CRISPR/Cas-based precision gene replacement in plants via homologous recombination-independent approaches

Precise gene or allele replacement is a desirable technology, but implementing it in plants remains challenging. CRISPR-Cas-based approaches, such as gene targeting (GT) and prime editing (PE), have opened up new possibilities for precise gene replacement in plants. However, their editing size and efficiency still need improvement. Recently, strategies using canonical nonhomologous end-joining (cNHEJ) and microhomology-mediated end joining (MMEJ) have been considered promising alternatives for precise gene replacement in yeast and mammals. However, these approaches have not been extensively explored and applied to plants. Here, we proposed and tested a CRISPR-Cas-based tool, termed PREMJ (precision gene replacement via microhomology-mediated end joining), for precision gene replacement in plants. The PREMJ strategy employing 20-bp microhomology MMEJ donors ([~]100 bp lengths) and a canonical nonhomologous end-joining (cNHEJ) inhibitor, NU7441, produced high targeted gene replacement efficiencies, up to 1.60 {+/-} 0.14, 4.47 {+/-} 1.98, and 8.98 {+/-} 4.73 % in protoplasts of tomato, lettuce, and cabbage, respectively. Our data also revealed the critical impacts of the microhomology length and NU7441 concentration on PREMJ-based precision gene editing in plants. Although obtaining edited plants remains challenging due to inefficient protoplast regenerations and Agrobacterium-mediated delivery, PREMJ may significantly contribute to precision gene editing in plants competent to PREMJ complex delivery and plant regeneration. Key MessageWe designed and tested a method, termed PREMJ, for precise gene replacement using the CRISPR-Cas-mediated double-stranded break (DSB) formation and repairing the DSBs via microhomology-mediated end joining (MMEJ) with MMEJ donor template carrying desired base changes and microhomologies flanking the DSB ends. PREMJ showed feasibility in precise gene replacement in protoplasts of tomatoes, lettuce, and cabbage, albeit its efficacy in Agrobacterium-mediated plant transformation requires further optimization.