Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

Bothmer, Anne; Phadke, Tanushree; Barrera, Luis; Margulies, Carrie M; Lee, Christina S.; Buquicchio, Frank A.; Moss, Sean; Abdulkerim, Hayat; Selleck, William; Jayaram, Hariharan; Myer, Vic E.; Cotta‐Ramusino, Cecilia

doi:10.1038/ncomms13905

Cited by 170 publications

(203 citation statements)

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“…A number of ‘alternative’ HDR pathways do not involve RAD51-mediated strand invasion [reviewed by (86)], among them HDR supported by SSO donors. At nicks, HDR by SSO donors requires RPA, but is stimulated dramatically (10-fold or more) upon depletion of RAD51 itself or factors that promote loading of RAD51 on DNA, including BRCA2 and its binding partners PALB2 and SHFM1; or by expression of the dominant negative RAD51K133R mutant or the inhibitory BRC3 peptide (9,10,87). …”

Section: Discussionmentioning

confidence: 99%

“…The human hemogloblin beta (HBB) and delta (HBD) genes are homologous direct repeats of nearly identical sequence spaced 6 kb apart, reminiscent of the organization of target and donor sequences in the DR-GFP reporter. HDR at paired nicks targeted to the HBB gene was analyzed in human U2OS osteosarcoma cells (87) which provide a convenient model for studying targeted HDR although they do not express globin genes. Paired Cas9D10A or Cas9N863A nickases were targeted to sites spaced to generate ends 47 nt in length and bearing 5′ or 3′ overhangs respectively (Figure 6).…”

Section: Discussionmentioning

confidence: 99%

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Initiation of homologous recombination at DNA nicks

Maizels

Davis

2018

Nucleic Acids Research

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Discontinuities in only a single strand of the DNA duplex occur frequently, as a result of DNA damage or as intermediates in essential nuclear processes and DNA repair. Nicks are the simplest of these lesions: they carry clean ends bearing 3′-hydroxyl groups that can undergo ligation or prime new DNA synthesis. In contrast, single-strand breaks also interrupt only one DNA strand, but they carry damaged ends that require clean-up before subsequent steps in repair. Despite their apparent simplicity, nicks can have significant consequences for genome stability. The availability of enzymes that can introduce a nick almost anywhere in a large genome now makes it possible to systematically analyze repair of nicks. Recent experiments demonstrate that nicks can initiate recombination via pathways distinct from those active at double-strand breaks (DSBs). Recombination at targeted DNA nicks can be very efficient, and because nicks are intrinsically less mutagenic than DSBs, nick-initiated gene correction is useful for genome engineering and gene therapy. This review revisits some physiological examples of recombination at nicks, and outlines experiments that have demonstrated that nicks initiate homology-directed repair by distinctive pathways, emphasizing research that has contributed to our current mechanistic understanding of recombination at nicks in mammalian cells.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Initiation of homologous recombination at DNA nicks

Maizels

Davis

2018

Nucleic Acids Research

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“…It has been suggested that different types of cuts engage different repair pathways, because 5' overhanging ends yield higher levels of HDR than 3' overhangs 5 . The fact that Trichostatin A and MLN4924 increase TNS with Cas9n and Cpf1 but has no effect with Cas9 suggests that blunt ends introduced by Cas9 may be repaired by different mechanisms versus 5' overhanging DNA ends introduced by Cpf1 or Cas9n.…”

mentioning

confidence: 99%

“…If the donor DNA provided in the experiment carries mutations, these will be introduced into the genome (precise genome editing). Repair with homologous ssDNA or dsDNA has been suggested to engage different pathways 5 . We will refer to Targeted Nucleotide Substitutions using ssDNA donors as 'TNS' and targeted insertion of cassettes .…”

mentioning

confidence: 99%

Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Riesenberg

Maričić

2017

Preprint

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A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks (DSBs) which are then repaired by homology-directed repair (HDR) using synthetic or cloned DNA donor molecules carrying desired mutations. However, another pathway, the non-homologous end joining (NHEJ) pathway competes with HDR for repairing DNA breaks in cells. To increase the frequency of precise genome editing in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) we have tested the capacity of a number of small molecules to enhance HDR or inhibit NHEJ. We identify molecules that increase the frequency of precise genome editing including some that have additive effects when applied together. Using a mixture of such molecules, the 'CRISPY' mix, we achieve 2.8-to 6.7-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes, to our knowledge the highest editing efficiency in hiPSCs described to date. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.9-to 4.0-fold, allowing almost 20% of chromosomes to be edited. Main textThe bacterial nuclease CRISPR/Cas9 is now frequently used to accurately cut chromosomal DNA sequences in eukaryotic cells. The resulting DNA breaks are repaired by two competing pathways: NHEJ and HDR (Fig. 1). In NHEJ, the first proteins to bind the cut DNA ends are Ku70/Ku80, followed by DNA protein kinase catalytic subunit (DNA-PKcs) Since NHEJ of Cas9-induced DSBs is error prone and frequently introduces short insertions and deletions (indels) at the cut site, it is useful for knocking out a targeted gene. In contrast, HDR allows precise repair of a DSB by using a homologous donor DNA. If the donor DNA provided in the experiment carries mutations, these will be introduced into the genome (precise genome editing). Repair with homologous ssDNA or dsDNA has been suggested to engage different pathways 5 . We will refer to Targeted Nucleotide Substitutions using ssDNA donors as 'TNS' and targeted insertion of cassettes . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/136374 doi: bioRxiv preprint first posted online May. 10, 2017; 3 using dsDNA donors as knock-ins, respectively. In order to introduce a DSB Cas9 requires the nucleotide sequence NGG (a "PAM" site) in the target DNA. Targeting of Cas9 is further determined by a guide RNA (gRNA) complementary to 20 nucleotides adjacent to the PAM site. However, the Cas9 may also cut the genome at sites that carry sequence similarity to the gRNA 6 . One strategy to reduce such off-target cuts is to use a mutated Cas9 that introduces single-stranded nicks instead of DSBs (Cas9n) 7 . Using two gRNAs to introduce two nicks on opposite DNA strands in close proximity to each other will result in a staggered D...

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“…On the other hand, other repair pathways such as NHEJ, MMEJ, and single-strand annealing (SSA) are considerably active during the majority of the cell cycle. In addition, single-strand DNA (ssDNA) can also be incorporated at the DSB site via single-strand template repair (SSTR), which is thought to be independent from HR, because SSTR is not Rad51-dependent (Bothmer et al 2017;Richardson et al 2017). In this review, we summarize these HR-independent gene knock-in systems developed recently.…”

Section: Introductionmentioning

confidence: 99%