CRISPR–Cas9 gene editing induced complex on-target outcomes in human cells

•Unwanted on-target mutation occurs after CRISPR–Cas9 cleavage.•Assessment of comprehensive on-target outcomes is necessary.•Clinical genomic engineering requires quality controls to address safety concerns. CRISPR–Cas9 is a powerful tool for editing the genome and holds great promise for gene therapy applications. Initial concerns of gene engineering focus on off-target effects. However, in addition to short indel mutations (often <50 bp), an increasing number of studies have revealed complex on-target results after double-strand break repair by CRISPR–Cas9, such as large deletions, gene rearrangement, and loss of heterozygosity. These unintended mutations are potential safety concerns in clinical gene editing. Here, in this review, we summarize the significant findings of CRISPR–Cas9-induced on-target deleterious outcomes and discuss putative ways to achieve safe gene therapy. CRISPR–Cas9 is a powerful tool for editing the genome and holds great promise for gene therapy applications. Initial concerns of gene engineering focus on off-target effects. However, in addition to short indel mutations (often <50 bp), an increasing number of studies have revealed complex on-target results after double-strand break repair by CRISPR–Cas9, such as large deletions, gene rearrangement, and loss of heterozygosity. These unintended mutations are potential safety concerns in clinical gene editing. Here, in this review, we summarize the significant findings of CRISPR–Cas9-induced on-target deleterious outcomes and discuss putative ways to achieve safe gene therapy. Clustered, regularly interspaced, short palindromic repeats (CRISPR)–Cas9 has been a promising tool for gene engineering, for example, in correcting disease-associated mutant alleles in somatic or stem cells [1Lander ES. The heroes of CRISPR.Cell. 2016; 164: 18-28Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar]. Cas9 is a single endonuclease evolved in bacteria and archaea to function as a natural adaptive immune system [2Deltcheva E Chylinski K Sharma CM et al.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.Nature. 2011; 471: 602-607Crossref PubMed Scopus (1558) Google Scholar,3Gasiunas G Barrangou R Horvath P Siksnys V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proc Natl Acad Sci USA. 2012; 109: E2579-E2586Crossref PubMed Scopus (1530) Google Scholar]. Cas9 programmed with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) (Cas9–sgRNA ribonucleoprotein complex) has HNH and RuvC nuclease domains to cleave target DNA, generating two blunt ends of double-strand breaks (DSBs), usually 3 bp upstream of a protospacer adjacent motif (PAM, NGG for SpCas9 from Streptococcus pyogenes) sequence [3Gasiunas G Barrangou R Horvath P Siksnys V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proc Natl Acad Sci USA. 2012; 109: E2579-E2586Crossref PubMed Scopus (1530) Google Scholar,4Jinek 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 (8553) Google Scholar]. Two elements are important for the cleavage by Cas9: (1) the requirement for a guide RNA that base pairs with the target sequence; (2) the necessity of the PAM site for target identification. Cas9 nickase is an enzyme that is inactivated in either nuclease domain. Inactivation of both the HNH and RuvC domains can generate catalytically dead Cas9 (dCas9), which maintains the capacity to bind to the cognate DNA without cutting it [5Nishimasu H Ran FA Hsu PD et al.Crystal structure of Cas9 in complex with guide RNA and target DNA.Cell. 2014; 156: 935-949Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar,6Cong L Ran FA Cox D et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9381) Google Scholar]. Nickases are engineered and used for applications such as base editors (BEs) and prime editors (PEs) [7Komor AC Kim YB Packer MS Zuris JA Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (2105) Google Scholar, 8Gaudelli NM Komor AC Rees HA et al.Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.Nature. 2017; 551: 464-471Crossref PubMed Scopus (1529) Google Scholar, 9Rees HA Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells.Nat Rev Genet. 2018; 19: 770-788Crossref PubMed Scopus (605) Google Scholar, 10Anzalone AV Randolph PB Davis JR et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (1216) Google Scholar]. Both BEs and PEs can edit DNA without generating DSBs. dCas9 can be used for transcriptional regulation and epigenetic modifications [11Shalem O Sanjana NE Zhang F. High-throughput functional genomics using CRISPR–Cas9.Nat Rev Genet. 2015; 16: 299-311Crossref PubMed Scopus (701) Google Scholar, 12Thakore PI Black JB Hilton IB Gersbach CA. Editing the epigenome: technologies for programmable transcription and epigenetic modulation.Nat Methods. 2016; 13: 127-137Crossref PubMed Scopus (260) Google Scholar, 13Pickar-Oliver A Gersbach CA. The next generation of CRISPR–Cas technologies and applications.Nat Rev Mol Cell Biol. 2019; 20: 490-507Crossref PubMed Scopus (477) Google Scholar]. CRISPR–Cas9 cleavage of one target site often induces nucleotide insertions and deletions. Simultaneous editing of two sites has been used to generate inversion of a DNA segment or chromosomal translocation [14Shou J Li J Liu Y Wu Q. Precise and predictable CRISPR chromosomal rearrangements reveal principles of Cas9-mediated nucleotide insertion.Mol Cell. 2018; 71 (e4): 498-509Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar]. DSB-Induced repair outcomes with similar sequences can result from distinct mechanisms [15Hussmann JA Ling J Ravisankar P et al.Mapping the genetic landscape of DNA double-strand break repair.Cell. 2021; 184 (e25): 5653-5669Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. These processes are regulated mainly by several types of competing DNA repair machinery [16Nambiar TS Baudrier L Billon P Ciccia A. CRISPR-based genome editing through the lens of DNA repair.Mol Cell. 2022; 82: 348-388Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar], including nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-directed repair (HDR). DSBs, one of the most hazardous forms of DNA damage, are a potent source of genome instability. Thus, to sustain an intact genome, cells with DSBs require rapid ligation of broken DNA ends and repair the break as quickly as possible. This is largely accomplished by a pathway termed canonical nonhomologous end joining (c-NHEJ), which is a rapid yet often erroneous repair process. During this process, key NHEJ molecules, such as Ku70, Ku80, and DNA ligase IV, are recruited to the ends of breaks, leading to nonmutated repair or producing mutated alleles with short insertion or deletions (indels) [17Scully R Panday A Elango R Willis NA. DNA double-strand break repair-pathway choice in somatic mammalian cells.Nat Rev Mol Cell Biol. 2019; 20: 698-714Crossref PubMed Scopus (405) Google Scholar]. When perfect end joining occurs, the reconstituted Cas9–sgRNA cognate sequence can be recut in the persistent presence of Cas9–sgRNA. However, end joining can also result in an insertion or deletion mutant outcome, preventing subsequent recognition and recutting by the nuclease [18Brinkman EK Chen T de Haas M Holland HA Akhtar W van Steensel B. Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks.Mol Cell. 2018; 70 (e6): 801-813Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar]. Furthermore, when the sgRNA guides Cas9 to target an open reading frame (ORF), the indel outcome may generate a frameshift mutation that abrogates protein function [19Wang T Wei JJ Sabatini DM Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system.Science. 2014; 343: 80-84Crossref PubMed Scopus (1775) Google Scholar,20Shalem O Sanjana NE Hartenian E et al.Genome-scale CRISPR-Cas9 knockout screening in human cells.Science. 2014; 343: 84-87Crossref PubMed Scopus (2908) Google Scholar] and create a premature stop codon that may trigger nonsense-mediated mRNA decay (NMD) [21Brogna S Wen J. Nonsense-mediated mRNA decay (NMD) mechanisms.Nat Struct Mol Biol. 2009; 16: 107-113Crossref PubMed Scopus (336) Google Scholar,22Popp MW Maquat LE. Leveraging rules of nonsense-mediated mRNA decay for genome engineering and personalized medicine.Cell. 2016; 165: 1319-1322Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar]. Recently, multiple reports have shown that editing outcomes are nonrandom [23van Overbeek M Capurso D Carter MM et al.DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks.Mol Cell. 2016; 63: 633-646Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar] and can be predicted [24Allen F Crepaldi L Alsinet C et al.Predicting the mutations generated by repair of Cas9-induced double-strand breaks.Nat Biotechnol. 2019; 37: 64-72Crossref Scopus (181) Google Scholar]. The mechanism mainly relies on target sequences and is less affected by cell types. Particularly in the position where the −4 bp upstream PAM sequence is a T (thymine), +T editing is the most frequent outcome of SpCas9 [25Fu YW Dai XY Wang WT et al.Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.Nucleic Acids Res. 2021; 49: 969-985Crossref PubMed Scopus (20) Google Scholar]. As such, most insertions result from NHEJ repair after Cas9 editing. Our unpublished work shows that this is a unique feature of SpCas9, and other Cas9 orthologs may behave differently. Due to its efficacy and predominant nature in bridging blunt ends, this end-joining machinery has been exploited to insert a long sequence using AAV [26Zhang JP Cheng XX Zhao M et al.Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse.Genome Biol. 2019; 20: 276Crossref PubMed Scopus (20) Google Scholar] as well as a double-strand oligonucleotide (dsODN) [27Auer TO Duroure K De Cian A Concordet JP Del Bene F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.Genome Res. 2014; 24: 142-153Crossref PubMed Scopus (437) Google Scholar,28Suzuki K Tsunekawa Y Hernandez-Benitez R et al.In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.Nature. 2016; 540: 144-149Crossref PubMed Scopus (604) Google Scholar], at the target site, as in the assessment of off-targets using the GUIDE-seq assay [29Tsai SQ Zheng Z Nguyen NT et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.Nat Biotechnol. 2015; 33: 187-197Crossref PubMed Scopus (1180) Google Scholar]. The default engagement of c-NHEJ can be disrupted by DNA end resection. When resection occurs at the breaks, the Mre11–Rad50–Nbs1 (MRN) complex [30Sfeir A Symington LS. Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway?.Trends Biochem Sci. 2015; 40: 701-714Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar] may be recruited, which hampers repair by the NHEJ pathway. In such scenario, microhomology-mediated end joining (MMEJ), also called alternative end joining (alt-EJ), is activated. MMEJ is considered to use stretches of microhomology of 2–25 bp on each side of the DSBs to realign the broken ends [30Sfeir A Symington LS. Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway?.Trends Biochem Sci. 2015; 40: 701-714Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar]. However, we found that a single C or G at both ends can also mediate significant levels of MMEJ editing [25Fu YW Dai XY Wang WT et al.Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.Nucleic Acids Res. 2021; 49: 969-985Crossref PubMed Scopus (20) Google Scholar]. The microhomologies also define the boundaries for DNA segment rejoining, leading to deletion of one microhomology and the regions between the two microhomologies. The MMEJ occurrence depends on the proximity to the break ends, and the length and G/C proportion of the microhomology, which determine the binding energy. The longer the mismatched nucleotides between the two homologies, the lower is the efficiency of MMEJ, as it will need to recruit flap endonucleases to remove DNA flaps. Therefore, a "strong" microhomology arm can generate a highly efficient MMEJ-mediated precise deletion [31Iyer S Suresh S Guo D et al.Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.Nature. 2019; 568: 561-565Crossref PubMed Scopus (55) Google Scholar,32Wang L Li L Ma Y et al.Reactivation of γ-globin expression through Cas9 or base editor to treat β-hemoglobinopathies.Cell Res. 2020; 30: 276-278Crossref PubMed Scopus (26) Google Scholar]. This phenomenon can be exploited to bring about a predictable and ideal editing outcome. To obtain more precise results, either single-nucleotide changes or large gene cassette insertion/deletion at specific sites, one needs to harness the HDR pathway by providing a donor template [33Porteus MH. Towards a new era in medicine: therapeutic genome editing.Genome Biol. 2015; 16: 1-12Crossref Scopus (43) Google Scholar,34Dever DP Porteus MH. The changing landscape of gene editing in hematopoietic stem cells: a step towards Cas9 clinical translation.Curr Opinion Hematol. 2017; 24: 481Crossref PubMed Scopus (46) Google Scholar]. An HDR donor contains an insertion sequence flanked by right and left homology arms (HAs), whose sequences are preferably identical to 300–1000 bp surrounding the DSB site. HDRs can make small genetic edits [35Chu VT Weber T Wefers B et al.Increasing the efficiency of homology-directed repair for CRISPR–Cas9-induced precise gene editing in mammalian cells.Nat Biotechnol. 2015; 33: 543-548Crossref PubMed Scopus (755) Google Scholar] and create precise insertion of long fragments [35Chu VT Weber T Wefers B et al.Increasing the efficiency of homology-directed repair for CRISPR–Cas9-induced precise gene editing in mammalian cells.Nat Biotechnol. 2015; 33: 543-548Crossref PubMed Scopus (755) Google Scholar], such as a gene of interest or a fluorescent reporter. Although end-joining processes (including NHEJ and MMEJ) are efficient in most cell types regardless of the cell cycle, potent HDR editing occurs predominantly in the S/G2 phases of the cell cycle [36Takata M Sasaki MS Sonoda E et al.Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.EMBO J. 1998; 17: 5497-5508Crossref PubMed Scopus (985) Google Scholar]. It has a relatively lower efficiency than NHEJ. However, HDR-mediated gene engineering has the advantage of enabling a large fragment DNA knockin with donor templates delivered in the form of plasmids [37Li XL Li GH Fu J et al.Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.Nucl Acids Res. 2018; 46: 10195-10215Crossref PubMed Scopus (52) Google Scholar, 38Zhang JP Li XL Li GH et al.Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.Genome Biol. 2017; 18: 35Crossref PubMed Scopus (214) Google Scholar, 39Wen W Cheng X Fu Y et al.High-level precise knockin of iPSCs by simultaneous reprogramming and genome editing of human peripheral blood mononuclear cells.Stem Cell Rep. 2018; 10: 1821-1834Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar], adeno-associated virus (AAV) vectors [40Kuo CY Long JD Campo-Fernandez B et al.Site-specific gene editing of human hematopoietic stem cells for X-linked hyper-IgM syndrome.Cell Rep. 2018; 23: 2606-2616Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 41Vakulskas CA Dever DP Rettig GR et al.A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.Nat Med. 2018; 24: 1216-1224Crossref PubMed Scopus (318) Google Scholar, 42Pavel-Dinu M Wiebking V Dejene BT et al.Gene correction for SCID-X1 in long-term hematopoietic stem cells.Nat Commun. 2019; 10: 1634Crossref PubMed Scopus (82) Google Scholar, 43Ferrari S Jacob A Beretta S et al.Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking.Nat Biotechnol. 2020; 38: 1298-1308Crossref PubMed Scopus (33) Google Scholar], or long ssDNA HDR donors [44Quadros RM Miura H Harms DW 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 (195) Google Scholar]. To promote HDR-mediated gene editing efficiency, a "double-cut" donor flanked by sgRNA–PAM sequences with HA can increase the HDR efficiency up to 10-fold [38Zhang JP Li XL Li GH et al.Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.Genome Biol. 2017; 18: 35Crossref PubMed Scopus (214) Google Scholar]. This design synchronizes the availability of both DSBs at the genome target and the linearized donor templates. In human induced pluripotent stem cells (iPSCs), a 20%–30% HDR-mediated knockin can be obtained using double-cut donors with 300- or 600-bp-long HAs [38Zhang JP Li XL Li GH et al.Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.Genome Biol. 2017; 18: 35Crossref PubMed Scopus (214) Google Scholar]. In K562 cells, a myelogenous leukemia cell line, the use of the double-cut HDR donor plasmid results in >50% editing efficacy (unpublished). With the development strategies of HDR-mediated knockin and blood cell reprogramming, blood cells can be reprogrammed into iPSCs and edited simultaneously at high efficiency in one step [39Wen W Cheng X Fu Y et al.High-level precise knockin of iPSCs by simultaneous reprogramming and genome editing of human peripheral blood mononuclear cells.Stem Cell Rep. 2018; 10: 1821-1834Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar]. To repair dsDNA damage induced by CRISPR–Cas9, these three major DNA repair pathways play a complementary role [45Tatiossian KJ Clark RDE Huang C Thornton ME Grubbs BH Cannon PM. Rational selection of CRISPR-Cas9 guide RNAs for homology-directed genome editing.Mol Ther. 2021; 29: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar,46Roidos P Sungalee S Benfatto S et al.A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice.Nat Commun. 2020; 11: 4077Crossref Scopus (16) Google Scholar]. NHEJ is a rapid and fast pathway, often leading to a +A/T editing outcome at the cutting site [25Fu YW Dai XY Wang WT et al.Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.Nucleic Acids Res. 2021; 49: 969-985Crossref PubMed Scopus (20) Google Scholar]. On the other hand, HDR and MMEJ are activated more slowly than NHEJ [25Fu YW Dai XY Wang WT et al.Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.Nucleic Acids Res. 2021; 49: 969-985Crossref PubMed Scopus (20) Google Scholar], mainly because HDR and MMEJ entail an Mre11-dependent DSB end resection process, yet NHEJ takes effect by directly joining two "clean" ends [47Truong LN Li Y Shi LZ et al.Microhomology-mediated end joining and homologous recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.Proc Natl Acad Sci USA. 2013; 110: 7720-7725Crossref PubMed Scopus (282) Google Scholar]. Of interest, HDR outcompetes MMEJ [47Truong LN Li Y Shi LZ et al.Microhomology-mediated end joining and homologous recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.Proc Natl Acad Sci USA. 2013; 110: 7720-7725Crossref PubMed Scopus (282) Google Scholar], likely because the presence of long homology in HDR stabilizes the annealing of donor template and the DSB-proximal genome sequence. As such, inhibition of the NHEJ pathway increases the chances of being repaired by MMEJ, leading to more deletions, small and large [48Yeh CD Richardson CD Corn JE. Advances in genome editing through control of DNA repair pathways.Nat Cell Biol. 2019; 21: 1468-1478Crossref PubMed Scopus (127) Google Scholar,49Wen W Quan ZJ Li SA et al.Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.Genome Biol. 2021; 22: 236Crossref PubMed Scopus (6) Google Scholar]. Similarly, inhibition of the NHEJ pathway is a common strategy to increase HDR efficiency [25Fu YW Dai XY Wang WT et al.Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.Nucleic Acids Res. 2021; 49: 969-985Crossref PubMed Scopus (20) Google Scholar,50Riesenberg S Maricic T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells.Nat Commun. 2018; 9: 2164Crossref PubMed Scopus (74) Google Scholar,51Maruyama T Dougan SK Truttman MC Bilate AM Ingram JR Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.Nat Biotechnol. 2015; 33: 538-542Crossref PubMed Scopus (721) Google Scholar]. Alternatively, selecting a sgRNA with lower-level NHEJ and higher-level MMEJ editing outcomes will increase relative HDR efficiency [45Tatiossian KJ Clark RDE Huang C Thornton ME Grubbs BH Cannon PM. Rational selection of CRISPR-Cas9 guide RNAs for homology-directed genome editing.Mol Ther. 2021; 29: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar]. Recent advances in CRISPR–Cas9 technology have permitted efficient DNA modifications. Studies on the gene editing of human T cells [52Stadtmauer EA Fraietta JA Davis MM et al.CRISPR-engineered T cells in patients with refractory cancer.Science. 2020; 367: eaba7365Crossref Scopus (478) Google Scholar], hematopoietic stem cells [53Wu Y Zeng J Roscoe BP et al.Highly efficient therapeutic gene editing of human hematopoietic stem cells.Nat Med. 2011; 25: 776-783Crossref Scopus (179) Google Scholar], induced pluripotent stem cells [37Li XL Li GH Fu J et al.Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.Nucl Acids Res. 2018; 46: 10195-10215Crossref PubMed Scopus (52) Google Scholar], and even human embryos [54Ma H Marti-Gutierrez N Park SW et al.Correction of a pathogenic gene mutation in human embryos.Nature. 2017; 548: 413-419Crossref PubMed Scopus (563) Google Scholar] have paved the way for clinical gene therapies. In addition to CRISPR-induced small indels and template-dependent repairs at on-target sites, several unintended outcomes, such as large deletions and complex genomic rearrangements, have been reported after Cas9–sgRNA cleavages [55Hendel A Kildebeck EJ Fine EJ 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 (84) Google Scholar, 56Kosicki M Tomberg K Bradley A. 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, 57Adikusuma F Piltz S Corbett MA et al.Large deletions induced by Cas9 cleavage.Nature. 2018; 560: E8-E9Crossref PubMed Scopus (122) Google Scholar, 58Song Y Liu Z Zhang Y et al.Large-fragment deletions induced by Cas9 cleavage while not in the BEs system.Mol Ther Nucl Acids. 2020; 21: 523-526Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. On-target effects, such as indels, are usually determined by sequencing a short polymerase chain reaction (PCR) product by either Sanger sequencing [59Brinkman EK van Steensel B. Rapid quantitative evaluation of CRISPR genome editing by TIDE and TIDER.in: Luo Y CRISPR Gene Editing. Humana Press, New York1961: 29-44Google Scholar,60Bloh K Kanchana R Bialk P et al.Deconvolution of complex DNA repair (DECODR): establishing a novel deconvolution algorithm for comprehensive analysis of CRISPR-edited sanger sequencing data.CRISPR J. 2021; 4: 120-131Crossref Scopus (11) Google Scholar] or Illumina sequencing [61Park J Lim K Kim JS Bae S. Cas-analyzer: an online tool for assessing genome editing results using NGS data.Bioinformatics. 2017; 33: 286-288Crossref PubMed Scopus (185) Google Scholar,62Clement K Rees H Canver MC et al.CRISPResso2 provides accurate and rapid genome editing sequence analysis.Nat Biotechnol. 2019; 37: 224-226Crossref PubMed Scopus (307) Google Scholar]. However, the short length has eluded the detection of large deletions because large fragment deletions have depleted one or two primer-binding sites. The advances of third-generation sequencing technologies, including Oxford Nanopore and PacBio, have made it possible to reveal complex on-target mutations. These single molecular high-throughput technologies have the advantage of sequencing long nucleotides >20 kb. Coupling long-range PCR with third-generation sequencing enables quantitation of the editing alleles with long deletions [56Kosicki M Tomberg K Bradley A. 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]. Deletion of many kilobases occurs after CRISPR–Cas9 gene editing in mouse and human cells. This extensive on-target genomic damage is a common outcome independent of loci and cell lines [49Wen W Quan ZJ Li SA et al.Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.Genome Biol. 2021; 22: 236Crossref PubMed Scopus (6) Google Scholar,56Kosicki M Tomberg K Bradley A. 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, 57Adikusuma F Piltz S Corbett MA et al.Large deletions induced by Cas9 cleavage.Nature. 2018; 560: E8-E9Crossref PubMed Scopus (122) Google Scholar, 58Song Y Liu Z Zhang Y et al.Large-fragment deletions induced by Cas9 cleavage while not in the BEs system.Mol Ther Nucl Acids. 2020; 21: 523-526Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. The recent development of the GREPore-seq workflow, which combines long-range PCR with Nanopore sequencing, enables scalable and quantitative assessment of large deletions cost-effectively [Quan et al., 2021, unpublished, doi: https://doi.org/10.1101/2021.12.13.472514]. However, because of the intrinsic limitations of long-range PCR, megadeletions >10 kb have not been fully illustrated, and their occurrence may depend on the unique features of the targets. That said, deletions exceeding 1 kb are observed at considerably lower frequencies than shorter deletions (100–500 bp) [49Wen W Quan ZJ Li SA et al.Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.Genome Biol. 2021; 22: 236Crossref PubMed Scopus (6) Google Scholar]. Chromosome rearrangement can occur when a cell simultaneously generates two or more DSBs. For example, under replication stress, fork collapse induces the formation of a DSB at the stalled fork; thus, rearrangement or translocation may sporadically arise in the presence of multiple stalled forks [63Howarth KD Pole JCM Beavis JC et al.Large duplications at reciprocal translocation breakpoints that might be the counterpart of large deletions and could arise from stalled replication bubbles.Genome Res. 2011; 21: 525-534Crossref PubMed Scopus (27) Google Scholar]. Double-strand cut by CRISPR–Cas9 can presumably increase chromosome rearrangement frequencies, particularly when cutting occurs at both an on-target and one or more off-targets. Simultaneously editing two or more sites will exacerbate this problem, for instance, in cases where multiple genes need to be depleted to generate universal "off-the-shelf" CAR-T cells, such as genes related to immune compatibility (B2M [64Gornalusse GG Hirata RK Funk SE et al.HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells.Nat Biotechnol. 2017; 35: 765-772Crossref PubMed Scopus (254) Google Scholar], CIITA [65DeSandro A Nagarajan UM Boss JM. The bare lymphocyte syndrome: molecular clues to the transcriptional regulation of major histocompatibility complex class II genes.Am J Hum Genet. 1999; 65: 279-286Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar], TRAC, or TRBC [66Torikai H Reik A Liu PQ et al.A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR.Blood. 2012; 119: 5697-5705Crossref PubMed Scopus (336) Google Scholar]) and immune checkpoints such as PDCD1 [67Ren J Liu X Fang C Jiang S June CH Zhao Y. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition.Clin Cancer Res. 2017; 23