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

Nat Chem Biol. 2020 Apr;16(4):387-390. doi: 10.1038/s41589-019-0432-1. Epub 2019 Dec 23.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 5' Flanking Region / genetics
  • CRISPR-Cas Systems / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA / genetics
  • Gene Knock-In Techniques / methods*
  • Genome / genetics
  • HEK293 Cells
  • Humans
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Sequence Homology, Nucleic Acid

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • DNA