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

Abstract

Summary The development of CRISPR‐based editors recognizing distinct protospacer‐adjacent motifs (PAMs), or having different spacer length/structure requirements broadens the range of possible genomic applications. We evaluated the natural and engineered variants of Cas12a (FnCas12a and LbCas12a) and Cas9 for their ability to induce mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a‐induced mutations in the wheat genome, 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 guides 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 and showed substantial increase after subjecting transgenic plants to high‐temperature treatment. We successfully applied LbCas12a‐MGE for generating heritable mutations in a gene controlling grain size and weight in wheat. We showed that the range of editable loci in the wheat genome could be further 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 a set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to modification using the original SpCas9 nuclease.

Multiplexed promoter and gene editing in wheat using a virus‐based guide RNA delivery system

“…This could partially be overcome by arraying sgRNAs in a single viral vector because prior studies suggest that no processing signals are required to separate sgRNA units in the viral transcripts (Cody et al ., 2017 ; Ellison et al ., 2020 ). 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 (Wang et al ., 2021 ; Zetsche et al ., 2015 ), which utilize much shorter guides, could also be used for expanding the multiplexing capacity of the BSMV‐sgRNA‐based editing system.…”

“…pooling only two BSMV‐sgRNAs targeting sites flanking the region of interest). Alternatively, as was discussed above, constructs incorporating multiple guides into a single viral transcript need to be designed (Cody et al ., 2017 ; Ellison et al ., 2020 ; Wang et al ., 2021 ; Zetsche et al ., 2015 ).…”

“…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 (Gil‐Humanes et al ., 2017 ; Liang et al ., 2017 ; Sánchez‐León et al ., 2018 ; Wang et al ., 2014 ; 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 , 2021 ). Moreover, the genetic transformation protocols developed for wheat, and for many other crops, are restricted to few varieties showing high regenerative capabilities (Debernardi et al ., 2020 ).…”

Dissecting Plant Gene Functions Using CRISPR Toolsets for Crop Improvement

“…Just like the ultramini CasMINI system, the amino acid length of Cas12f (formerly known as Cas14) is only 40% of the length of Cas9, and it consists of only 529 amino acids. Such gene editing reagents will be delivered using AAV (adeno-associated virus). − Of course, such a smaller and tighter DNA-bound engineered Cas protein is bound to trigger a new round of large-scale technological innovation. But now, breeders who master CRISPR-Cas9 and related toolsets should quickly apply it to plant genome research, crop breeding, and other related fields of plants.…”

Evaluation of Factors Affecting In Planta Gene Editing Efficiency in Wheat (Triticum aestivum L.)

“…16−19 Although the activity of gRNAs could be quickly evaluated in vivo using protoplasts assay, it has a limited correlation with editing efficiency observed in transgenic wheat plants both for the Cas9-and Cas12a-based nucleases. 20 Therefore, further assessment of factors affecting in planta editing efficiency in wheat is needed to increase the efficiency and predictability of the GE experiments. Previously, it has been shown that GE efficiency could be improved in several species (Arabidopsis, citrus, rice, and maize) through the exposure of plants carrying either the Cas9 or Cas12a transgene to elevated temperatures.…”