The SpRY Cas9 variant release the PAM sequence constraint for genome editing in the model plant Physcomitrium patens

Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. In this study, the "DNA Repair and Genome Engineering" DRAGON team assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, a SpRYCas9i variant was design, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. The results show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and no off-target activity for the sgRNAs tested was detected in this study. These findings contribute to advancing versatile genome editing techniques in plants.

Research developed at the Institute Jean-Pierre Bourgin for Plant Sciences.