Advancing PAM-less genome editing in soybean using CRISPR-SpRY

Abstract

Abstract Although CRISPR-Cas9 technology has been rapidly applied in soybean genetic improvement, it is difficult to achieve the targeted editing of the specific loci in the soybean complex genome due to the limitations of the classical protospacer adjacent motif (PAM). Here, we developed a PAM-less genome editing system mediated by SpRY in soybean. By performing targeted editing of representative agronomic trait targets in soybean and evaluating the results, we demonstrate that the SpRY protein can achieve efficient targeted mutagenesis at relaxed PAM sites in soybean. Furthermore, the SpRY-based cytosine base editor SpRY-hA3A and the adenine base editor SpRY-ABE8e both can accurately induce C-to-T and A-to-G conversion in soybean, respectively. Thus, our data illustrate that the SpRY toolbox can edit the soybean genomic sequence in a PAM-free manner, breaking restrictive PAM barriers in the soybean genome editing technology system. More importantly, our research enriches soybean genome editing tools, which has important practical application value for precise editing and molecular design in soybean breeding.