PEM-seq comprehensively quantifies DNA repair outcomes during gene-editing and DSB repair

Liu, Yang; Yin, Jianhang; Gan, Tingting; Liu, Mengzhu; Xin, Changchang; Zhang, Weiwei; Hu, Jiazhi

doi:10.1016/j.xpro.2021.101088

Cited by 21 publications

(24 citation statements)

References 17 publications

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“…We next employed a previously developed primer-extension-mediated sequencing (PEM-seq) method to profile the editing outcomes of Cas12f nucleases, assessing both intended small insertions/deletions (indels) and unwanted chromosomal rearrangements such as large deletions, off-target translocations, and general translocations (Fig. 2a ) 30 , 38 . We designed 12 target sites within or adjacent to the human VEGFA , HBB , IFNγ , COL8A1 , NLRC4 , NUDT16 , CLIC4 , LNX1 , FGF18 , P2RX5-TAX1BP3 , and KLHL29 genes and used the nine abovementioned CRISPR-Cas nucleases to target these sites in HEK293T cells.…”

Section: Resultsmentioning

confidence: 99%

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Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption

Xin

Yin

et al. 2022

Nat Commun

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Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.

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Section: Resultsmentioning

confidence: 99%

“…Each PEM-seq DNA library was constructed according to the standard procedure 30 , 38 , for which 20 μg of genomic DNA from different Cas nuclease-edited samples is generally required. The primer control of each target site was generally applied to the genome of wild-type HEK293T cells transfected by a Cas nuclease without sgRNA targeting.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption

Xin

Yin

et al. 2022

Nat Commun

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“…PEM-seq analysis. The PEM-seq libraries were generated as described previously 9,55 . In general, biotinylated primer was set within 150 bp from Cas9 target site to achieve primer extension.…”

Section: Methodsmentioning

confidence: 99%

Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing

Yin

Xin

et al. 2022

Nat Commun

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The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

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“…To monitor CRISPR‐guided AID‐generated S region breaks, a bait site cleaved by SaCas9 at Iγ3 was used (Appendix Table S2). PEM‐Seq libraries were performed as previously described (Yin et al , 2019; Liu et al , 2022) to quantitatively recover the Cas9‐generated translocations. In brief, primer extension was performed with biotinylated primers as listed in Appendix Table S2.…”

Section: Methodsmentioning

confidence: 99%

“…Enriched biotinylated PCR products were ligated to the bridge adapters, and two subsequent PCR reactions were performed to amplify products for sequencing. Sequences were mapped to mm10 reference via the SuperQ pipeline (Yin et al , 2019; Liu et al , 2022). In the PEM‐Seq assay, a random barcode was introduced to remove PCR repeats and quantify translocation junctions.…”

Section: Methodsmentioning

confidence: 99%

C‐terminal deletion‐induced condensation sequesters AID from IgH targets in immunodeficiency

et al. 2022

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In activated B cells, activation‐induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C‐terminal peptide. In immunodeficient‐patient cells expressing mutant AID lacking its C‐terminus, a catalytically active AID‐delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID‐delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID‐delC proteins form condensates both in vivo and in vitro, dependent on its N‐terminus and on a surface arginine‐rich patch. Co‐expression of AID‐delC and wild‐type AID leads to an unbalanced nuclear AID‐delC/AID ratio, with AID‐delC proteins able to trap wild‐type AID in condensates, resulting in a dominant‐negative phenotype that could contribute to immunodeficiency. The co‐condensation model of mutant and wild‐type proteins could be an alternative explanation for the dominant‐negative effect in genetic disorders.

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PEM-seq comprehensively quantifies DNA repair outcomes during gene-editing and DSB repair

Cited by 21 publications

References 17 publications

Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption

Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption

Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing

C‐terminal deletion‐induced condensation sequesters AID from IgH targets in immunodeficiency

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