Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

Abstract

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9-guide RNA (gRNA) and LbCas12a-CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium-mediated transformation. On-target mutation analysis showed that 90%-100% of the Cas9-edited T0 plants carried indel mutations and 63%-77% of them were homozygous or biallelic mutants. In contrast, 0%-60% of Cas12a-edited T0 plants had on-target mutations. We then conducted CIRCLE-seq analysis to identify genome-wide potential off-target sites for Cas9. A total of 18 and 67 potential off-targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off-target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.

Assessment of Cas12a‐mediated gene editing efficiency in plants

“…Together, there are now enough data to conclude that the robustness of LbCas12a is a general feature in plants. Regarding the comparison between LbCas12a and SpCas9 for stable genome editing, recent experiments in maize showed higher editing rates with SpCas9 as compared to LbCas12a targeted to the glossy2 locus (Lee et al ., ), in line with the data obtained in our tomato experiment. However, these observations reflect only the results obtained with a very limited number of genome targets, and therefore, it would be premature to draw conclusions from these findings, as our transient expression data show a strong locus‐dependent effect on RGENs activities, with LbCas12a producing higher mutagenesis rates in the majority of the loci assayed.…”

Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

“…Therefore, it is quite necessary to detect off-target mutation at a genome-wide level. Recently, a highly sensitive screen for genome-wide CRISPR/ Cas9 nuclease off-target effect was reported in maize genome editing using CIRCLE-seq method (Lee et al, 2018). A very meaningful off-target analysis was conducted in Cas9-and Cpf1edited rice through whole genome sequencing (Tang et al, 2018).…”

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

“…Cas12a family proteins are generally smaller than most of the Cas9 orthologs because they lack an HNH domain (Yamano et al ., ). The CRISPR/Cas12a system was first used for targeted genome editing in mammalian cells (Kim et al ., ), and has since been successfully used for genome editing in plants, such as Arabidopsis thaliana (Tang et al ., ), rice ( Oryza sativa ) (Hu et al ., ; Tang et al ., ; Wang et al ., ; Xu et al ., ; Li et al ., ), soybean ( Glycine max ) (Kim et al ., ), tobacco ( Nicotiana tabacum ) (Kim et al ., ), maize ( Zea mays ) (Lee et al ., ), and the unicellular green alga ( Chlamydomonas reinhardtii ) (Ferenczi et al ., ).…”