Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases

Abstract

The development of CRISPR-based editors having different protospacer adjacent motif (PAM) recognition specificities, or guide RNA length/structure requirements broadens the range of possible genome editing applications. Here, we evaluated the natural and engineered variants of Cas12a (FnCas12a from Francisella novicida and LbCas12a from Lachnospiraceae bacterium) and Cas9 for wheat genome editing efficiency and ability to induce heritable mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a was able to induce mutations in the wheat genome in the current study, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides. A LbCas12a-MGE construct was successfully applied for generating heritable mutations in a gene controlling grain size and weight in wheat. We show that the range of editable loci in the wheat genome could be expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports the set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to Cas9-based modification.

Multiplexed promoter and gene editing in wheat using the virus-based guide RNA delivery system

“…The usage of both the β- and γ-chains of the BSMV genome for subcloning the sgRNAs or development of the BSMV-sgRNA system based on the Cas12a editors (Zetsche et al . 2015; Wang et al . 2021), which utilize much shorter guides, are two paths to explore for expanding the multiplexing capacity of the BSMV-sgRNA-based editing system.…”

“…To detect target editing events, a 2 cm-long segment of the 4 th leaf was sampled about two weeks after inoculation when BSMV-PDS-inoculated control plants showed photobleached leaf symptoms. DNA was isolated using the TPS buffer (100 mM Tris-HCl, 10 mM EDTA, 1 M KCl, pH8.0) as described (Wang et al . 2021).…”

“…2019), they show relatively low editing efficiency and required transformation of a large number of plants or screening of large populations in the next generation of transgenic plants to recover desired mutations (Wang et al . 2018a, 2021). Moreover, the genetic transformation protocols developed for wheat, as well as for many other crops, are restricted to few varieties showing high regenerative capabilities (Debernardi et al .…”

“…In many crops, including wheat, the realization of the full potential of CRISPR technology is hindered by a combination of methodological challenges. While CRISPR technologies based on the Cas9 and Cas12a editors have been successfully applied to edit single and multiple genes in the wheat genome (Wang et al 2014;Gil-Humanes et al 2017;Liang et al 2017;Sánchez-León et al 2018;Zhang et al 2019), they show relatively low editing efficiency and required transformation of a large number of plants or screening of large populations in the next generation of transgenic plants to recover desired mutations (Wang et al 2018a(Wang et al , 2021.…”

Molecular evolution and functional modification of plant miRNAs with CRISPR

“…In the practice of gene editing, vector construction for CRISPR/Cas systems is easy, but for ZFNs and TALENs, construction is timeand labor-consuming [83]. In addition, many natural Cas proteins and optimized Cas variants have been employed to improve the targeting specificity and broaden the targeting range [84][85][86][87][88][89]. Therefore, CRISPR/Cas is becoming the most popular system in biotechnological applications due to its accuracy, efficiency, and cost-effectiveness [7,8,10,18,30,32,80,90,91].…”