Cas12a (formerly Cpf1), a type V-A CRISPR effector RNA-guided DNA endonuclease, has been widely used for plant genome editing in recent years. Cas12b is a type V-B CRISPR effector RNA-guided DNA endonuclease. Like Cas12a, Cas12b recognizes T-rich PAMs and generates staggered DNA double strand breaks. While Cas12a only requires crRNA to function, Cas12b requires both crRNA and tracrRNA or their engineered fusion known as single guide RNA (sgRNA). This feature resembles the CRISPR-Cas9 system and enables guide RNA engineering, making Cas12b advantageous over Cas12a in certain genome engineering applications. Here, we describe our recent efforts on developing a new plant genome engineering platform based on Cas12b. We first compared multiple Cas12b orthologs of different bacterial origins for genome editing in rice, an important crop. Among them, we identified a potent ortholog for targeted mutagenesis, which was further demonstrated in multiplexed genome editing in stable transgenic lines. Next, we engineered three Cas12b based repressors and showed that they could mediate targeted transcriptional repression at different levels. Finally, we compared over a dozen transcription activation systems based on Cas12b in plants. We found the most potent transcription activation system relies on both Cas12b protein and engineered sgRNAs for the recruitment of different transactional activators. With the demonstration of Cas12b for genome editing, CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), our work comprehensively establishes Cas12b as the third promising CRISPR system, after Cas9 and Cas12a, for plant genome engineering.
Coauthors: Meiling Ming – University of Maryland; Qiurong Ren – University of Electronic Science and Technology of China; Yao He – University of Electronic Science and Technology of China; Yingxiao Zhang – University of Maryland; Shishi Liu – University of Electronic Science and Technology of China; Zhaohui Zhong – University of Electronic Science and Technology of China; Jiaheng Wang – University of Electronic Science and Technology of China; Aimee Malzahn – University of Maryland; Jun Wu – Nanjing Agricultural University; Xuelian Zheng – University of Electronic Science and Technology of China; Yong Zhang – University of Electronic Science and Technology of China; Yiping Qi – University of Maryland
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