Plant Genomics

Biography

Biography: Akira Endo

Abstract

Cas9 is a RNA-guided endonuclease (RGN) that belongs to CRISPR/Cas system, functioning in an adaptive immune system of bacteria. CRISPR/Cas9 has been intensively developed and applied to genome editing in various organisms. Most widely used Cas9 is from Streptcoccus pyogenes (SpCas9) since it robust genome editing activity. SpCas9 gene is 4.1 kb in size and protein recognizes NGG as protospacer adjacent motif (PAM) which is located just next to target sequence. The size and PAM of SpCas9 could restrict the utilization of virus vector and number of target sequence, respectively. To expand the utility of Cas9 protein in plant genome engineering, we tried to adapt two different RGNs to genome editing in plant. One is Cas9 from Staphylococcus aureus (SaCas9). The feature of SaCas9 is the smallness in size. SpCas9 consists of 1368 amino acids. In contrast, the number of amino acids of SaCas9 is 1053. Therefore, SaCas9 could be mounts on virus vector. The other is the CRISPR from Prevotella and Francisella 1 (Cpf1) which is an emerging RGN. Cpf1 has two distinct properties that distinguish it from Cas9. First, Cpf1 utilizes a thymidine (T)-rich PAM, in contrast to the guanidine (G)-rich PAM preferred by Cas9. Thus, Cpf1 can target T-rich regions that Cas9 hardly access. Second, upon cleavage of the target sequence, Cpf1 produces a staggered (sticky) DNA end with a 5´ overhang while Cas9 generates a blunt DNA end. Sticky DNA ends are thought to be applicable to precise genome engineering via a precise end-joining pathway. To evaluate whether these RGNs can be adapted to plant genome editing, we applied SaCas9 and FnCpf1 to targeted mutagenesis in tobacco and rice. Our results demonstrate that SaCas9 and FnCpf1 can be applied successfully to genome engineering in plants.