Development and activity evaluation of a highly efficient CRISPR-Cas genome editing system in larch

The CRISPR/Cas system has been widely used among plant species. However, due to the long life cycle, complex genetic background, high levels of genomic heterozygosity, and difficulties in stable transformation, genome editing has not been thoroughly explored in larch [Larix kaempferi (Lamb.) Carr.]. In this study, we evaluated a highly efficient CRISPR/Cas genome editing system in larch that utilizes endogenous promoters. We optimized the critical parameters in larch protoplast preparation and transformation. As a result, this process achieved more than 90% active cells and a transient transformation efficiency of 40%, meeting the requirements for gene editing system evaluation. Using integrated whole-genome and transcriptome sequencing analysis, we screened highly expressed mRNA transcripts and cloned 41 candidate promoters. Based on protoplast transient expression detection, we identified a highly expressed endogenous promoter in larch, designated LarPE004. We compared the single and multiple gene knockout capabilities of the LarPE004-driven single transcription unit CRISPR-Cas9 (STU-Cas9) and two transcription unit CRISPR-Cas9 (TTU-Cas9) systems. Our findings showed that the LarPE004::STU-Cas9 system was more efficient than the LarPE004::TTU-Cas9 system and significantly outperformed the CaMV 35S- and ZmUbi1-driven STU-Cas9 systems. Moreover, the LarPE004::STU-Cas9 system demonstrated improved capabilities for multiple gene editing in larch. The LarPE004::STU-SpRY system based on Cas9 mutant protein SpRY facilitated the efficient editing of various PAM sites in larch. Combining these findings, we developed LarPE004::STU-Cas9, an efficient CRISPR-Cas9 genome system for single and multiple gene editing, establishing a foundation for functional genomics research and molecular breeding in larch.