Here, we report a rapid CRISPR\u2013Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5\u2032-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65\/40% knock-in rates for 0.7\/2.5\u2009kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5\u2032-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells. A simple and effective strategy is introduced to increase CRISPR\u2013Cas9-mediated gene knock-in rates by using 5\u2032-modified double-stranded DNA donors with short homology arms.<\/p>\n","protected":false},"excerpt":{"rendered":"
Here, we report a rapid CRISPR\u2013Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5\u2032-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65\/40% knock-in rates for 0.7\/2.5\u2009kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5\u2032-end modification led to up to a fivefold increase in gene knock-in rates at various […]<\/p>\n","protected":false},"featured_media":0,"template":"","meta":{"msr-url-field":"","msr-podcast-episode":"","msrModifiedDate":"","msrModifiedDateEnabled":false,"ep_exclude_from_search":false,"_classifai_error":"","msr-author-ordering":null,"msr_publishername":"","msr_publisher_other":"","msr_booktitle":"","msr_chapter":"","msr_edition":"","msr_editors":"","msr_how_published":"","msr_isbn":"","msr_issue":"4","msr_journal":"Nature Chemical 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