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An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms.

ABSTRACT: Here, we report a rapid CRISPR-Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5'-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5?kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5'-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.

SUBMITTER: Yu Y

PROVIDER: S-EPMC7085973 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms.

Yu Yi Y Guo Yijun Y Tian Qiqi Q Lan Yuanqing Y Yeh Hugh H Zhang Meng M Tasan Ipek I Jain Surbhi S Zhao Huimin H

Nature chemical biology 20191223 4

Here, we report a rapid CRISPR-Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5'-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5'-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells. ...[more]

PMID: 31873222

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