The recent advancement in developing CRISPR/Cas9 system and various derivative tools such as base editors (BEs) has accelerated basic plant science research and crop improvement by creating multiple types of genetic variations. However, the use of Cas9 protein is frequently limited by the requirement of G/C-rich protospacer-adjacent motif (PAM) sequences, especially in triticeae plants, many of which are important food and forage crops carrying large and complex genomes.

In this study, we developed efficient gene and base editing tools by CRISPR/CasΦ2 system in wheat and rye. By sequentially testing crRNA expression, NLS incorporation and protein variants, we efficiently improved the efficiency of gene editing from near-background level up to 30% in transgenic wheat, with the mutations all being nucleotides deletions. Using the paired opposite crRNAs strategy in wheat and rye protoplasts, we have further improved the editing efficiency with ~1.5 fold higher than that produced with single crRNA. To develop CRISPR/CasΦ2-mediated base editors, we generated catalytically inactive dCasΦ2 by mutating the active sites of the RuvC domain (D394, E606, and D695), creating dCasΦ2-CBE and dCasΦ2-ABE.

Both base editors exhibited efficient C-to-T or A-to-G editing with very low levels of unwanted indels in wheat and rye. And the deamination window spanned from protospacer positions C2 to C17 or A9 to A11. Deep sequencing analysis indicated the high specificity in the editing activity of CRISPR/CasΦ2-mediated gene and base editing in transgenic wheat mutants.

With its unique properties, i.e., efficient use of TTN PAM and alternative base editing window, CRISPR/CasΦ2 provides a complementary genome engineering tool, which may find wide applications in future research on CRISPR/CasΦ2-mediated genome modifications, particularly for wheat and related triticeae crops.