Black goji berry (Lycium ruthenicum Murr.) is a valuable functional food and traditional medicinal plant owing to its rich content of anthocyanins, trace minerals, vitamins, and polysaccharides. However, limited genetic manipulation tools have hindered functional genomic studies and trait improvement in this species. In this study, we optimized the genetic transformation system for L. ruthenicum, achieving a remarkably high transformation efficiency of 95.4%. Based on this system, we developed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene knockout approach, using the Arabidopsis U6 (AtU6) promoter to drive sgRNA expression and the cauliflower mosaic virus 35S (35S) promoter to drive Cas9 expression, achieving editing efficiencies of 68.8% at the phytoene desaturase (LrPDS) locus and 96.0% at the betaine aldehyde dehydrogenase (LrBADH2) locus. Furthermore, we established an adenine base editing (ABE) system using the ribosomal protein subunit 5A (RPS5A) promoter to drive tRNA adenine deaminase-8e (TadA-8e-nSpCas9) cassette expression, achieving an editing efficiency of 72.2% at the LrPDS locus. To broaden protospacer adjacent motif (PAM) compatibility, we introduced the PAM-relaxed variant SpRY, enabling successful A-to-G editing at an acetolactate synthase (LrALS) target site containing a noncanonical NAN PAM, with an efficiency of 5.3%. Additionally, we developed a multiplex ABE system based on the tRNA-processing strategy, which enabled simultaneous editing at 2 independent loci with an efficiency of 33.3%. Our study establishes a robust genome editing toolkit for L. ruthenicum, offering valuable tools for functional gene analysis and molecular breeding in this economically important species.
