Expanding plant genome editing scope and profiles with CRISPR-FrCas9 systems targeting palindromic TA sites
CRISPR-Cas9 is widely used for genome editing, but its PAM sequence requirements limit efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. We first optimize FrCas9's sgRNA scaffold for efficient editing across different temperatures. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites which editing outcomes sharing the same characteristics of a typicalCRISPR-Cas9 system. FrCas9 induces high efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9 derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole genome sequencing- based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants however causes detectable guide RNA-independent off-target mutations mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with expanded targeting scope.