CRISPR/Cas9 increases mitotic gene conversion in human cells

Javidi-Parsijani, Parisa; Lyu, Pin; Makani, Vishruti; Sarhan, Walaa; Yoo, Kyung Whan; El‐Korashi, Lobna A.; Atala, Anthony; Lü, Biao

doi:10.1038/s41434-020-0126-z

Cited by 22 publications

(24 citation statements)

References 48 publications

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“…4 Gene conversion via CRISPR-Cas9 between a 101 bp homologous region in HBD and HBB has also been reported; notably, these genes were also the first reported gene conversion event in humans. 3,39 To our knowledge, this is the first report of pseudogene-mediated gene conversion during the CRISPR-Cas9 editing process. Our results demonstrate the need for rigorous screening in studies which rely on gene editing, and analysis of the region flanking the expected cut site for homology and possible gene conversion.…”

Section: Discussionmentioning

confidence: 94%

Pseudogene-mediated gene conversion after CRISPR-Cas9 editing demonstrated by partialCD33conversion withSIGLEC22P

Shaw

Estus

2021

Preprint

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Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocytes. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.

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Section: Discussionmentioning

confidence: 94%

Pseudogene-mediated gene conversion after CRISPR-Cas9 editing demonstrated by partialCD33conversion withSIGLEC22P

Shaw

Estus

2021

Preprint

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“…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][47][48]. 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].…”

Section: Unknown Unintended Consequences Of Genome Editingmentioning

confidence: 99%

“…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][47][48]. 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]. In particular, because conversion tracks may expand well beyond the targeted region, the resulting loss of heterozygosity represents an additional risk for clinical application [31,49].…”

Section: Unknown Unintended Consequences Of Genome Editingmentioning

confidence: 99%

Anticipating and Identifying Collateral Damage in Genome Editing

Burgio

Teboul

2020

Trends in Genetics

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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: A Transformative TechnologyThe 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 [1-3]. The promise of applicability is turning into reality, as illustrated by the first nonrandomized Phase I clinical trial i in which the use of clustered regularly interspaced short palindromic repeats (CRISPR)-engineered T cells was recently found to be safe [4]. To date, N20 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 [1,5]).Genome editing is generally based on either zinc finger nucleases [6], transcription activator-like effector nucleases [7], or the CRISPR/CRISPR-associated (Cas) system (see Glossary) [8]. 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][10][11] or nonhomologous end-joining [12]. Base editors [13] and prime editors [14] 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. Genome Editing: A Disruptive but Still Erratic TechnologyThe 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 ri...

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“…We propose that DAJIN is a novel validation tool for long-range sequencing at the single base level to capture genome editing events. Recently, DNA cleavage in cultured cells and zygotes has been shown to induce gene conversions mediated by homologous chromosomes or homologous sequences on the same chromosome [11,12]. DAJIN might contribute to a better understanding of the consequences of editing events at the targeted locus.…”

Section: Table S3mentioning

confidence: 99%

“…Short-range assessments with targeted PCR amplification and Sanger sequencing likely to miss long-range mutation events, which may result in pathogenic phenotypes through unintended changes in gene expression [10]. Furthermore, there is a possibility of gene conversion between homologous regions following genomic DNA cleavage [11][12][13]. Although targeted long-read sequencing allows the detection of complex ontarget mutations over several kilobases [9,14], this method has instrumental limitations such as error rates that need to be overcome to validate the target locus to a singlebase level [15].…”

Section: Introductionmentioning

confidence: 99%

Multiplex genotyping method to validate the multiallelic genome editing outcomes using machine learning-assisted long-read sequencing

Kuno

Ikeda

Ayabe

et al. 2020

Preprint

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Genome editing induces various on-target mutations. Accurate identification of mutations in founder mice and cell clones is essential to perform reliable genome editing experiments. However, no genotyping method allows the comprehensive analysis of diverse mutations. We developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify diverse mutations, including point mutations, deletions, inversions, and knock-in. Our genotyping method with DAJIN can handle approximately 100 samples within a day and may become a new standard for validating genome editing outcomes.

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CRISPR/Cas9 increases mitotic gene conversion in human cells

Cited by 22 publications

References 48 publications

Pseudogene-mediated gene conversion after CRISPR-Cas9 editing demonstrated by partialCD33conversion withSIGLEC22P

Pseudogene-mediated gene conversion after CRISPR-Cas9 editing demonstrated by partialCD33conversion withSIGLEC22P

Anticipating and Identifying Collateral Damage in Genome Editing

Multiplex genotyping method to validate the multiallelic genome editing outcomes using machine learning-assisted long-read sequencing

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