Genome Editing Technologies: Defining a Path to Clinic
Authors: Jacqueline Corrigan-Curay, Marina O'Reilly, Donald B Kohn, Paula M Cannon, Gang Bao, Frederic D Bushman, Dana Carroll, Toni Cathomen, J Keith Joung, David Roth, Michel Sadelain, Andrew M Scharenberg, Christof von Kalle, Feng Zhang, Robert Jambou, Eugene Rosenthal, Morad Hassani, Aparna Singh, Matthew H Porteus
Journal: Molecular Therapy (2015)

Recently developed genomic editing technologies have the potential to be powerful tools for gene therapy because of their ability to inactivate genes, correct mutated sequences, or insert intact genes. While the genomic editing field is advancing at an exceptionally rapid pace, there remain key issues regarding development of appropriate preclinical assays to evaluate off-target effects and establish safety. In order to begin a dialogue on these issues, the National Institutes of Health (NIH) Office of Science Policy, in collaboration with several NIH-funded investigators and the NIH Recombinant DNA Advisory Committee, organized a workshop on 10 June 2014, in Bethesda, Maryland, to provide a forum to educate the scientific and oversight communities and the public on different genome editing technologies, clinical experiences to date, and the preclinical assays being developed to examine the precision of these tools and their suitability for clinical application. Targeted genome modification by designer nucleases is an emerging technology that can be used to investigate gene function and could also be used to treat genetic or acquired diseases. A wide range of genome alterations has been achieved by these nucleases, including localized mutagenesis, local and dispersed sequence replacements, large and small insertions and deletions, and even chromosomal translocations. The nuclease approach to targeted genome editing has been applied successfully to more than 50 different organisms, including crop plants, livestock, and humans.1Carroll D Genome engineering with targetable nucleases.Annu Rev Biochem. 2014; 83: 409-439Crossref PubMed Scopus (381) Google Scholar Recently developed genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) are being investigated as promising tools for human gene therapy. ZFNs are the first class of nucleases to have reached the clinic in phase I trials for HIV.2Tebas P Stein D Tang WW Frank I Wang SQ Lee G et al.Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.N Engl J Med. 2014; 370: 901-910Crossref PubMed Scopus (1008) Google Scholar One key issue that will have to be addressed as these technologies move into clinical trials is whether technology-specific preclinical evaluations are available that can establish safety. Although these are targeted editing tools, their precision, and specifically the degree to which there are off-target actions and the clinical implications of such activity are important questions for the field. Overview of genome editing technologies The fundamental process common to all of these technologies is the use of nucleases to make site-specific double-stranded breaks (DSBs) in the genome. Several approaches to genome editing have been developed; this summary describes them briefly. ZFNs. ZFNs are the most clinically advanced nuclease platform. Each zinc-finger consists of ~30 amino acids that fold into a conserved ββα configuration.3Gaj T Gersbach CA Barbas 3rd, CF ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol. 2013; 31: 397-405Abstract Full Text Full Text PDF PubMed Scopus (2479) Google Scholar Each "finger" recognizes about three or four base pairs of DNA using at least six amino acids through contacts between specific residues in the second α-helix, also known as the "recognition helix" terminus. Three to six individual fingers can be linked to enable construction of arrays that recognize longer sequences of 9–18 base pairs (bp). Of note, 18 bp of DNA sequence can confer specificity within 68 billion base pairs of DNA. Further specificity can be engineered by changing critical residues within the recognition helices. In addition, individual fingers in an array can potentially interact and make base-specific contacts into the sequences recognized by adjacent fingers, and optimization of binding can therefore be potentially altered by interactions between individual fingers. The nuclease domain of the zinc-finger is derived from the C-terminus of the FokI restriction endonuclease. FokI only cuts DNA when it dimerizes, so two sets of zinc-fingers are required (see Figure 1a). The natural enzyme generates a 5′ overhang. However, one can engineer these enzymes such that within the dimer, one side of the nuclease domain is catalytically active while its twin is catalytically inactive, which will generate a single-stranded nick rather than a DSB. TALENS. TALENs are similar in architecture to ZFNs except that they use a different DNA-binding domain. They consist of arrays of single protein modules that each recognize a single DNA base pair and that are derived from transcription activator-like effectors (TALEs), factors encoded by plant pathogenic bacteria4Bogdanove AJ Voytas DF ). TAL effectors: customizable proteins for DNA targeting.Science. 2011; 333: 1843-1846Crossref PubMed Scopus (743) Google Scholar (see Figure 1b). Each of these modules is about 34 amino acids long, and they are nearly identical except for the identities of amino acids at positions 12 and 13, which together are known as the "repeat variable di-residue". To engineer DNA-binding domains with novel DNA-binding specificities, individual TALE repeats are assembled into an array that is designed to recognize the target DNA sequence. Although the single-nucleotide specificity of TALE repeats potentially offers greater design flexibility than do zinc-fingers, their highly repetitive nature presents technical challenges in assembling DNA-encoding arrays of these domains. Different strategies have been developed to facilitate rapid assembly of DNA-encoding TALE repeats, including the "Golden Gate" assembly method5Cermak T Starker CG Voytas DF Efficient design and assembly of custom TALENs using the Golden Gate platform.Methods Mol Biol. 2015; 1239: 133-159Crossref PubMed Scopus (34) Google Scholar and a system called FLASH.6Reyon D Maeder ML Khayter C Tsai SQ Foley JE Sander JD et al.Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.Curr Protoc Mol Biol. 2013; (Chapter 12: Unit 12.16)Crossref PubMed Scopus (29) Google Scholar TALE repeat arrays that recognize 13–20 bp can be constructed. The nuclease domain used in TALENs is also from FokI; however, in contrast to ZFNs and for reasons that are not yet understood, TALENs cannot be manipulated to create nicks rather than DSBs. CRISPR/CRISPR-associated (Cas) protein 9. CRISPR/Cas9 is distinct from the previous engineered endonucleases in that it uses an RNA-guided system to perform genome editing. This platform, derived from a bacterial innate immune system, was described relatively recently, but progress on its development has been rapid. It has captured considerable attention due to the relative ease of engineering its RNA-based targeting component. In bacteria, type II CRISPR systems process foreign sequences from invading phages or plasmids into small segments that are then introduced into the CRISPR array, which contains the regularly interspaced palindromic repeats. These snippets of foreign DNA become templates for CRISPR RNA (crRNA), which now contains a variable sequence from the invading DNA. This crRNA then hybridizes with a trans-activating RNA (tracrRNA), and the RNAs form complexes with the Cas9 protein. The next time this foreign sequence is detected, it is cleaved and degraded (see Figure 1c).7Sander JD Joung JK CRISPR-Cas systems for editing, regulating and targeting genomes.Nat Biotechnol. 2014; 32: 347-355Crossref PubMed Scopus (2121) Google Scholar Recognition of the target DNA sequence is mediated between the genomic DNA target and by a 20-nucleotide sequence in the crRNA. Another feature of this system is that the Cas9 protein is directed to cleave the complementary target-DNA sequence if it is adjacent to a short sequence known as the protospacer adjacent motif (PAM). The PAM sequence commonly used from Streptococcus pyogenes has the sequence [N]GG, although [N]AG can also be used. Many bacteria have the Cas9 system, but not all of them possess the same PAM sequences, providing some additional variability. In addition, mutations in the PAM sequence will prevent the Cas9 protein from causing a break at that site. In 2012 it was shown that the crRNA and the tracrRNA can be combined into a single RNA molecule known as a guide RNA (or gRNA) that can still engage the Cas9 protein.8Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9321) Google Scholar As discussed later, gRNAs recognize 20-bp target sites but can also recognize off-target sites bearing up to six mismatches and/or bulges. Similar to ZFNs, it is possible to make a nick rather than a break with the CRISPR/Cas9 system. However, unlike the ZFNs and TALENs, the double-strand cut made by CRISPR/Cas9 leaves a blunt end. Homing endonucleases (meganucleases). These are natural proteins called endodeoxyribonucleases that recognize long (>12 bp) DNA sequences with high specificity. Described as genetic parasites, they target the recognition site in an allele, make a break in that allele, and target the transfer of their own protein-coding sequence into that allele by homologous recombination. They generate a DSB with a four-base 3′ overhang, which is proposed to be a natural substrate for the homologous recombination machinery. Initial genome editing manipulations were done using the I-CreI and the I-SceI endonucleases. Engineering these nucleases is difficult primarily because both recognition and enzymatic activity are intertwined within the protein, often making it difficult to alter one without having an effect on the other. However, these nucleases are small and can be easily contained in commonly used vectors. In sum, they offer potentially high specificity, but barriers to their engineering are high.9Silva G Poirot L Galetto R Smith J Montoya G Duchateau P et al.Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy.Curr Gene Ther. 2011; 11: 11-27Crossref PubMed Scopus (259) Google Scholar Impact of double-stranded breaks The mechanism unifying each of these technologies is the ability to make targeted DSBs in genomic DNA. The outcome of targeted cleavage depends on cellular repair pathways of DSBs, which are potentially lethal to the cell unless they are repaired quickly. The principal mechanisms of repair are nonhomologous end joining (NHEJ) and homology-dependent repair (HDR) (see Figure 2). NHEJ often results in sequence changes at the cut site, most commonly variable-length insertions or deletions, referred to as "indels," which can be detected through sequencing or mismatch cleavage assays that use enzymes such as CelI or T7 endonuclease I and more recently a new method called TIDE (tracking of indels by decomposition).10Brinkman EK Chen T Amendola M van Steensel B Easy quantitative assessment of genome editing by sequence trace decomposition.Nucleic Acids Res. 2014; 42: e168Crossref PubMed Scopus (1147) Google Scholar There are subtle differences among the NHEJ products generated by each of the above nucleases. Small deletions and insertions are common in NHEJ mutations, but the average deletion size is somewhat larger with TALENs and ZFNs compared to CRISPRs, and insertions are somewhat more frequent with ZFNs.1Carroll D Genome engineering with targetable nucleases.Annu Rev Biochem. 2014; 83: 409-439Crossref PubMed Scopus (381) Google Scholar Rarely are insertions large enough that the source of the inserted sequences can be identified.11Hendel A Kildebeck EJ Fine EJ Clark JT Punjya N Sebastiano V et al.Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing.Cell Rep. 2014; 7: 293-305Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Such findings highlight that the mechanisms of DSB repair are still not completely understood. HDR can incorporate user-provided sequence changes from a donor DNA. NHEJ dominates in almost all systems, but there may be ways to shift the balance toward HDR through downregulation of enzymes that are involved in the NHEJ pathway or modulating the stage of the cell cycle. In addition, altering engineered nucleases to produce a single-stranded nick rather than a DSB will favor HDR, but at an overall lower absolute frequency of repair.12Gilbert LA Larson MH Morsut L Liu Z Brar GA Torres SE et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.Cell. 2013; 154: 442-451Abstract Full Text Full Text PDF PubMed Scopus (2253) Google Scholar,13Wang J Friedman G Doyon Y Wang NS Li CJ Miller JC et al.Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme.Genome Res. 2012; 22: 1316-1326Crossref PubMed Scopus (110) Google Scholar If the goal is to incorporate new genetic material rather than just to disrupt a particular sequence, the nature of the donor DNA will also influence the success of the approach. With long double-stranded donor DNAs, successful incorporation requires several hundred base pairs of homology on both sides of the nuclease-induced break.11Hendel A Kildebeck EJ Fine EJ Clark JT Punjya N Sebastiano V et al.Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing.Cell Rep. 2014; 7: 293-305Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar,14Beumer KJ Carroll D Targeted genome engineering techniques in Drosophila.Methods. 2014; 68: 29-37Crossref PubMed Scopus (53) Google Scholar Short single-stranded donor DNAs can also be used, but such templates would be limited to applications requiring small changes close to the DSB, such as correction of point mutations. In some systems (e.g., Drosophila), sequences throughout the length of a long donor can be captured at the target, but in other cases (e.g., cultured mammalian cells) only sequences close to the break are routinely incorporated, although sequences several kilobases away can occasionally be incorporated.15Elliott B Richardson C Winderbaum J Nickoloff JA Jasin M Gene conversion tracts from double-strand break repair in mammalian cells.Mol Cell Biol. 1998; 18: 93-101Crossref PubMed Scopus (259) Google Scholar,16Stark JM Jasin M Extensive loss of heterozygosity is suppressed during homologous repair of chromosomal breaks.Mol Cell Biol. 2003; 23: 733-743Crossref PubMed Scopus (93) Google Scholar Further knowledge of the activities that control this feature, known as "conversion tract length," is needed as the field moves toward gene insertion applications. Improving specificity through design Because genome editing is directed at specific sites, this technology offers greater precision compared to other approaches to long-term gene modification, e.g., delivery of a gene by an integrating viral vector. Most integrating viral vectors have a largely random integration pattern. However, despite the elegant precision that these editing tools offer, off-target effects are likely, depending upon the construct and the length of the target site. Ideally, the insertions, deletions, inversions, and translocations that may result from NHEJ or HDR at these off-target sites must be minimized before moving to the clinic. Improving on the natural design. much has been learned regarding how to use these tools to target specific sequences, and the designs of these naturally occurring tools are being modified to improve the specificity. At the workshop, Keith Joung reviewed his research with different platforms—ZFNs, TALENs, and CRISPR/Cas9 nucleases—in which he generally has seen stronger binding to on-target sites compared with off-target sites, but there is still considerable activity at off-target sequences. Focusing on the CRISPR/Cas9 platform, he examined the off-target effects of first-generation CRISPR/Cas9 agents that were directed by six different gRNAs and demonstrated that there were a number of off-target sites (harboring as many as five mismatches relative to the on-target site) and that the rate of mutagenesis at these sites could be as high as that seen at the on-target site.17Fu Y Foden JA Khayter C Maeder ML Reyon D Joung JK et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (2207) Google Scholar This analysis was done across different cell types, and the mutations sometimes fell within the coding sequences of the genome. This finding has led to the development of second-generation CRISPR/Cas9 agents with modifications to increase specificity. Intuitively one might conclude that increasing the length of the binding site for the nuclease would increase specificity, but Dr Joung found that for the CRISPR/Cas 9 system, specificity can be improved by truncating the 5′ end of the gRNA by as many as three nucleotides. This truncation generally does not impair the ability of a gRNA to direct on-target site cleavage but appears to make the gRNA/Cas9 complex more sensitive to mismatches and therefore reduces off-target site cleavage.18Fu Y Reyon D Joung JK Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.Methods Enzymol. 2014; 546: 21-45Crossref PubMed Scopus (31) Google Scholar This truncation approach does not work for all gRNAs but appears to work well for the vast majority of gRNAs tested to date. Another strategy is to combine the specificity from different platforms. For example, it is possible to combine the dimerization-dependent FokI nuclease used in ZFNs and TALENs with the CRISPR/Cas9 system. By fusing the FokI nuclease to a catalytically inactive Cas9, one can use the gRNAs to direct binding but require dimerization for cleavage.19Tsai SQ Zheng Z Nguyen NT Liebers M Topkar VV Thapar V et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.Nat Biotechnol. 2014; 33: 187-197Crossref PubMed Scopus (1287) Google Scholar,20Guilinger JP Thompson DB Liu DR Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.Nat Biotechnol. 2014; 32: 577-582Crossref PubMed Scopus (638) Google Scholar In one experiment in which five off-target sites were previously observed with the use of a specific gRNA, deep sequencing was used to confirm that the frequency of indel mutations induced by the dimeric CRISPR (or RNA-guided FokI nuclease) was not greater than background at all five off-target sites, indicating the ability of this platform to eliminate activity at the off-target sites of a single gRNA. This hybrid platform provides an increase in the length of the binding site by utilizing both gRNAs in a dimeric configuration and reduction in potential off-target sites.19Tsai SQ Zheng Z Nguyen NT Liebers M Topkar VV Thapar V et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.Nat Biotechnol. 2014; 33: 187-197Crossref PubMed Scopus (1287) Google Scholar Interestingly, in that experiment the authors also failed to see detectable evidence of off-target activity for these dimeric RNA-guided FokI nucleases, even at the most closely mismatched sites in the genome. Another approach being explored is to mutate the Cas9 protein so that it will only create a nick rather than a DSB. By then pairing two such mutated Cas9 proteins with gRNAs that are offset, one can create DSBs while increasing the specificity of cleavage and significantly reducing off-target breaks.21Ran FA Hsu PD Lin CY Gootenberg JS Konermann S Trevino AE et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell. 2013; 154: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (2334) Google Scholar Essentially this approach attempts to increase the specificity in the same way that dimerizing nucleases does but with the caveat that monomeric nickases still have the potential to induce mutations.19Tsai SQ Zheng Z Nguyen NT Liebers M Topkar VV Thapar V et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.Nat Biotechnol. 2014; 33: 187-197Crossref PubMed Scopus (1287) Google Scholar Further research is needed to determine whether these strategies can be combined to enhance specificity further. A recent study describes a method for gRNA design that significantly enhanced the frequency of genome editing by Cas9 in Caenorhabditis elegans. The key innovation was to design gRNAs with a GG motif at the 3′ end of their target-specific sequences. All guides designed for all targets supported robust genome editing, both imprecise NHEJ events and precise, template HDR events.22Farboud B Meyer BJ Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.Genetics. 2015; (e-pub ahead of print 18 February 2015)PubMed Google Scholar Dr Scharenberg reviewed work he has done with Cellectis Therapeutics and Seattle Children's Research Institute combining a meganuclease, or homing endonuclease, with a TAL array.23Boissel S Jarjour J Astrakhan A Adey A Gouble A Duchateau P et al.megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.Nucleic Acids Res. 2014; 42: 2591-2601Crossref PubMed Scopus (131) Google Scholar The goal is to combine the high binding specificity of the TAL with the high cleavage specificity of the meganuclease. By fusing a site-specific meganuclease to a TAL array that binds adjacent to the meganuclease target site (thus tethering the meganuclease adjacent to its desired target site), one can increase cleavage activity at that target site and minimize off-target activity, as tethering will not occur at off-target sites lacking an adjacent sequence capable of being bound by the TAL array. The homing endonuclease has to be engineered, but the specificity can be improved through the engineering of the TAL, which is easier. Scharenberg and colleagues have explored this construct (called a megaTAL) in T cells, in which the goal is to engineer the T cell to express a specific chimeric antigen receptor and at the same time disrupt the native T-cell receptor alpha (TCRα). This approach would allow for allogeneic designer T cells, currently being tested for a number of oncology applications, to function without the risk of graft-vs.-host disease (GVHD). The activity and specificity of the meganuclease targeting TCRα was assessed both with and without fusion to a TAL array that could bind a DNA sequence upstream from the cleavage site for the TCRα-specific meganuclease. Cleavage of the TCRα gene using the meganuclease alone was ~1.6% but was increased by 20-fold with the megaTAL construct. The megaTAL was then tested with co-transfection of each nuclease with Trex2, a 3′ endonuclease that can trim back the 3′ overhangs that homing endonucleases make, thereby markedly accentuating the generation of indels. Addition of Trex2 further increased the rate of disruption, yielding rates of TCRα disruption consistently exceeding 70%.23Boissel S Jarjour J Astrakhan A Adey A Gouble A Duchateau P et al.megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.Nucleic Acids Res. 2014; 42: 2591-2601Crossref PubMed Scopus (131) Google Scholar This research underscores that the specificity of these tools could be enhanced further by combining them to take advantage of their respective specificity and ease of engineering. Nonetheless, an assessment of safety and specificity using preclinical assays will still be necessary, no matter which nuclease platform is utilized. Identifying safe harbors. An alternative strategy to achieve safe, targeted gene delivery and limit off-target activity is to identify sites in the human genome that are at minimal risk of causing insertional oncogenesis upon integration of foreign DNA, while being accessible to a highly specific nuclease with minimal off-target activity. Such "genomic safe harbors" may be extragenic sites that are remote from a gene or genomic regulatory sequence, or intragenic sites (within a gene) whose disruption is deemed to be tolerable. Drawing from human clinical trial data on integration sites for retroviral and lentiviral vectors, several researchers24Papapetrou EP Lee G Malani N Setty M Riviere I Tirunagari LM et al.Genomic safe harbors permit high beta-globin transgene expression in thalassemia-induced pluripotent stem cells.Nat Biotechnol. 2011; 29: 73-78Crossref PubMed Scopus (248) Google Scholar,25Sadelain M Papapetrou EP Bushman FD Safe harbours for the integration of new DNA in the human genome.Nat Rev Cancer. 2012; 12: 51-58Crossref Scopus (147) Google Scholar have proposed the following criteria that could constitute an extragenic safe harbor for DNA integration: A safe harbor should be (i) outside a gene transcription unit; (ii) located >50 kilobases (kb) from the 5′ end of any gene; (iii) located >300 kb from cancer-related genes; (iv) located >300 kb from any identified microRNA; and (v) outside ultra-conserved regions and long noncoding RNAs. In studies of lentiviral vector integrations in transduced induced pluripotent stem cells, analysis of over 5,000 integration sites revealed that ~17% of integrations occurred in safe harbors. The vectors that integrated into these safe harbors were able to express therapeutic levels of β-globin from their transgene without perturbing endogenous gene expression.24Papapetrou EP Lee G Malani N Setty M Riviere I Tirunagari LM et al.Genomic safe harbors permit high beta-globin transgene expression in thalassemia-induced pluripotent stem cells.Nat Biotechnol. 2011; 29: 73-78Crossref PubMed Scopus (248) Google Scholar Several candidate genomic safe-harbor sites (GSHs) have been explored, including AAVS1, CCR5, and the ROSA26 locus. Although there are clinical data for CCR5 knockout in T cells and other data showing the safety of integration into AAVS1 in human cultured T cells, these sites have not been validated as universal GSHs.25Sadelain M Papapetrou EP Bushman FD Safe harbours for the integration of new DNA in the human genome.Nat Rev Cancer. 2012; 12: 51-58Crossref Scopus (147) Google Scholar In addition, it is not known whether the gene-rich loci of these sites, including some oncogenes, will limit their use when targeting other cell lines. Of course, much remains to be learned about sites that are identified as being GSHs. Non-protein-coding sequences may be more prevalent than currently known, and some data suggest that there may be low levels of transcription in intragenic sites. Validation of such sites will require measuring the effect of the integrated transgene on neighboring gene expression. This can be more easily accomplished in cell types that can be cloned such as pluripotent stem cells or T lymphocytes, and may not be feasible in cells such as neural cells or hematopoietic stem cells. Moreover, true GSHs would have to tolerate (i.e., without unintended transformation) the integration of a number of different elements, including promoters, enhancers, and chromatin determinants. Finally, validation of the safety of integration at these safe harbors should be done in animals; however, there are challenges in developing appropriate animal models.25Sadelain M Papapetrou EP Bushman FD Safe harbours for the integration of new DNA in the human genome.Nat Rev Cancer. 2012; 12: 51-58Crossref Scopus (147) Google Scholar Identifying and evaluating the impact of off-target activity Although innovative designs and exploration of safe harbors are certainly important strategies, any clinical development strategy will have to include identification and evaluation of potential off-target sites. A complete catalog of off-target sites might be accomplished using whole-genome sequencing to look for evidence of indels and translocations. However, this approach would also be very costly, especially for less frequent events, and the sequencing itself has an error rate.26Tsai SQ Iafrate AJ Joung JK Genome editing: a tool for research and therapy: towards a functional understanding of variants for molecular diagnostics using genome editing.Nat Med. 2014; 20: 1103-1104Crossref PubMed Scopus (11) Google Scholar For example, if the off-target cleavage occurs at a frequency of 0.1% per genome, at least 1,000 genomes may have to be sequenced to capture such a low-frequency event, adding a significant cost to the analysis. Furthermore, as DSBs and repair can occur in cells as a result of culture conditions alone (even in the absence of exogenous nucleases), there is the challenge of distinguishing the actions of nucleases from naturally occurring background DNA breaks and the spontaneous formation of small indels. In addition, with ongoing deep-sequencing projects, it is now recognized that any individual's genome can contain up to 750,000 unique indels.27Snyder M Du J Gerstein M Personal genome sequencing: current approaches and challenges.Genes Dev. 2010; 24: 423-431Crossref PubMed Scopus (113) Google Scholar As a result of these limitations, many groups have used a focused approach to base prediction of potential off-target sites sequence similarity to the on-target site followed by experimental confirmation to validate those predictions. However, the significant disadvantage of such a focused approach is that it will potentially miss other potential off-target sites that could have clinical significance. Bioinformatics tools are being developed to predict off-target sites. A number of web-based tools have been developed, such as Predicted Report of Genome-wide Nuclease Off-Target Sites (PROGNOS, http://baolab.bme.gatech.edu/R