Optimizing Rice Genomics: Employing the Hypercompact Cas12j2 System for Targeted Transcriptional Regulation and Epigenome Modification.

ABSTRACT

In the burgeoning field of genome engineering, the CRISPR-Cas systems have emerged as pivotal tools for precise genetic modifications in various organisms, including humans, animals, and plants. One significant obstacle in this arena is the substantial size of Cas proteins, such as SpCas9, which is approximately 190 kDa, complicating their delivery, particularly via viral vectors. To overcome this challenge, our research introduces the hypercompact Cas12j2 system, a groundbreaking development with a size of merely ~80 kDa, originally identified in Biggiephage. We demonstrate its application in plant genome editing, with a particular focus on rice. In this context, we have successfully adapted Cas12j2 for gene activation, achieving significant increases in gene expression, specifically up to a tenfold activation for OsER1 and a fourfold activation for OsNRT1.1A in stable transgenic rice plants. Moreover, we have ventured beyond mere gene editing to develop a Cas12j2-based approach for targeted epigenome editing, particularly in the context of DNA methylation. This was demonstrated through the targeted methylation of the OsGBSS1 promoter, as verified by Next-Generation Sequencing of bisulfite sequencing PCR products. This chapter presents a detailed protocol about utilizing the hypercompact Cas12j2 system in conjunction with specific effectors, such as transcriptional activation or repression domains, or methylation domains, to achieve targeted gene transcriptional regulation and epigenome modification in rice.