Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems

Yingxiao Zhang,Han Yang,Lidiya Franklin,Ysa Le,Yalin Cheng ,Changtian Pan,Stephen M Mount,Qiudeng Que,Xuelian Zheng,Shishi Liu,Xu Tang,Lan Huang,Desuo Yin,Jianping Zhou,Jeremy D Selengut,Qing Fang,Jiaheng Wang,Bailey Mccoy,Li Tian,Yuxin Zhao,Qiao Ren ,Mingzhu Yuan,Yong Zhang,Aimee Malzahn ,Yiping Qi

doi:10.1038/s41467-021-22330-w

Abstract

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.

Highlights

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions
While most Cas12a studies in plants pursued targeted mutagenesis by non-homologous end joining (NHEJ) DNA repair, precise genome editing based on homologydirected repair (HDR) has been demonstrated[20,22,31,32,33]
The second limitation is that current Cas12a orthologs in use are not effective at lower temperatures[24,43], though this could partially be addressed by protein engineering[15,26]

Summary

Introduction

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. With low biallelic editing efficiency, it is laborious or impractical to obtain multigene knockouts in crops through subsequent breeding To address these issues, we comprehensively assess nine Cas12a orthologs and engineered their variants in rice for efficient genome editing with expanded targeting ranges. We establish a Cas12a toolbox for efficient multiplexed genome engineering with improved target accessibility and scalability

Methods

Results

Conclusion