Anticipating and Identifying Collateral Damage in Genome Editing
Authors: Gaëtan Burgio, Lydia Teboul
Journal: Trends in Genetics (2020)

Genome editing has a transformative potential in healthcare or to improve crops or livestock. However, the use of Cas9 or other nucleases can yield unpredictable events at the target site or off target.To overcome these challenge, it is critical to understand and accurately predict the whole range of possible editing outcomes.The key to success is to combine molecular assays to evaluate the sequence changes at the target site and to quantify the number of copies of segments deleted/inserted across the genome.For all applications, thorough evaluation of these outcomes is essential to identify all collateral damage from nuclease activity and for a real appraisal of the benefits and risks associated with applying this technology. Genome editing has powerful applications in research, healthcare, and agriculture. However, the range of possible molecular events resulting from genome editing has been underestimated and the technology remains unpredictable on, and away from, the target locus. This has considerable impact in providing a safe approach for therapeutic genome editing, agriculture, and other applications. This opinion article discusses how to anticipate and detect those editing events by a combination of assays to capture all possible genomic changes. It also discusses strategies for preventing unwanted effects, critical to appraise the benefit or risk associated with the use of the technology. Anticipating and verifying the result of genome editing are essential for the success for all applications. Genome editing has powerful applications in research, healthcare, and agriculture. However, the range of possible molecular events resulting from genome editing has been underestimated and the technology remains unpredictable on, and away from, the target locus. This has considerable impact in providing a safe approach for therapeutic genome editing, agriculture, and other applications. This opinion article discusses how to anticipate and detect those editing events by a combination of assays to capture all possible genomic changes. It also discusses strategies for preventing unwanted effects, critical to appraise the benefit or risk associated with the use of the technology. Anticipating and verifying the result of genome editing are essential for the success for all applications. The application of genome editing is transforming agriculture, biomedical research, and healthcare. The many proposed purposes include the generation of more productive or robust crops and farm animals, animal hosts for the production of tissues for graft purposes and therapies that use ex vivo or somatic tissue engineering [1.Pickar-Oliver A. Gersbach C.A. The next generation of CRISPR-Cas technologies and applications.Nat. Rev. Mol. Cell Biol. 2019; 20: 490-507Crossref PubMed Scopus (471) Google Scholar, 2.Hajizadeh Dastjerdi A. et al.The expanding class 2 CRISPR toolbox: diversity, applicability, and targeting drawbacks.BioDrugs Clin. Immunother. Biopharm. Gene Ther. 2019; 33: 503-513Google Scholar, 3.Zhang Y. et al.Applications and potential of genome editing in crop improvement.Genome Biol. 2018; 19: 210Crossref PubMed Scopus (142) Google Scholar]. The promise of applicability is turning into reality, as illustrated by the first nonrandomized Phase I clinical triali in which the use of clustered regularly interspaced short palindromic repeats (CRISPR)-engineered T cells was recently found to be safe [4.Stadtmauer E.A. et al.CRISPR-engineered T cells in patients with refractory cancer.Science. 2020; 367eaba7365Crossref PubMed Scopus (470) Google Scholar]. To date, >20 Phase I/II human clinical trials are underway for a broad range of diseases, including cancers, β-thalassemia, sickle cell disease, and Duchenne muscular dystrophy (summarized and discussed in [1.Pickar-Oliver A. Gersbach C.A. The next generation of CRISPR-Cas technologies and applications.Nat. Rev. Mol. Cell Biol. 2019; 20: 490-507Crossref PubMed Scopus (471) Google Scholar,5.Hamilton J.R. Doudna J.A. Knocking out barriers to engineered cell activity.Science. 2020; 367: 976-977Crossref PubMed Scopus (8) Google Scholar]). Genome editing is generally based on either zinc finger nucleases [6.Urnov F.D. et al.Highly efficient endogenous human gene correction using designed zinc-finger nucleases.Nature. 2005; 435: 646-651Crossref PubMed Scopus (1293) Google Scholar], transcription activator-like effector nucleases [7.Miller J.C. et al.A TALE nuclease architecture for efficient genome editing.Nat. Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1509) Google Scholar], or the CRISPR/CRISPR-associated (Cas) system (see Glossary) [8.Jinek M. et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8500) Google Scholar]. These molecules act by inducing a double-stranded cut in a specific DNA sequence, which results in a genetic alteration as the gap is being repaired. In the clinic, the initial applications aim for deletions of genomic DNA intervals and do not yet involve precision at the nucleotide level; thus, these can be executed through the sole delivery of a genome-editing nuclease. However, for more precise editing, such as the generation of point mutations or more intricate changes, or even accurate deletion of a genomic segment, single- or double-stranded DNA templates are also delivered, together with the nucleases, to direct the repair to result in a given sequence by homology directed repair (HDR) [9.Wang H. et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell. 2013; 153: 910-918Abstract Full Text Full Text PDF PubMed Scopus (2469) Google Scholar, 10.Quadros R.M. et al.Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.Genome Biol. 2017; 18: 92Crossref PubMed Scopus (192) Google Scholar, 11.Gu B. et al.Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.Nat. Biotechnol. 2018; 36: 632-637Crossref PubMed Scopus (130) Google Scholar] or nonhomologous end-joining [12.Danner E. et al.A homology independent sequence replacement strategy in human cells using a CRISPR nuclease.bioRxiv. 2020; (Published online May 12, 2020. https://doi.org/10.1101/2020.05.11.088252)Google Scholar]. Base editors [13.Komor A.C. et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (2090) Google Scholar] and prime editors [14.Anzalone A.V. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (1200) Google Scholar] are alternative strategies for more precise editing. Overall, the range of genome editing tools is ever increasing and their transformative potential across a range of fields of application is immense. The safety of genome-editing technologies is just as critical as their efficiency for their successful application in health or agriculture. Common to all fields of application are the risks associated with undesired genetic changes that can be triggered by genome editing. The potential for unwanted off-target nuclease activity was recognized early in the application of the CRISPR/Cas9 system as a genome-editing tool [15.Fu Y. et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat. Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (2081) Google Scholar]. The frequency of such events and the attached risks were the subjects of much debate [16.Iyer V. et al.No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice.PLoS Genet. 2018; 14e1007503Crossref PubMed Scopus (69) Google Scholar, 17.Feng Z. et al.Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 4632-4637Crossref PubMed Scopus (481) Google Scholar, 18.Smith C. et al.Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs.Cell Stem Cell. 2014; 15: 12-13Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar]. The general consensus is that, with careful molecular design, off-target events are rare and generally can be segregated away from the allele of interest in genome edited animals, unless the off-target region is in linkage disequilibrium with the target site; however, they are potentially more pernicious in cultured cells or in a somatic delivery system [19.Kim S-T. et al.Response to "Unexpected mutations after CRISPR–Cas9 editing in vivo".Nat. Methods. 2018; 15: 239-240Crossref PubMed Google Scholar, 20.Lareau C.A. et al.Response to "Unexpected mutations after CRISPR–Cas9 editing in vivo".Nat. Methods. 2018; 15: 238Crossref PubMed Google Scholar, 21.Lescarbeau R.M. et al.Response to "Unexpected mutations after CRISPR–Cas9 editing in vivo".Nat. Methods. 2018; 15237–237Crossref Google Scholar, 22.Nutter L.M.J. et al.Response to "Unexpected mutations after CRISPR–Cas9 editing in vivo".Nat. Methods. 2018; 15: 235-236Crossref PubMed Google Scholar, 23.Wilson C.J. et al.Response to "Unexpected mutations after CRISPR–Cas9 editing in vivo".Nat. Methods. 2018; 15: 236-237Crossref PubMed Google Scholar]. In those cases, it is particularly essential that off-target events are captured [24.Teboul L. et al.Variability in genome editing outcomes: challenges for research reproducibility and clinical safety.Mol. Ther. 2020; 28: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. However, on-target effects and ectopic insertions of donor template are less predictable and have often been underestimated. Nevertheless, on-target effects of genome-editing enzymes are also now better documented. These can take many forms: single nucleotide variations, indels, large and/or complex genomic rearrangements, segmental duplications, chromosomal translocation, terminal chromosomal truncation up to several megabases, or loss of one or both arms of a chromosome [25.Owens D.D.G. et al.Microhomologies are prevalent at Cas9-induced larger deletions.Nucleic Acids Res. 2019; 47: 7402-7417Crossref PubMed Scopus (41) Google Scholar, 26.Kosicki M. et al.Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.Nat. Biotechnol. 2018; 36: 765-771Crossref PubMed Scopus (46) Google Scholar, 27.Birling M-C. et al.Efficient and rapid generation of large genomic variants in rats and mice using CRISMERE.Sci. Rep. 2017; 743331–43331Crossref Scopus (47) Google Scholar, 28.Boroviak K. et al.Revealing hidden complexities of genomic rearrangements generated with Cas9.Sci. 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These effects are intimately linked to the kinetics of the enzyme's interaction with the DNA, and with the DNA repair pathways [32.Sternberg S.H. et al.Conformational control of DNA target cleavage by CRISPR-Cas9.Nature. 2015; 527: 110-113Crossref PubMed Scopus (329) Google Scholar,33.Clarke R. et al.Enhanced bacterial immunity and mammalian genome editing via RNA-polymerase-mediated dislodging of Cas9 from double-strand DNA breaks. Mol.Cell. 2018; 71: 42-55Scopus (61) Google Scholar]. This results in multiple cutting at the target site and can lead to the alteration of a larger than expected segment by ~1–2 kb up to 50 kb at a frequency of ~15–20% of the target DNA in somatic cells [25.Owens D.D.G. et al.Microhomologies are prevalent at Cas9-induced larger deletions.Nucleic Acids Res. 2019; 47: 7402-7417Crossref PubMed Scopus (41) Google Scholar,26.Kosicki M. et al.Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.Nat. Biotechnol. 2018; 36: 765-771Crossref PubMed Scopus (46) Google Scholar,29.Cullot G. et al.CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.Nat. Commun. 2019; 10: 1136Crossref PubMed Scopus (120) Google Scholar]. In early embryos, this additionally translates to mosaicism of the mutated alleles [34.Mizuno S. et al.Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system.Mamm. Genome. 2014; 25: 327-334Crossref PubMed Scopus (79) Google Scholar, 35.Renaud J-B. et al.Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases.Cell Rep. 2016; 14: 2263-2272Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 36.Mianné J. et al.Analysing the outcome of CRISPR-aided genome editing in embryos: screening, genotyping and quality control.Methods. 2017; 121–122: 68-76Crossref PubMed Scopus (45) Google Scholar]. Similarly, repair with a template can result in a variety of sequence changes even when the insertion of the repair template is on target (Figure 1). It can yield unpredictable and sometimes complex events at the target site, such as partial insertion of the template, sequence duplications, inversions or rearrangements of the template in combination with endogenous sequences [35.Renaud J-B. et al.Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases.Cell Rep. 2016; 14: 2263-2272Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,37.Mianné J. et al.Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair.Genome Med. 2016; 816–16Crossref Scopus (77) Google Scholar, 38.Lanza D.G. et al.Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles.BMC Biol. 2018; 16: 69Crossref PubMed Scopus (32) Google Scholar, 39.Codner G.F. et al.Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.BMC Biol. 2018; 16: 70Crossref PubMed Scopus (41) Google Scholar], as well as ectopic insertions of the repair template. The delivery of a template for repair or vectors for nuclease expression can also yield ectopic insertions that could affect the safety of the approach [40.Chen K. et al.CRISPR/Cas genome editing and precision plant breeding in agriculture.Annu. Rev. Plant Biol. 2019; 70: 667-697Crossref PubMed Scopus (481) Google Scholar,41.Skryabin B.V. et al.Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9-mediated genome editing events.Sci. Adv. 2020; 6eaax2941Crossref PubMed Scopus (28) Google Scholar]. The recent example of genome editing of the POLLED allele in cattle [42.Carlson D.F. et al.Production of hornless dairy cattle from genome-edited cell lines.Nat. Biotechnol. 2016; 34: 479-481Crossref PubMed Scopus (172) Google Scholar], in which the full outcome had not been identified from the first analysis of the edited cows [43.Young A.E. et al.Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.Nat. Biotechnol. 2020; 38: 225-232Crossref PubMed Scopus (35) Google Scholar], illustrates the difficulties involved in thoroughly identifying ectopic insertions of the repair template. Additional studies show that pervasive insertion of the donor template across the genome can remain undetected with conventional methods, such as PCR and Sanger sequencing [39.Codner G.F. et al.Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.BMC Biol. 2018; 16: 70Crossref PubMed Scopus (41) Google Scholar]. These examples underline questions relating to the prevalence and type of on-target modifications and ectopic insertions of the donor DNA following codelivery with nucleases. They also articulate the importance of using the appropriate assays to evaluate the correctness of the resulting genome-editing event(s). (See Box 1). As genome editing is increasingly used, further unexpected and potentially negative outcomes of its application are still being uncovered. For example, the perdurance and transmission of DNA double-stranded breaks (DSBs) is a phenomenon so far overlooked and forms a molecular basis for several mixed alleles arising from a single cutting event [31.Zuccaro M.V. et al.Reading frame restoration at the EYS locus, and allele-specific chromosome removal after Cas9 cleavage in human embryos.bioRxiv. 2020; (Published online June 18, 2020. https://doi.org/10.1101/2020.06.17.149237)Google Scholar,44.Aitken S.J. et al.Pervasive lesion segregation shapes cancer genome evolution.Nature. 2020; 583: 265-270Crossref PubMed Scopus (19) Google Scholar]. Also, the occurrence of potentially extensive gene conversion [45.Chen J-M. et al.Gene conversion: mechanisms, evolution and human disease.Nat. Rev. Genet. 2007; 8: 762-775Crossref PubMed Scopus (432) Google Scholar] of edited alleles went unnoticed until recently. This involves the transfer of DNA from one genomic location to another by homologous recombination. Examples of gene conversion are the transfer of the DNA from delta-hemoglobin to beta-hemoglobin in the case of dividing or nondividing cells, or transfer to the use of the paternal allele as a repair template in the case of embryonic cells [46.Slightom J.L. et al.Human fetal G gamma- and A gamma-globin genes: complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes.Cell. 1980; 21: 627-638Abstract Full Text PDF PubMed Scopus (571) Google Scholar, 47.Javidi-Parsijani P. et al.CRISPR/Cas9 increases mitotic gene conversion in human cells.Gene Ther. 2020; 27: 281-296Crossref PubMed Scopus (13) Google Scholar, 48.Chandrasegaran S. et al.Genome editing of human embryos: to edit or not to edit, that is the question.J. Clin. Invest. 2017; 127: 3588-3590Crossref PubMed Scopus (2) Google Scholar]. Gene conversion could result in a partial or full repair of the allele; it was hypothesized that such a mechanism could be utilized as an internal template repair for precision editing, but this is still disputed [47.Javidi-Parsijani P. et al.CRISPR/Cas9 increases mitotic gene conversion in human cells.Gene Ther. 2020; 27: 281-296Crossref PubMed Scopus (13) Google Scholar]. In particular, because conversion tracks may expand well beyond the targeted region, the resulting loss of heterozygosity represents an additional risk for clinical application [31.Zuccaro M.V. et al.Reading frame restoration at the EYS locus, and allele-specific chromosome removal after Cas9 cleavage in human embryos.bioRxiv. 2020; (Published online June 18, 2020. https://doi.org/10.1101/2020.06.17.149237)Google Scholar,49.Liang D. et al.Frequent gene conversion in human embryos induced by double strand breaks.bioRxiv. 2020; (Published online June 20, 2020. https://doi.org/10.1101/2020.06.19.162214)Google Scholar]. Finally, and importantly, it is now becoming increasingly apparent that genomic segments can be inadvertently altered at comparatively large distances from the cutting site [29.Cullot G. et al.CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.Nat. Commun. 2019; 10: 1136Crossref PubMed Scopus (120) Google Scholar,50.Simeonov D.R. et al.A large CRISPR-induced bystander mutation causes immune dysregulation.Commun. Biol. 2019; 270–70Crossref Scopus (12) Google Scholar]. The frequency of such outcomes and the genetic range susceptible to alteration following genome-editing intervention remain to be fully appraised. All of these poorly understood consequences pertain to changes to the DNA sequence. Other potential unknown consequences of on- and off-target effects could have an entirely different molecular basis, such as deregulation of the chromatin environment or the 3D organization of the nucleus, which could change the genome stability or gene expression. The incidence of such potential consequences is as yet largely unexplored, In all instances, the challenge is to fully apprehend the editing outcomes that may have adverse consequences. This is likely to require the application of a suite of molecular techniques to interrogate the different artefactual features that can be encountered in genome editing. These features can be diverse in scale (single base to megabase) and may involve additional template insertions. All of these outcomes can occur at the targeted site or ectopically. Second, to add more complexity, the extent and nature of lesions vary considerably depending on whether the editing occurs in nondividing somatic, dividing somatic, or germinal (early embryonic) cells. In the case of euploid clonal cell populations or the progeny of founder animals, there are only two alleles for each autosomal locus and, therefore, a maximum of two variants may need to be identified. By contrast, animals born from genome editing of early embryos are generally mosaic [34.Mizuno S. et al.Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system.Mamm. Genome. 2014; 25: 327-334Crossref PubMed Scopus (79) Google Scholar,35.Renaud J-B. et al.Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases.Cell Rep. 2016; 14: 2263-2272Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,37.Mianné J. et al.Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair.Genome Med. 2016; 816–16Crossref Scopus (77) Google Scholar]. Modification of pools of cultured cells yield heterogenous cell populations [18.Smith C. et al.Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs.Cell Stem Cell. 2014; 15: 12-13Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar], and tissues modified by somatic modification [51.Laoharawee K. et al.Dose-dependent prevention of metabolic and neurologic disease in murine MPS II by ZFN-mediated in vivo genome editing.Mol. Ther. 2018; 26: 1127-1136Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar] represent yet a larger degree of genetic complexity. In all these examples, multiple and potentially diverse allelic variants are represented at different frequencies. It is critical to elaborate a clear strategy that takes into account these different degrees of genetic complexity to uncover, fully characterize, or ideally prevent, these unwanted events. The stakes are high because the impact of incomplete characterization of editing effects is potentially important in all areas of application: in the laboratory, the risk is of irreproducible or artefactual research. Therefore, information obtained with genetically edited founder animals (likely to be mosaic) must be interpreted on the basis of the intrinsic genetic complexity of these animals and the genetic content of progeny must be extensively revalidated. Equally, interpretation of data obtained with edited culture cell pools (where repair may result in many different alleles with various rates) requires an understanding of the genetic composition of these complex cell populations, such as mosaicism [25.Owens D.D.G. et al.Microhomologies are prevalent at Cas9-induced larger deletions.Nucleic Acids Res. 2019; 47: 7402-7417Crossref PubMed Scopus (41) Google Scholar]. When editing is used for the production of agricultural products (plants or animals), it remains unclear whether uncontrolled outcomes may pose a risk to the users. Such variability may prevent licensing for commercialization by regulators or negatively affect the confidence of consumers in the safety of those products [43.Young A.E. et al.Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.Nat. Biotechnol. 2020; 38: 225-232Crossref PubMed Scopus (35) Google Scholar,52.Grohmann L. et al.Detection and identification of genome editing in plants: challenges and opportunities.Front. Plant Sci. 2019; 10: 236Crossref PubMed Scopus (44) Google Scholar]. For use in the clinic, in tissue engineering or by somatic delivery, the degree of variability of genomic outcome that may be acceptable in terms of safety remains to be appreciated and may not represent an insurmountable obstacle [4.Stadtmauer E.A. et al.CRISPR-engineered T cells in patients with refractory cancer.Science. 2020; 367eaba7365Crossref PubMed Scopus (470) Google Scholar]. However, the range of edited sequences that can result from a given therapeutic intervention must still be thoroughly understood to evaluate the associated benefit–risk balance [24.Teboul L. et al.Variability in genome editing outcomes: challenges for research reproducibility and clinical safety.Mol. Ther. 2020; 28: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. Finally, an inability to fully validate the consequences of CRISPR/Cas activity throughout the embryo represents a practical barrier to germline editing in the clinic [53.Fogarty N.M.E. et al.Genome editing reveals a role for OCT4 in human embryogenesis.Nature. 2017; 550: 67Crossref PubMed Scopus (212) Google Scholar]. In all applications of genome editing (whether in biomedical research, agricultural production, or the clinic) a thorough evaluation of these outcomes is necessary for a realistic appraisal of the benefit–risk ratio associated with the use of the technology. The required level of investigation depends on the specific application, but all demand the ability to anticipate the whole range of potential (wanted and unwanted) consequences of each genome-editing intervention. Capturing the variability of genome-editing outcomes requires increasing investment in resources as the attention extends away from the target site: (i) as a minimum, amplification and sequencing of the target site and of chromosomally linked potential off-target sites should be achieved; (ii) quantification of the number of copies of deleted segments or donor template to capture on target duplications and ectopic integrations should be included. (This is also essential for all applications.); (iii) the use of more elaborate assays to inform on potential larger-scale chromosomal rearrangements is desirable, because an increasing number of examples have been identified in which additional sequence changes away from the cutting site have been found; and (v) where required, analysis should be extended to the whole genome to predict or capture potential off-target sites. Equally, a pragmatic approach to the interrogation of genome editing takes into account the likely genetic complexity of the edited material (whether all cells have identical genomes or constitute a genetically diverse population) and the context of application (see Box 1). For example, mosaic founder small laboratory animals will be bred, thus allowing for the segregation of most unwanted edits at the next generation. Therefore, it is only essential to search for the presence of an allele of interest and linked off-target effects. Definitive characterization of the model can await transmission of the allele of interest to the subsequent generation. By contrast, the whole gamut of mutations arising from CRISPR/Cas activity is to be considered when this technique is used in large livestock (because associated financial and welfare costs are high and timelines extended by long gestations) or for somatic treatment in the clinic [24.Teboul L. et al.Variability in genome editing outcomes: challenges for research reproducibility and clinical safety.Mol. Ther. 2020; 28: 1422-1431Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,43.Young A.E. et al.Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.Nat. Biotechnol. 2020; 38: 225-232Crossref PubMed Scopus (35) Google Scholar]. Therefore, depending on the genome-editing application, the strategy for validation will either aim to identify the presence of a specific variant, seek to capture complexity, or definitively ascertain an entire genetic make-up. In summary, the genotyping strategy will take into account the ability of each molecular assay to cope with the genetic complexity of the material and will customize effort for the context of utilization.Box 1Factors that Should Be Assessed when Validating Genome-Editing Outcomes1.What is/are the new genetic modification(s) on target? What is the length of the interval potentially altered by the intervention?2.Are there sequence changes in potential off-target sites? Are these sites physically linked to the target locus?3.What is the number of copies of the mobilized segments (deleted or introduced as template)?4.Can the purpose of the application cope with potential unwanted changes in the genome? 1.What is/are the new genetic modification(s) on target? What is the length of the interval potentially altered by the intervention?2.Are there sequence changes in potential off-target sites? A