PAM-Less CRISPR-SpRY Genome Editing in Plants.
Methods in molecular biology (Clifton, N.J.), 2653: 3-19 PMID 36995616 DOI 10.1007/978-1-0716-3131-7_1
Abstract
Engineered SpCas9 variant, SpRY, has been demonstrated to facilitate protospacer adjacent motif (PAM) unrestricted targeting of genomic DNA in various biological systems. Here we describe fast, efficient, and robust preparation of SpRY-derived genome and base editors that can be easily adapted to target various DNA sequences in plants due to modular Gateway assembly. Presented are detailed protocols for preparing T-DNA vectors for genome and base editors and for assessing genome editing efficiency through transient expression of these reagents in rice protoplasts.