Identification of U6 Promoter and Establishment of Gene-Editing System in Larix kaempferi (Lamb.) Carr

This study aimed to establish a CRISPR/Cas9 gene-editing system for <i>Larix kaempferi</i> (Lamb.) Carr. (Japanese larch). We screened <i>L. kaempferi</i> U6 promoters and used them to drive sgRNA expression in the CRISPR/Cas9 gene-editing system. The <i>L. kaempferi</i> embryogenic callus was used as the receptor material for genetic transformation, and the frequency and types of gene editing were then analyzed. The results showed various mutations in the transgenic materials, including base substitutions and deletions, and the editing frequency ranged from 5% to 14.29%. In summary, we established a CRISPR/Cas9 gene-editing system for <i>L. kaempferi</i>. Our results demonstrate that the CRISPR/Cas9 system can efficiently edit genes in <i>L. kaempferi</i>, with significantly higher editing frequencies observed when sgRNA expression is driven by endogenous <i>LaU6</i> promoters compared to the exogenous promoter ProAtU6-26. This work provides technical support for the study of <i>L. kaempferi</i> gene functions and genetic improvement.