Mechanisms of precise genome editing using oligonucleotide donors

Kan, Yinan; Ruis, Brian L.; Takasugi, Taylor; Hendrickson, Eric A.

doi:10.1101/gr.214775.116

Cited by 82 publications

(118 citation statements)

References 69 publications

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“…Overall, while there are nuclease- and cell-type-specific differences HDR efficiencies, the relative orientation of the donor does not have a consistent impact on HDR-based editing. This is consistent with previously described ssDNA donor strand biases in HDR efficiencies, which are generally locus- and cell type-specific 19 .…”

Section: Resultssupporting

confidence: 92%

“…For short insertions (<200 nt), ssDNA oligonucleotides harboring the mutation, as well as flanking homology arms that range from 35-60 nucleotides, are introduced into cells along with Cas9 protein and guide RNA. 15, 18, 19 When modifications longer than 200bp are desired, double-stranded DNA (dsDNA) templates such as plasmids or PCR products are typically used as donor templates. However, these double-stranded templates are often associated with high cellular toxicity and off-target integration events.…”

Section: Introductionmentioning

confidence: 99%

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Efficient Homology-directed Repair with Circular ssDNA Donors

Iyer

Mir

Vega-Badillo

et al. 2019

Preprint

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32While genome editing has been revolutionized by the advent of CRISPR-based 33 nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair 34(HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer 35 from low HDR efficiencies in many cell types, as well as integration at unintended sites. 36In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with 37 minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently 38 and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors 39 serve as efficient HDR templates when used with Cas9 or Cas12a, with integration 40 frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative 41 efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a 42 nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-43

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Section: Resultssupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Efficient Homology-directed Repair with Circular ssDNA Donors

Iyer

Mir

Vega-Badillo

et al. 2019

Preprint

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“…If flaps formed during HDR are indeed cleaved, this suggests that repair of some SSB with blocked or damaged ends may occur by flap cleavage that would remove the damaged 5′ or 3′ end, permitting HDR without canonical SSB repair. It has been reported that donor SSOs used by the cI pathway can become physically incorporated into the chromosome (83), but it has not been established whether this accounts for a significant fraction of recombinants.…”

Section: Discussionmentioning

confidence: 99%

“…MMR is not required for HDR at nicks, which is active in MMR-deficient cell lines, including the embryonic kidney derived cell line HEK293T (9,10) and the HCT116 colon cancer line (83). The absence of Ku70 does not affect the frequency of HDR at nicks, suggesting that there is no obligatory DSB intermediate (84), presuming that Ku loads on replicative DSBs.…”

Section: Discussionmentioning

confidence: 99%

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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“…Davis and Maizels 2016) and in the context of repair from Cas9 DSBs (Kan et al 2017;Paix et al 2017)) and meganucleases (Kan et al 2014).…”

Section: Precise Repair From Homologous Single-stranded Oligonucleotimentioning

confidence: 99%

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Farboud

Severson

Meyer

2018

Preprint

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The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and non-model organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes, both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-stranded breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans. We devised strategies to insert long (10 kb) exogenous sequences or incorporate multiple nucleotide substitutions at considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex-dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. Our studies revealed a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9 targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.

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Mechanisms of precise genome editing using oligonucleotide donors

Cited by 82 publications

References 69 publications

Efficient Homology-directed Repair with Circular ssDNA Donors

Efficient Homology-directed Repair with Circular ssDNA Donors

Initiation of homologous recombination at DNA nicks

Strategies for Efficient Genome Editing Using CRISPR-Cas9

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