Precise and broad scope genome editing based on high-specificity Cas9 nickases
Nucleic Acids Research, vol. 49, pp. 1173–1198
Abstract
RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.
Authors 6
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Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
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Stanford Medicine · Stanford University
Affiliation as printed
Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University School of Medicine, 269 Campus Dr. Stanford, CA 94305, USA
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Stanford Medicine · Stanford University
Affiliation as printed
Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University School of Medicine, 269 Campus Dr. Stanford, CA 94305, USA
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Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
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