Cited by 1,098CRISPR and Genetic EngineeringRNA regulation and diseaseDNA Repair Mechanisms

Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA

Richardson, Christopher D, Ray, Graham J, DeWitt, Mark A, Curie, Gemma L, Corn, Jacob E·2016·Nature Biotechnology

Abstract

Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower. Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime ∼6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3' end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it.

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Crossref · 10.1038/nbt.3481

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33 of 33 references verified0 citation errors · 0 not checkable

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Authors
Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L., & Corn, J. E.
Affiliations
Innovative Genomics Institute (US)
Corresponding author
Corn, Jacob E
Venue
Nature Biotechnology
Published
2016-01-20
Volume
34
Issue
3
Pages
339-344
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
OpenAlex
W2260480416
Cited by
1,098
References
33
Citations by year
2026: 47 · 2025: 69 · 2024: 80 · 2023: 92 · 2022: 130 · 2021: 126 · 2020: 124 · 2019: 129 · 2018: 123 · 2017: 115 · 2016: 61 · 2013: 1
FWCI
74.12
Type
journal article
Language
en
Open access
closed
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
homology-directed repair, CRISPR/Cas9, genome editing, human cells
MeSH terms
Cell Line, DNA, Single-Stranded, Genetic Engineering, Humans, Genome, RNA Editing, DNA Breaks, Double-Stranded, DNA End-Joining Repair, Recombinational DNA Repair, CRISPR-Cas Systems

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