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

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. A simple and effective strategy is introduced to increase CRISPR–Cas9-mediated gene knock-in rates by using 5′-modified double-stranded DNA donors with short homology arms.