CRISPR/Cas-based precision gene replacement in plants via microhomology-mediated end joining

Highly efficient precision gene editing is ultimately a desirable technology, but implementation remains challenging, especially in plants. Here, we tested a novel CRISPR-Cas-based tool for precision gene replacement in plants employing a microhomology-mediated end joining (MMEJ) mechanism. The MMEJ-mediated precise gene replacement employing 20-bp microhomology MMEJ donors (~100 bp lengths) and a cNHEJ inhibitor, NU7441, produced high targeted editing efficiencies, up to 1.60 ± 0.14, 4.47 ± 1.98, and 8.98 ± 4.73 % in tomato, lettuce, and cabbage, respectively. Our data also revealed the critical impacts of the microhomology length and NU7441 concentration on MMEJ-based precision gene editing in plants.