to consider the empirical evidence provided by evolutionary biology, developmental biology and other disciplines.

Lay Summary: This Commentary explores genetic enhancement in an evolutionary context. We highlight the multiple effects associated with germline heritable genetic intervention, the need to consider the unit of impact to human populations and their natural environment, and propose that a practicable distinction between ‘therapy’ and ‘enhancement’ is needed.

There are countless examples where technology has contributed to ameliorate the lives of people by improving their inherent or acquired capabilities. For example, over time, there have been biomedical interventions attempting to restore functions that are deficient, such as vision, hearing or mobility. If we consider human vision, substantial advances started from the time spectacles were developed (possibly in the 13th century), continuing in the last few years, with researchers implanting artificial retinas to give blind patients partial sight [ 1–3 ]. Recently, scientists have also successfully linked the brain of a paralysed man to a computer chip, which helped restore partial movement of limbs previously non-responsive [ 4 , 5 ]. In addition, synthetic blood substitutes have been created, which could be used in human patients in the future [ 6–8 ].

The progress being made by technology in a restorative and therapeutic context could in theory be applied in other contexts to treat non-pathological conditions. Many of the technologies and pharmaceutical products developed in a medical context to treat patients are already being used by humans to ‘enhance’ some aspect of their bodies, for example drugs to boost brain power, nutritional supplements, brain stimulating technologies to control mood or growth hormones for children of short stature. Assistive technology for disabled people, reproductive medicine and pharmacology, beside their therapeutic and restorative use, have a greater potential for human ‘enhancement’ than currently thought. There are also dual outcomes as some therapies can have effects that amount to an enhancement as for example, the artificial legs used by the South African sprinter Oscar Pistorius providing him with a competitive advantage.

This commentary will provide general ethical considerations on human enhancement, and within the several forms of so-called human biomedical enhancement, it will focus on genetic engineering, particularly on germline (heritable) genetic interventions and on the insights evolutionary biology can provide in rationalizing its likely impact. These insights are a subject often limited in discussions on genetic engineering and human enhancement in general, and its links to ethical, philosophical and policy discussions, in particular [ 9 ]. The rapid advances in genetic technology make this debate very topical. Moreover, genes are thought to play a very substantial role in biological evolution and development of the human species, thus making this a topic requiring due consideration. With this commentary, we explore how concepts based in evolutionary biology could contribute to better assess the implications of human germline modifications, assuming they were widely employed. We conclude our brief analysis by summarizing key issues requiring resolution and potential approaches to progress them. Overall, the aim is to contribute to the debate on human genetic enhancement by looking not only at the future, as it is so often done, but also at our evolutionary past.

The noun ‘enhancement’ comes from the verb ‘enhance’, meaning ‘to increase or improve’. The verb enhance can be traced back to the vulgar Latin inaltiare and late Latin inaltare (‘raise, exalt’), from ‘ altare ’ (‘make high’) and altus (‘high’), literally ‘grown tall’. For centuries human enhancement has populated our imagination outlined by stories ranging from the myths of supernormal strengths and eternal life to the superpowers illustrated by the 20th century comic books superheroes. The desire of overcoming normal human capacities and the transformation to an almost ‘perfect’ form has been part of the history of civilization, extending from arts and religion to philosophy. The goal of improving the human condition and health has always been a driver for innovation and biomedical developments.

In the broadest sense, the process of human enhancement can be considered as an improvement of the ‘limitations’ of a ‘natural version’ of the human species with respect to a specific reference in time, and to different environments, which can vary depending on factors such as, for example, climate change. The limitations of the human condition can be physical and/or mental/cognitive (e.g. vision, strength or memory). This poses relevant questions of what a real or perceived human limitation is in the environment and times in which we are living and how it can be shifted over time considering social norms and cultural values of modern societies. Besides, the impact that overcoming these limitations will have on us humans, and the environment, should also be considered. For example, if we boost the immune system of specific people, this may contribute to the development/evolution of more resistant viruses and bacteria or/and lead to new viruses and bacteria to emerge. In environmental terms, enhancing the longevity of humans could contribute to a massive increase in global population, creating additional pressures on ecosystems already under human pressure.

Two decades ago, the practices of human enhancement have been described as ‘biomedical interventions that are used to improve human form or functioning beyond what is necessary to restore or sustain health’ [ 10 ]. The range of these practices has now increased with technological development, and they are ‘any kind of genetic, biomedical, or pharmaceutical intervention aimed at improving human dispositions, capacities, or well-being, even if there is no pathology to be treated’ [ 11 ]. Practices of human enhancement could be visualized as upgrading a ‘system’, where interventions take place for a better performance of the original system. This is far from being a hypothetical situation. The rapid progress within the fields of nanotechnology, biotechnology, information technology and cognitive science has brought back discussions about the evolutionary trajectory of the human species by the promise of new applications which could provide abilities beyond current ones [ 12 , 13 ]. If such a possibility was consciously embraced and actively pursued, technology could be expected to have a revolutionary interference with human life, not just helping humans in achieving general health and capabilities commensurate with our current ones but helping to overcome human limitations far beyond of what is currently possible for human beings. The emergence of new technologies has provided a broader range of potential human interventions and the possibility of transitioning from external changes to our bodies (e.g. external prosthesis) to internal ones, especially when considering genetic manipulation, whose changes can be permanent and transmissible.

The advocates of a far-reaching human enhancement have been referred to as ‘transhumanists’. In their vision, so far, humans have largely worked to control and shape their exterior environments (niche construction) but with new technologies (e.g. biotechnology, information technology and nanotechnology) they will soon be able to control and fundamentally change their own bodies. Supporters of these technologies agree with the possibility of a more radical interference in human life by using technology to overcome human limitations [ 14–16 ], that could allow us to live longer, healthier and even happier lives [ 17 ]. On the other side, and against this position, are the so-called ‘bioconservatives’, arguing for the conservation and protection of some kind of ‘human essence’, with the argument that it exists something intrinsically valuable in human life that should be preserved [ 18 , 19 ].

There is an ongoing debate between transhumanists [ 20–22 ] and bioconservatives [ 18 , 19 , 23 ] on the ethical issues regarding the use of technologies in humans. The focus of this commentary is not centred on this debate, particularly because the discussion of these extreme, divergent positions is already very prominent in the public debate. In fact, it is interesting to notice that the ‘moderate’ discourses around this topic are much less known. In a more moderate view, perhaps one of the crucial questions to consider, independently of the moral views on human enhancement, is whether human enhancement (especially if considering germline heritable genetic interventions) is a necessary development, and represents an appropriate use of time, funding and resources compared to other pressing societal issues. It is crucial to build space for these more moderate, and perhaps less polarized voices, allowing the consideration of other positions and visions beyond those being more strongly projected so far.

Ethical and societal discussions on what constitutes human enhancement will be fundamental to support the development of policy frameworks and regulations on new technological developments. When considering the ethical implications of human enhancement that technology will be available to offer now and in the future, it could be useful to group the different kinds of human enhancements in the phenotypic and genetic categories: (i) strictly phenotypic intervention (e.g. ranging from infrared vision spectacles to exoskeletons and bionic limbs); (ii) somatic, non-heritable genetic intervention (e.g. editing of muscle cells for stronger muscles) and (iii) germline, heritable genetic intervention (e.g. editing of the C–C chemokine receptor type 5 (CCR5) gene in the Chinese baby twins, discussed later on). These categories of enhancement raise different considerations and concerns and currently present different levels of acceptance by our society. The degree of ethical, societal and environmental impacts is likely to be more limited for phenotypic interventions (i) but higher for genetic interventions (ii and iii), especially for the ones which are transmissible to future generations (iii).

The rapid advances in technology seen in the last decades, have raised the possibility of ‘radical enhancement’, defined by Nicholas Agar, ‘as the improvement of human attributes and abilities to levels that greatly exceed what is currently possible for human beings’ [ 24 ]. Genetic engineering offers the possibility of such an enhancement by providing humans a profound control over their own biology. Among other technologies, genetic engineering comprises genome editing (also called gene editing), a group of technologies with the ability to directly modify an organism’s DNA through a targeted intervention in the genome (e.g. insertion, deletion or replacement of specific genetic material) [ 25 ]. Genome editing is considered to achieve much greater precision than pre-existing forms of genetic engineering. It has been argued to be a revolutionary tool due to its efficiency, reducing cost and time. This technology is considered to have many applications for human health, in both preventing and tackling disease. Much of the ethical debate associated with this technology concerns the possible application of genome editing in the human germline, i.e. the genome that can be transmitted to following generations, be it from gametes, a fertilized egg or from first embryo divisions [ 26–28 ]. There has been concern as well as enthusiasm on the potential of the technology to modify human germline genome to provide us with traits considered positive or useful (e.g. muscle strength, memory and intelligence) in the current and future environments.

Genetic engineering: therapy or enhancement and predictability of outcomes

To explore some of the possible implications of heritable interventions we will take as an example the editing (more specifically ‘deletion’ using CRISPR genome editing technology) of several base pairs of the CCR5 gene. Such intervention was practised in 2018 in two non-identical twin girls born in China. Loss of function mutations of the CCR5 had been previously shown to provide resistance to HIV. Therefore, the gene deletion would be expected to protect the twin baby girls from risk of transmission of HIV which could have occurred from their father (HIV-positive). However, the father had the infection kept under control and the titre of HIV virus was undetectable, which means that risk of transmission of HIV infection to the babies was negligible [ 29 ].

From an ethical ground, based on current acceptable practices, this case has been widely criticized by the scientific community beside being considered by many a case of human enhancement intervention rather than therapy [ 29 , 30 ]. One of the questions this example helps illustrate is that the ethical boundary between a therapy that ‘corrects’ a disorder by restoring performance to a ‘normal’ scope, and an intervention that ‘enhances’ human ability outside the accepted ‘normal’ scope, is not always easy to draw. For the sake of argument, it could be assumed that therapy involves attempts to restore a certain condition of health, normality or sanity of the ‘natural’ condition of a specific individual. If we take this approach, the question is how health, normality and sanity, as well as natural per se, are defined, as the meaning of these concepts shift over time to accommodate social norms and cultural values of modern societies. It could be said that the difficulty of developing a conceptual distinction between therapy and enhancement has always been present. However, the potential significance of such distinction is only now, with the acceleration and impact of technological developments, becoming more evident.

Beyond ethical questions, a major problem of this intervention is that we do not (yet?) know exactly the totality of the effects that the artificial mutation of the CCR5 may have, at both the genetic and phenotypic levels. This is because we now know that, contrary to the idea of ‘one gene-one trait’ accepted some decades ago, a gene—or its absence—can affect numerous traits, many of them being apparently unrelated (a phenomenon also known as pleiotropy). That is, due to constrained developmental interactions, mechanisms and genetic networks, a change in a single gene can result in a cascade of multiple effects [ 31 ]. In the case of CCR5, we currently know that the mutation offers protection against HIV infection, and also seems to increase the risk of severe or fatal reactions to some infectious diseases, such as the influenza virus [ 32 ]. It has also been observed that among people with multiple sclerosis, the ones with CCR5 mutation are twice as likely to die early than are people without the mutation [ 33 ]. Some studies have also shown that defective CCR5 can have a positive effect in cognition to enhance learning and memory in mice [ 34 ]. However, it’s not clear if this effect would be translated into humans. The example serves to illustrate that, even if human enhancement with gene editing methods was considered ethically sound, assessing the totality of its implications on solid grounds may be difficult to achieve.

Genetic engineering and human evolution: large-scale impacts

Beyond providing the opportunity of enhancing human capabilities in specific individuals, intervening in the germline is likely to have an impact on the evolutionary processes of the human species raising questions on the scale and type of impacts. In fact, the use of large-scale genetic engineering might exponentially increase the force of ‘niche construction’ in human evolution, and therefore raise ethical and practical questions never faced by our species before. It has been argued that natural selection is a mechanism of lesser importance in the case of current human evolution, as compared to other organisms, because of advances in medicine and healthcare [ 35 ]. According to such a view, among many others advances, natural selection has been conditioned by our ‘niche-construction’ ability to improve healthcare and access to clean water and food, thus changing the landscape of pressures that humans have been facing for survival. An underlying assumption or position of the current debate is that, within our human species, the force of natural selection became minimized and that we are somehow at the ‘end-point’ of our evolution [ 36 ]. If this premise holds true, one could argue that evolution is no longer a force in human history and hence that any human enhancement would not be substituting itself to human evolution as a key driver for future changes.

However, it is useful to remember that, as defined by Darwin in his book ‘On the Origin of the Species’, natural selection is a process in which organisms that happen to be ‘better’ adapted to a certain environment tend to have higher survival and/or reproductive rates than other organisms [ 37 ]. When comparing human evolution to human genetic enhancement, an acceptable position could be to consider ethically sound those interventions that could be replicated naturally by evolution, as in the case of the CCR5 gene. Even if this approach was taken, however, it is important to bear in mind that human evolution acts on human traits sometimes increasing and sometimes decreasing our biological fitness, in a constant evolutionary trade-off and in a contingent and/or neutral—in the sense of not ‘progressive’—process. In other worlds, differently from genetic human enhancement, natural selection does not ‘ aim ’ at improving human traits [ 38 ]. Human evolution and the so-called genetic human enhancement would seem therefore to involve different underlying processes, raising several questions regarding the implications and risks of the latter.

But using genetic engineering to treat humans has been proposed far beyond the therapeutic case or to introduce genetic modifications known to already occur in nature. In particular, when looking into the views expressed on the balance between human evolution and genetic engineering, some argue that it may be appropriate to use genetic interventions to go beyond what natural selection has contributed to our species when it comes to eradicate vulnerabilities [ 17 ]. Furthermore, when considering the environmental, ecological and social issues of contemporary times, some suggest that genetic technologies could be crucial tools to contribute to human survival and well-being [ 20–22 ]. The possible need to ‘engineer’ human traits to ensure our survival could include the ability to allow our species to adapt rapidly to the rate of environmental change caused by human activity, for which Darwinian evolution may be too slow [ 39 ]. Or, for instance, to support long-distance space travel by engineering resistance to radiation and osteoporosis, along with other conditions which would be highly advantageous in space [ 40 ].

When considering the ethical and societal merits of these propositions, it is useful to consider how proto-forms of enhancement has been approached by past human societies. In particular, it can be argued that humans have already employed—as part of our domestication/‘selective breeding’ of other animals—techniques of indirect manipulation of genomes on a relatively large scale over many millennia, albeit not on humans. The large-scale selective breeding of plants and animals over prehistoric and historic periods could be claimed to have already shaped some of our natural environment. Selective breeding has been used to obtain specific characteristics considered useful at a given time in plants and animals. Therefore, their evolutionary processes have been altered with the aim to produce lineages with advantageous traits, which contributed to the evolution of different domesticated species. However, differently from genetic engineering, domestication possesses inherent limitations in its ability to produce major transformations in the created lineages, in contrast with the many open possibilities provided by genetic engineering.

When considering the impact of genetic engineering on human evolution, one of questions to be considered concerns the effects, if any, that genetic technology could have on the genetic pool of the human population and any implication on its resilience to unforeseen circumstances. This underlines a relevant question associated with the difference between ‘health’ and biological fitness. For example, a certain group of animals can be more ‘healthy’—as domesticated dogs—but be less biologically ‘fit’ according to Darwin’s definition. Specifically, if such group of animals are less genetically diverse than their ancestors, they could be less ‘adaptable’ to environmental changes. Assuming that, the human germline modification is undertaken at a global scale, this could be expected to have an effect, on the distribution of genetically heritable traits on the human population over time. Considering that gene and trait distributions have been changing under the processes of evolution for billions of years, the impact on evolution will need to be assessed by analysing which genetic alterations have been eventually associated with specific changes within the recent evolutionary history of humans. On this front, a key study has analysed the implications of genetic engineering on the evolutionary biology of human populations, including the possibility of reducing human genetic diversity, for instance creating a ‘biological monoculture’ [ 41 ]. The study argued that genetic engineering will have an insignificant impact on human diversity, while it would likely safeguard the capacity of human populations to deal with disease and new environmental challenges and therefore, ensure the health and longevity of our species [ 41 ]. If the findings of this study were considered consistent with other knowledge and encompassing, the impact of human genetic enhancements on the human genetic pool and associated impacts could be considered secondary aspects. However, data available from studies on domestication strongly suggests that domestication of both animals and plans might lead to not only decreased genetic diversity per se, but even affect patterns of variation in gene expression throughout the genome and generally decreased gene expression diversity across species [ 42–44 ]. Given that, according to recent studies within the field of biological anthropology recent human evolution has been in fact a process of ‘self-domestication’ [ 45 ], one could argue that studies on domestication could contribute to understanding the impacts of genetic engineering.

Beyond such considerations, it is useful to reflect on the fact that human genetic enhancement could occur on different geographical scales, regardless of the specific environment and geological periods in which humans are living and much more rapidly than in the case of evolution, in which changes are very slow. If this was to occur routinely and on a large scale, the implications of the resulting radical and abrupt changes may be difficult to predict and its impacts difficult to manage. This is currently highlighted by results of epigenetics studies, and also of the microbiome and of the effects of pollutants in the environment and their cumulative effect on the development of human and non-human organisms alike. Increasingly new evidence indicates a greater interdependence between humans and their environments (including other microorganisms), indicating that modifying the environment can have direct and unpredictable consequences on humans as well. This highlight the need of a ‘systems level’ approach. An approach in which the ‘bounded body’ of the individual human as a basic unit of biological or social action would need to be questioned in favour of a more encompassing and holistic unit. In fact, within biology, there is a new field, Systems Biology, which stresses the need to understand the role that pleiotropy, and thus networks at multiple levels—e.g. genetic, cellular, among individuals and among different taxa—play within biological systems and their evolution [ 46 ]. Currently, much still needs to be understood about gene function, its role in human biological systems and the interaction between genes and external factors such as environment, diet and so on. In the future if we do choose to genetically enhance human traits to levels unlikely to be achieved by human evolution, it would be crucial to consider if and how our understanding of human evolution enable us to better understand the implications of genetic interventions.

New forms of human enhancement are increasingly coming to play due to technological development. If phenotypic and somatic interventions for human enhancement pose already significant ethical and societal challenges, germline heritable genetic intervention, require much broader and complex considerations at the level of the individual, society and human species as a whole. Germline interventions associated with modern technologies are capable of much more rapid, large-scale impacts and seem capable of radically altering the balance of humans with the environment. We know now that beside the role genes play on biological evolution and development, genetic interventions can induce multiple effects (pleiotropy) and complex epigenetics interactions among genotype, phenotype and ecology of a certain environment. As a result of the rapidity and scale with which such impact could be realized, it is essential for ethical and societal debates, as well as underlying scientific studies, to consider the unit of impact not only to the human body but also to human populations and their natural environment (systems biology). An important practicable distinction between ‘therapy’ and ‘enhancement’ may need to be drawn and effectively implemented in future regulations, although a distinct line between the two may be difficult to draw.

In the future if we do choose to genetically enhance human traits to levels unlikely to be achieved by human evolution, it would be crucial to consider if and how our understanding of humans and other organisms, including domesticated ones, enable us to better understand the implications of genetic interventions. In particular, effective regulation of genetic engineering may need to be based on a deep knowledge of the exact links between phenotype and genotype, as well the interaction of the human species with the environment and vice versa .

For a broader and consistent debate, it will be essential for technological, philosophical, ethical and policy discussions on human enhancement to consider the empirical evidence provided by evolutionary biology, developmental biology and other disciplines.

This work was supported by Fundação para a Ciência e a Tecnologia (FCT) of Portugal [CFCUL/FIL/00678/2019 to M.A.].

Conflict of interest : None declared.

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Principles of genetic engineering.

1. Introduction

2. principles of genetic engineering, 2.1. types of genetic modifications, 2.2. genetic engineering with crispr/cas9, 2.3. locus-specific genetic engineering vectors in mouse and rat zygotes, 2.4. gene editing in immortalized cell lines, 2.5. viruses and transposons as genetic engineering vectors, 2.6. genetic engineering using retroviruses, 2.7. gene targeting using adeno-associated virus, 3. conclusions, author contributions, conflicts of interest.

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Lanigan, T.M.; Kopera, H.C.; Saunders, T.L. Principles of Genetic Engineering. Genes 2020 , 11 , 291. https://doi.org/10.3390/genes11030291

Lanigan TM, Kopera HC, Saunders TL. Principles of Genetic Engineering. Genes . 2020; 11(3):291. https://doi.org/10.3390/genes11030291

Lanigan, Thomas M., Huira C. Kopera, and Thomas L. Saunders. 2020. "Principles of Genetic Engineering" Genes 11, no. 3: 291. https://doi.org/10.3390/genes11030291

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Medicine is at a turning point, on the cusp of major change as disruptive technologies such as gene, RNA, and cell therapies enable scientists to approach diseases in new ways. The swiftness of this change is being driven by innovations such as CRISPR gene editing , which makes it possible to correct errors in DNA with relative ease.

Progress in this field has been so rapid that the dialogue around potential ethical, societal, and safety issues is scrambling to catch up.

This disconnect was brought into stark relief at the Second International Summit on Human Genome Editing , held in Hong Kong in November, when exciting updates about emerging therapies were eclipsed by a disturbing announcement. He Jiankui, a Chinese researcher, claimed that he had edited the genes of two human embryos, and that they had been brought to term.

There was immediate outcry from scientists across the world, and He was subjected to intense social pressure, including the removal of his affiliations, for having allegedly disregarded ethical norms and his patients’ safety.

Yet as I. Glenn Cohen, faculty director of the Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics at Harvard Law School, has said, gene editing comes in many varieties, with many consequences. Any deep ethical discussion needs to take into account those distinctions.

Human genome editing: somatic vs. germline

The germline editing He claimed to have carried out is quite different from the somatic gene therapies that are currently changing the frontiers of medicine. While somatic gene editing affects only the patient being treated (and only some of his or her cells), germline editing affects all cells in an organism, including eggs and sperm, and so is passed on to future generations. The possible consequences of that are difficult to predict.

Somatic gene therapies involve modifying a patient’s DNA to treat or cure a disease caused by a genetic mutation. In one clinical trial, for example, scientists take blood stem cells from a patient, use CRISPR techniques to correct the genetic mutation causing them to produce defective blood cells, then infuse the “corrected” cells back into the patient, where they produce healthy hemoglobin. The treatment changes the patient’s blood cells, but not his or her sperm or eggs.

Germline human genome editing, on the other hand, alters the genome of a human embryo at its earliest stages. This may affect every cell, which means it has an impact not only on the person who may result, but possibly on his or her descendants. There are, therefore, substantial restrictions on its use.

Germline editing in a dish can help researchers figure out what the health benefits could be, and how to reduce risks. Those include targeting the wrong gene; off-target impacts, in which editing a gene might fix one problem but cause another; and mosaicism, in which only some copies of the gene are altered. For these and other reasons, the scientific community approaches germline editing with caution, and the U.S. and many other countries have substantial policy and regulatory restrictions on using germline human genome editing in people.

But many scientific leaders are asking: When the benefits are believed to outweigh the risks, and dangers can be avoided, should science consider moving forward with germline genome editing to improve human health? If the answer is yes, how can researchers do so responsibly?

CRISPR pioneer Feng Zhang of the Broad Institute of Harvard and MIT responded immediately to He’s November announcement by calling for a moratorium on implanting edited embryos in humans. Later, at a public event on “Altering the Human Genome” at the Belfer Center at Harvard Kennedy School (HKS), he explained why he felt it was important to wait:

“The moratorium is a pause. Society needs to figure out if we all want to do this, if this is good for society, and that takes time. If we do, we need to have guidelines first so that the people who do this work can proceed in a responsible way, with the right oversight and quality controls.”

Professors at the University’s schools of medicine, law, business, and government saw He’s announcement as a turning point in the discussion about heritable gene therapies and shared their perspectives on the future of this technology with the Gazette.

Here are their thoughts, issue by issue:

Aside from the safety risks, human genome editing poses some hefty ethical questions. For families who have watched their children suffer from devastating genetic diseases, the technology offers the hope of editing cruel mutations out of the gene pool. For those living in poverty, it is yet another way for the privileged to vault ahead. One open question is where to draw the line between disease treatment and enhancement, and how to enforce it, considering differing attitudes toward conditions such as deafness.

Robert Truog , director of the Center for Bioethics at Harvard Medical School (HMS), provided context:

“This question is not as new as it seems. Evolution progresses by random mutations in the genome, which dwarf what can be done artificially with CRISPR. These random mutations often cause serious problems, and people are born with serious defects. In addition, we have been manipulating our environment in so many ways and exposing ourselves to a lot of chemicals that cause unknown changes to our genome. If we are concerned about making precise interventions to cure disease, we should also be interested in that.

“To me, the conversation around Dr. He is not about the fundamental merits of germline gene editing, which in the long run will almost certainly be highly beneficial. Instead, it’s about the oversight of science. The concern is that with technologies that are relatively easy to use, like CRISPR, how does the scientific community regulate itself? If there’s a silver lining to this cloud, I think it is that the scientific community did pull together to be critical of this work, and took the responsibility seriously to use the tools available to them to regulate themselves.”

When asked what the implications of He’s announcement are for the emerging field of precision medicine, Richard Hamermesh, faculty co-chair of the Harvard Business School/Kraft Precision Medicine Accelerator, said:

“Before we start working on embryos, we have a long way to go, and civilization has to think long and hard about it. There’s no question that gene editing technologies are potentially transformative and are the ultimate precision medicine. If you could precisely correct or delete genes that are causing problems — mutating or aberrant genes — that is the ultimate in precision. It would be so transformative for people with diseases caused by a single gene mutation, like sickle cell anemia and cystic fibrosis. Developing safe, effective ways to use gene editing to treat people with serious diseases with no known cures has so much potential to relieve suffering that it is hard to see how anyone could be against it.

“There is also commercial potential and that will drive it forward. A lot of companies are getting venture funding for interesting gene therapies, but they’re all going after tough medical conditions where there is an unmet need — [where] nothing is working — and they’re trying to find gene therapies to cure those diseases. Why should we stop trying to find cures?

“But anything where you’re going to be changing human embryos, it’s going to take a long time for us to figure out what is appropriate and what isn’t. That has to be done with great care in terms of ethics.”

George Q. Daley  is dean of HMS, the Caroline Shields Walker Professor of Medicine, and a leader in stem cell science and cancer biology. As a spokesperson for the organizing committee of the Second International Summit on Human Genome Editing, he responded swiftly to He’s announcement in Hong Kong. Echoing those remarks, he said:

“It’s time to formulate what a clinical path to translation might look like so that we can talk about it. That does not mean that we’re ready to go into the clinic — we are not. We need to specify what the hurdles would be if one were to move forward responsibly and ethically. If you can’t surmount those hurdles, you don’t move forward.

“There are stark distinctions between editing genes in an embryo to prevent a baby from being born with sickle cell anemia and editing genes to alter the appearance or intelligence of future generations. There is a whole spectrum of considerations to be debated. The prospect includes an ultimate decision that we not go forward, that we decide that the benefits do not outweigh the costs.”

Asked how to prevent experiments like He’s while preserving academic freedom, Daley replied:

“For the past 15 years, I have been involved in efforts to establish international standards of professional conduct for stem cell research and its clinical translation, knowing full well that there could be — and has been — a growing number of independent practitioners directly marketing unproven interventions to vulnerable patients through the internet. We advocated so strongly for professional standards in an attempt to ward off the risks of an unregulated industry. Though imperfect, our efforts to encourage a common set of professional practices have been influential.

“You can’t control rogue scientists in any field. But with strongly defined guidelines for responsible professional conduct in place, such ethical violations like those of Dr. He should remain a backwater, because most practitioners will adhere to generally accepted norms. Scientists have a responsibility to come together to articulate professional standards and live by them. One has to raise the bar very high to define what the standards of safety and efficacy are, and what kind of oversight and independent judgment would be required for any approval.

“We have called for an ongoing international forum on human genome editing, and that could take many shapes. We’ve suggested that the national academies of more countries come together — the National Academy of Sciences in the U.S. and the Royal Society in the U.K. are very active here — because these are the groups most likely to have the expertise to convene these kinds of discussions and keep them going.”

Cohen , speaking to the legal consequences of germline human genome editing, said:

“I think we should slow down in our reaction to this case. It is not clear that the U.S. needs to react to Dr. He’s announcement with regulation. The FDA [Food and Drug Administration] already has a strong policy on germline gene editing in place. A rider in the Consolidated Appropriations Act of 2016 — since renewed — would have blocked the very same clinical application of human germline editing He announced, had it been attempted in the U.S.

“The scientific community has responded in the way I’d have liked it to. There is a difference between ‘governance’ and ‘self-governance.’ Where government uses law, the scientific community uses peer review, public censure, promotions, university affiliations, and funding to regulate themselves. In China, in Dr. He’s case, you have someone who’s (allegedly) broken national law and scientific conventions. That doesn’t mean you should halt research being done by everyone who’s law-abiding.

“Public policy or ethical discussion that’s divorced from how science is progressing is problematic. You need to bring everyone together to have robust discussions. I’m optimistic that this is happening, and has happened. It’s very hard to deal with a transnational problem with national legislation, but it would be great to reach international consensus on this subject. These efforts might not succeed, but ultimately they are worth pursuing.”

Professor Kevin Eggan of Harvard’s Department of Stem Cell and Regenerative Biology said, “The question we should focus on is: Will this be safe and help the health of a child? Can we demonstrate that we can fix a mutation that will cause a terrible health problem, accurately and without the risk of harming their potential child? If the answer is yes, then I believe germline human genome editing is likely to gain acceptance in time.

“There could be situations where it could help a couple, but the risks of something going wrong are real. But at this point, it would be impossible to make a risk-benefit calculation in a responsible manner for that couple. Before we could ever move toward the clinic, the scientific community must come to a consensus on how to measure success, and how to measure off-target effects in animal models.

“Even as recently as this past spring and fall, the results of animal studies using CRISPR — the same techniques Dr. He claimed to have used — generated a lot of confusion. There is disagreement about both the quality of the data and how to interpret it. Until we can come to agreement about what the results of animal experiments mean, how could we possibly move forward with people?

“As happened in England with mitochondrial replacement therapy, we should be able to come to both a scientific and a societal consensus of when and how this approach should be used. That’s missing.”

According to Catherine Racowsky, professor of obstetrics, gynecology and reproductive biology at Brigham and Women’s Hospital, constraints on the use of embryos in federally funded research pose barriers to studying the risks and benefits of germline editing in humans. She added:

“Until the work is done, carefully and with tight oversight, to understand any off-target effects of replacing or removing a particular gene, it is inappropriate to apply the technology in the clinical field. My understanding of Dr. He’s case is that there wasn’t a known condition in these embryos, and by editing the genes involved with HIV infection, he could also have increased the risks of susceptibility to influenza and West Nile viruses.

“We need a sound oversight framework, and it needs to be established globally. This is a technology that holds enormous promise, and it is likely to be applied to the embryo, but it should only be applied for clinical purposes after the right work has been done. That means we must have consensus on what applications are acceptable, that we have appropriate regulatory oversight, and, perhaps most importantly, that it is safe. The only way we’re going to be able to determine that these standards are met is to proceed cautiously, with reassessments of the societal and health benefits and the risks.”

Asked about public dialogue around germline human genome editing, George Church , Robert Winthrop Professor of Genetics at HMS, said:

“With in vitro  fertilization (IVF), ‘test tube babies’ was an intentionally scary term. But after Louise Brown, the first IVF baby, was born healthy 40 years ago, attitudes changed radically. Ethics flipped 180 degrees, from it being a horrifying idea to being unacceptable to prevent parents from having children by this new method. If these edited twins are proven healthy, very different discussions will arise. For example, is a rate of 900,000 deaths from HIV infection per year a greater risk than West Nile virus, or influenza? How effective is each vaccine?”

Science, technology, and society

Sheila Jasanoff , founding director of the Science, Technology, and Society program at HKS, has been calling for a “global observatory” on gene editing, an international network of scholars and organizations dedicated to promoting exchange across disciplinary and cultural divides. She said:

“The notion that the only thing we should care about is the risk to individuals is very American. So far, the debate has been fixated on potential physical harm to individuals, and not anything else. This is not a formulation shared with other countries in the world, including practically all of Europe. Considerations of risk have equally to do with societal risk. That includes the notion of the family, and what it means to have a ‘designer baby.’

“These were not diseased babies Dr. He was trying to cure. The motivation for the intervention was that they live in a country with a high stigma attached to HIV/AIDS, and the father had it and agreed to the intervention because he wanted to keep his children from contracting AIDS. AIDS shaming is a fact of life in China, and now it won’t be applied to these children. So, are we going to decide that it’s OK to edit as-yet-to-be children to cater to this particular idea of a society?

“It’s been said that ‘the genie is out of the bottle’ with germline human genome editing. I just don’t think that’s true. After all, we have succeeded in keeping ‘nuclear’ inside the bottle. Humanity doesn’t lack the will, intelligence, or creativity to come up with ways for using technology for good and not ill.

“We don’t require students to learn the moral dimensions of science and technology, and that has to change. I think we face similar challenges in robotics, artificial intelligence, and all kinds of frontier fields that have the potential to change not just individuals but the entirety of what it means to be a human being.

“Science has this huge advantage over most professional thought in that it has a universal language. Scientists can hop from lab to lab internationally in a way that lawyers cannot because laws are written in many languages and don’t translate easily. It takes a very long time for people to understand each other across these boundaries. A foundational concept for human dignity? It would not be the same thing between cultures.

“I would like to see a ‘global observatory’ that goes beyond gene editing and addresses emerging technologies more broadly.”

To learn more:

Technology and Public Purpose project, Belfer Center for Science and International Affairs, Harvard Kennedy School of Government, https://www.belfercenter.org/tapp/person

Concluding statement from the Second International Summit on Human Genome Editing. http://www8.nationalacademies.org/onpinews/newsitem.aspx?RecordID=11282018b

A global observatory for gene editing: Sheila Jasanoff and J. Benjamin Hurlbut call for an international network of scholars and organizations to support a new kind of conversation. https://www.nature.com/articles/d41586-018-03270-w

Building Capacity for a Global Genome Editing Observatory: Institutional Design. http://europepmc.org/abstract/MED/29891181

Glenn Cohen’s blog: How Scott Gottlieb is Wrong on the Gene Edited Baby Debacle. http://blog.petrieflom.law.harvard.edu/2018/11/29/how-scott-gottlieb-is-wrong-on-the-gene-edited-baby-debacle/

Gene-Editing: Interpretation of Current Law and Legal Policy. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651701/

Forum: Harvard T.H. Chan School of Public Health event on the promises and challenges of gene editing, May 2017: https://theforum.sph.harvard.edu/events/gene-editing/

Petrie-Flom Center Annual Conference: Consuming Genetics: Ethical and Legal Considerations of New Technologies: http://petrieflom.law.harvard.edu/events/details/2019-petrie-flom-center-annual-conference

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