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

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.

Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

“…The expression of the different Cas variants is driven either under PcUBi4-2 or pEC1.2 promoter [ 13 , 31 , 32 ]. Differently from Cas9 and intronized zCas9i , SaCas9 and AsCas12a have different requirements concerning the interaction between crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) and previously developed entry vectors, such as pEn-Sa_Chimera and pYPQ133-STU-As (Addgene ID 138100) can be used for gRNA cloning [ 13 , 54 ]. pEn-Sa_Chimera contains the Arabidopsis pU6-26 promoter, followed by the spacer for cloning the 20 nt sgRNA sequence using BbsI restriction sites and tracrRNA specific to SaCas9 .…”

“…This cassette is flanked by attL1-attL2 recombination sites for an easy single-step Gateway cloning into pRU320, pRU321 and pRU322 T-DNA vectors for fluorescent seed selection. For cloning into the vectors containing AsCas12a , pYPQ133-STU-As entry clone, developed by Zhang et al can be used [ 54 ]. pYPQ133-STU-As contains AsCas12a crRNA scaffold flanked by the hammer head (HH) and hepatitis delta virus (HDV) ribozymes and a sgRNA cloning site between HH and HDV.…”

Construct design for CRISPR/Cas-based genome editing in plants

“…The polycistronic mRNA containing multiple [123][124][125]. Among these gRNA processing systems, tRNA and Csy4 systems appear more effective for Cas9 and HH-HDV for Cas12a [113,126,127].…”

“…To date, up to 24 gRNAs have been expressed in plants from one construct, albeit at reduced efficiency [128]. It appears that the stoichiometry of the gRNA:Cas complex in the cell is important for efficient gene editing using multiplex CRISPR systems because an inappropriate concentration of each gRNA:Cas complex can lead to reduced editing events [127,128]. Although it is easy to express gRNAs at high levels using a strong ubiquitous promoter, coexistence of multiple gRNAs in a cell at the same time dilutes the concentration of each gRNA:Cas complex harboring a specific target sequence [109,129].…”

“…Therefore, strong ubiquitous expression of Cas gene is necessary to increase the functional concentration of each gRNA:Cas complex in plant cells to improve the efficiency of multiplex editing [109,131,132]. Using a dual Pol II prompter system with HH-HDV-based processing of crRNAs, 16 target sites can be simultaneously edited by Cas12a within one generation in rice [127]. A general guidance on the design of multiplex CRISPR constructs includes choosing the right: (i) Cas protein, such as SpCas9 or Cas12a for multiplex editing (Figure 1A); (ii) RNA polymerase II-based promoter to express multiplex gRNAs; and (iii) gRNA processing system based on either tRNA, Csy4, or HH-HDV enzyme.…”