Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

Gonçalves, Manuel A.F.V.; Nierop, Gijsbert P. van; Hilbrant, Maarten; Vries, Antoine A.F. de

doi:10.1093/nar/gkr1234

Cited by 17 publications

(19 citation statements)

References 45 publications

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“…We too used an EGFP-based reporter system with a short homologous region (795 bp; rNCO assay) and observed low efficiencies of targeted knockin by tandem paired nicking compared with those obtained by the Cas9-nuclease-based method. Several previous studies have described the enhancement of targeted knockin by creating two nicks, one on the donor and the other within the genome, at different positions (Chen et al, 2017;Davis and Maizels, 2014;Gonç alves et al, 2012;Nakajima et al, 2018). This strategy, however, requires careful donor design, including the incorporation of a foreign sequence or silent mutation into the donor, which are eventually integrated into the genome upon targeted knockin.…”

Section: Discussionmentioning

confidence: 99%

“…Although single nicks have been found to trigger HDR with efficiency generally lower than that triggered by DSBs (Bolukbasi et al, 2016;Cornu et al, 2017;Komor et al, 2017), further studies have shown that the efficiency of HDR can be improved by the use of two nickases so that they create no DSB in the genome. The majority of these studies created one nick on the donor DNA and the other within the genome, at different positions, by using two nickases (Chen et al, 2017;Davis and Maizels, 2014;Gonç alves et al, 2012;Nakajima et al, 2018). In this study, we explored a distinct and less-well-described strategy of using Cas9 nickases for targeted knockin, which is called ''tandem paired nicking'' (Chen et al, 2017) or ''tandem nicking'' (Nakajima et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53

et al. 2020

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Graphical Abstract Highlights d Tandem paired nicking promotes targeted knockin with substantial efficiency d Tandem paired nicking scarcely creates indels at the edited genomic loci d DNA recombination elicited by tandem paired nicking is analogous to Holliday's model d Targeted knockin by tandem paired nicking shows no reciprocal interference with p53 SUMMARYTargeted knockin mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knockin. Here, we demonstrate that tandem paired nicking, a method for targeted knockin involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knockin largely equivalent to that of the Cas9-nuclease-based approach. Tandem paired nicking seems to accomplish targeted knockin by DNA recombination analogous to Holliday's model and creates intended genomic changes without introducing additional nucleotide changes, such as silent mutations. Targeted knockin through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53

et al. 2020

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“…Concerted nicking of both donor and target DNA has been reported to stimulate HDR (30). This raises the possibility that the prevalence of nicks in the genome may stimulate HDR not only between sister chromatids, which would not alter genomic sequence, but also between homologous chromosomes, which could result in loss of heterozygosity, a common form of genomic instability in tumor cells.…”

Section: Nicked Dsdna Donors Promote Efficient Alternative Hdr At a Nmentioning

confidence: 99%

Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair

Davis¹,

Maizels

2014

Proc. Natl. Acad. Sci. U.S.A.

183

184

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DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9 D10A nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.NA nicks (single-strand breaks) are the most common form of DNA damage. Every day tens of thousands of DNA nicks occur and are repaired in each cell (1). Nicks can be caused by oxidative stress or ionizing radiation, which generates 30 nicks for every double-strand break (DSB). Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals, can damage a deoxyribose moiety to nick DNA directly, or modify DNA precursors (e.g., by converting guanine to 8-oxoguanine) and thereby overload downstream repair to create a burden of nicked DNA (1-4). Nicks are also intermediates in essential DNA metabolism and repair pathways, including base excision repair, nucleotide excision repair, mismatch repair, ribonucleoside monophosphate removal, and regulation of superhelicity by topoisomerases.Nicks are efficiently repaired by the single-strand break repair (SSBR) pathway, which assembles a repair complex at a nick in which X-ray repair cross-complementing protein 1 (XRCC1) is a critical but noncatalytic member (5-8). XRCC1 interacts with factors that clean up modified DNA ends to create a gap that is filled by polymerase POL β, or the replicative polymerases POL δ and e. LIG3 or other ligases then reseal the DNA backbone (5-7).Nicks can also initiate homology-directed repair (HDR) (9-12). This has drawn considerable interest as a strategy for gene therapy by targeted gene correction, because nicks cause less mutagenic end-joining (mutEJ) than do DSBs (13,14). However, the mechanism of HDR at nicks has not been defined, either in mammalian cells or in model organisms such as Saccharomyces cerevisiae. In particular, it is not known whether HDR at nicks proceeds via the canonical HDR pathway that has been characterized in detail at DNA DSBs, in which free single-stranded 3′ ends ...

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“…We too employed an EGFP-based reporter system with a short homologous region (795 bp; rNCO assay) and observed low efficiencies of targeted knock-in via tandem paired nicking compared with those obtained by the Cas9 nuclease-based method. Several previous studies have described enhancement of targeted knock-in by creating two nicks, one on the donor and the other within the genome, at different positions (Chen et al, 2017;Davis and -12 -Maizels, 2014;Goncalves et al, 2012;Nakajima et al, 2018). This strategy, however, requires careful donor design, including the incorporation of a foreign sequence or silent mutation into the donor, which are eventually integrated into the genome upon targeted knock-in.…”

Section: Discussionmentioning

confidence: 99%

“…Although single nicks have been found to trigger HDR with efficiency generally lower than that triggered by DSBs (Bolukbasi et al, 2016;Cornu et al, 2017;Komor et al, 2017), further studies have -4 -shown that the efficiency of HDR can be improved by the use of two nickases so that they create no DSB in the genome. The majority of these studies created one nick on the donor DNA and the other within the genome, at different positions, using two nickases (Chen et al, 2017;Davis and Maizels, 2014;Goncalves et al, 2012;Nakajima et al, 2018). In this study, we explored a distinct and less well described strategy of using Cas9 nickases for targeted knock-in, which is called 'tandem paired nicking' (Chen et al, 2017), or 'tandem nicking' (Nakajima et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Tandem paired nicking promotes precise genome editing with scarce interference by p53

Hyodo

Rahman

Karnan

et al. 2019

Preprint

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Targeted knock-in mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites.A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knock-in. Here, we demonstrate that tandem paired nicking, a method for targeted knock-in involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knock-in largely equivalent to that of the Cas9 nuclease-based approach. Tandem paired nicking seems to accomplish targeted knock-in via DNA recombination analogous to Holliday's model, and creates intended genetic changes in the genome without introducing additional nucleotide changes such as silent mutations. Targeted knock-in through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

show abstract

Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

Cited by 17 publications

References 45 publications

Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53

Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53

Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair

Tandem paired nicking promotes precise genome editing with scarce interference by p53

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