Multiplex genome editing in Arabidopsis thaliana using Mb3Cas12a

Abstract

The use of CRISPR-Cas proteins for the creation of multiplex genome engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR-Cas9 system for multiplex genome engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome editing in Arabidopsis thaliana. We identified optimized cis-regulatory sequences for driving expression of single-transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex genome engineering in plant-based systems.

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox

“…Potentially, more important is the ability of Cas12a to process pre-crRNA, which provides for multiplexing, that is, co-expression of several guides on a single transcript for compact delivery, expression under inducible or state-dependent promoters, or for monitoring of gene expression . Indeed, highly efficient, multiplex, simultaneous genome editing of dozens of targets using Cas12a has been repeatedly demonstrated. ,− Moreover, efficient simultaneous regulation of multiple genes in mammalian cells by multiplexing with inactivated Cas12a has been reported by several groups. − Several studies have reported high specificity of Cas12a, − with an extremely low off-target rate. Although the natural diversity of Cas12a is far lower than that of Cas9, Cas12a’s from different bacteria were found to be optimal for different applications in different in vitro and in vivo systems.…”

“… 106 Indeed, highly efficient, multiplex, simultaneous genome editing of dozens of targets using Cas12a has been repeatedly demonstrated. 104 , 107 − 110 Moreover, efficient simultaneous regulation of multiple genes in mammalian cells by multiplexing with inactivated Cas12a has been reported by several groups. 111 − 114 Several studies have reported high specificity of Cas12a, 115 − 118 with an extremely low off-target rate.…”

Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice

“…In fact, when solely assessed visually, nuclease activity appears to be reduced at room temperature (22 °C-28 °C) (Moreno-Mateos et al, 2017;LeBlanc et al, 2018;Malzahn et al, 2019;Milner et al, 2020;Schindele and Puchta, 2020), with Cas12a being the most negatively impacted. Editing efficiency is reported to improve when transformed plant tissue is incubated at higher temperature for a shorter period of time (Jordan et al, 2021;Kurokawa et al, 2021). In our work, when all edits were considered (i.e., including monoallelic edits detected only after amplicon sequencing), the beneficial effect of elevated temperature only held true for Mb3Cas12a.…”

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

“…In 2020, Hu et al used modified tRNA-crRNA arrays to not only effectively achieve multiple genome editing in rice, but also to successfully edit target loci that were not edited by crRNA arrays, improving the efficiency of gene editing [ 185 ]. In 2021, William et al performed multiple genome editing in Arabidopsis using Moraxella boroculi 3 Cas12a and successfully achieved the expression of a single transcript with as many as 13 crRNAs [ 186 ]. Temperature is also an essential factor in the efficiency of Cas12a editing.…”