Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing

Feng, Yixiang; Liu, Sicheng; Chen, Ruo-Dan; Xie, Anyong

doi:10.1631/jzus.b2000282

Cited by 24 publications

(18 citation statements)

References 92 publications

(211 reference statements)

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“…Instead, as one of the most frequent endogenous DNA lesions in mammalian cells, DNA nicks could be converted into one-ended DSBs by DNA replication and become a major inducer of mutational signatures associated with BRCA1 deficiency. By exploiting the long duration of Cas9-sgRNA post-cleavage target residence 45 , 50 , 51 , together with fast doubling time and short G1 phase of mESC, we increased the probability of a collision between nCas9-induced nicks and DNA replication forks, readily generating one-ended DSBs. Due to BRCA1 mutation, one-ended DSBs that are supposed to be efficiently repaired by BRCA1-mediated SCR could be left unrepaired, increasing the opportunity for alternative repair pathways (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

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DNA nicks induce mutational signatures associated with BRCA1 deficiency

et al. 2022

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Analysis of human cancer genome sequences has revealed specific mutational signatures associated with BRCA1-deficient tumors, but the underlying mechanisms remain poorly understood. Here, we show that one-ended DNA double strand breaks (DSBs) converted from CRISPR/Cas9-induced nicks by DNA replication, not two-ended DSBs, cause more characteristic chromosomal aberrations and micronuclei in Brca1-deficient cells than in wild-type cells. BRCA1 is required for efficient homologous recombination of these nick-converted DSBs and suppresses bias towards long tract gene conversion and tandem duplication (TD) mediated by two-round strand invasion in a replication strand asymmetry. However, aberrant repair of these nick-converted one-ended DSBs, not that of two-ended DSBs in Brca1-deficient cells, generates mutational signatures such as small indels with microhomology (MH) at the junctions, translocations and small MH-mediated TDs, resembling those in BRCA1-deficient tumors. These results suggest a major contribution of DNA nicks to mutational signatures associated with BRCA1 deficiency in cancer and the underlying mechanisms.

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Section: Discussionmentioning

confidence: 99%

“…Unlike classic DNA nicks that are readily exposed, nCas9-induced nicks are buried within the nCas9-sgRNA-DNA ternary complex 45 , 50 , 51 . It is unclear whether cells could sense this type of nicks before nCas9-sgRNA is dissociated spontaneously or by forces from nicked DNA targets.…”

Section: Discussionmentioning

confidence: 99%

DNA nicks induce mutational signatures associated with BRCA1 deficiency

et al. 2022

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“…The DSB repair pathway choice is governed by a host of factors, including cell cycle stage, DNA end configurations, surrounding chromatin context, and local DNA metabolism [ 10 ]. Uniqueness in DSB induction by CRISPR/Cas9 may also participate in this regulation [ 11 , 12 ]. In CRISPR/Cas9 genome editing, targeting Cas9 to a given site is mediated by several interactions, including the contacts between Cas9 and the protospacer adjacent motif (PAM) of the target, the base pairing of the sgRNA spacer with target strand and non-specific interactions between Cas9 and target DNA [ 2 ].…”

Section: Introductionmentioning

confidence: 99%

“…Owing to intrinsic disparity in the interactions that mediate the binding of Cas9-sgRNA to its target, the binding affinity of Cas9-sgRNA varies at different sites along with altered target residence. It is likely that Cas9-sgRNA could be spontaneously released from its target or may encounter local DNA replication, transcription, or chromatin remodeling, leading to release of Cas9-sgRNA from cleaved DNA and exposure of Cas9-induced DSBs [11,12,[19][20][21][22]. These DSBs are subsequently recognized and engaged with repair factors that determine a pathway choice.…”

Section: Introductionmentioning

confidence: 99%

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing

et al. 2022

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Background Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. Results In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. Conclusions Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

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“…Instead, as one of the most frequent endogenous DNA lesions in mammalian cells, DNA nicks could be converted into one-ended DSBs by DNA replication and become a major inducer of mutational signatures associated with BRCA1 de ciency. By exploiting long duration of Cas9-sgRNA post-cleavage target residence [43][44][45] , together with fast doubling time and short G1 phase of mESC, we increased the probability of a collision between nCas9-induced nicks and DNA replication forks, readily generating one-ended DSBs. Due to BRCA1 mutation, one-ended DSBs that are supposed to be e ciently repaired by BRCA1-mediated SCR could be left unrepaired, increasing the opportunity for alternative repair pathways 9,13 .…”

Section: Discussionmentioning

confidence: 99%

DNA nicks induce mutational signatures associated with BRCA1 deficiency

Feng

Liu

Chen

et al. 2022

Preprint

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Analysis of human cancer genome sequences has revealed specific mutational signatures associated with BRCA1-deficient tumors, but the underlying mechanisms remain poorly understood. Here, we show that one-ended DNA double strand breaks (DSBs) converted from CRISPR/Cas9-induced nicks by DNA replication, not two-ended DSBs, cause more characteristic chromosomal aberrations and micronuclei in Brca1-deficient cells than in wild-type cells. BRCA1 is required for efficient homologous recombination of these nick-converted DSBs and suppresses bias towards long tract gene conversion and tandem duplication (TD) mediated by two-round strand invasion in a replication strand asymmetry. However, aberrant repair of these nick-converted one-ended DSBs, not that of two-ended DSBs in Brca1-deficient cells, generates mutational signatures such as small indels with microhomology (MH) at the junctions, unbalanced translocations and small MH-mediated TDs, resembling those in BRCA1-deficient tumors. These results suggest a major contribution of DNA nicks to mutational signatures associated with BRCA1 deficiency in cancer and the underlying mechanisms.

show abstract

Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing

Cited by 24 publications

References 92 publications

DNA nicks induce mutational signatures associated with BRCA1 deficiency

DNA nicks induce mutational signatures associated with BRCA1 deficiency

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing

DNA nicks induce mutational signatures associated with BRCA1 deficiency

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