Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells.

Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient. Additionally, we developed an easy and effective Golden-Gate-based system for crRNA cloning. We compared LbCas12a to SpCas9 by investigating on-target efficacy and specificity at 35 overlapping target sites and 57 (LbCas12a) or 100 (SpCas9) predicted off-target sites. We found LbCas12a an efficient, robust addition to SpCas9, with similar overall though target-dependent efficiencies. LbCas12a induced more and larger deletions than SpCas9, which can be advantageous for specific genome editing applications. Off-target activity for LbCas12a was found at 10 out of 57 investigated sites. One or two mismatches were present distal from the PAM in all cases. We conclude that Cas12a-mediated genome editing is generally precise as long as such off-target sites can be avoided. In conclusion, we have determined the mutation pattern and efficacy of Cas12a-mediated CRISPR mutagenesis in tomato and developed a cloning system for the routine application of Cas12a for tomato genome editing.

High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells.

CRISPR/Cas9 technology has the potential to significantly enhance plant breeding. To determine the specificity and the mutagenic spectrum of SpCas9 in tomato, we designed 89 g(uide) RNAs targeting genes of the tomato MYB transcription factor family with varying predicted specificities. Plasmids encoding sgRNAs and Cas9 were introduced into tomato protoplasts, and target sites as well as 224 predicted off-target sites were screened for the occurrence of mutations using amplicon sequencing. Algorithms for the prediction of efficacy of the sgRNAs had little predictive power in this system. The analysis of mutations suggested predictable identity of single base insertions. Off-target mutations were found for 13 out of 89 sgRNAs and only occurred at positions with one or two mismatches (at 14 and 3 sites, respectively). We found that PAM-proximal mismatches do not preclude low frequency off-target mutations. Off-target mutations were not found at all 138 positions that had three or four mismatches. We compared off-target mutation frequencies obtained with plasmid encoding sgRNAs and Cas9 with those induced by ribonucleoprotein (RNP) transfections. The use of RNPs led to a significant decrease in relative off-target frequencies at 6 out of 17, no significant difference at 9, and an increase at 2 sites. Additionally, we show that off-target sequences with insertions or deletions relative to the sgRNA may be mutated, and should be considered during sgRNA design. Altogether, our data help sgRNA design by providing insight into the Cas9-induced double-strand break repair outcomes and the occurrence of off-target mutations.