Assessment of Cas12a‐mediated gene editing efficiency in plants

Abstract

Summary The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of −10 to −2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

Multiplexed conditional genome editing with Cas12a inDrosophila

“…These results confirm and expand previous observations by us and others that Cas12a induced mutations are significantly larger than those induced by Cas9 (Port and Bullock 2016;D. Kim et al 2016;Bernabé-Orts et al 2019). Larger deletions are often desirable as they have a higher chance to impair the function of genetic elements.…”

Improving CRISPR‐Cas‐mediated RNA targeting and gene editing using SPLCV replicon‐based expression vectors in Nicotiana benthamiana

“…The average relative amplification values of the target sites obtained using the qPCR‐based method were approximately 1.01 and 0.90 in NT‐infiltrated and TV‐infiltrated samples, respectively (Figures 1t and u; Peng et al , 2018). Such a notion suggests that the mutation frequency for TVs was approximately 10%, and this result was similar to a previous report (Bernabé‐Orts et al , 2019). However, the average relative amplification values of five target sites were approximately 0.65 in SV‐infiltrated samples (Figures 1t and u).…”

CRISPR-Cas nucleases and base editors for plant genome editing

“…Second, Cas12a requires a T-rich (5 0 -TTTV-3 0 or 5 0 -TTV-3 0 ) PAM, which is suitable for targeting AT-rich genomic regions. (Endo et al 2016b;Kim et al 2017b;Li et al 2018aLi et al , 2019aHu et al 2017;Xu et al 2017;Tang et al 2017;Wang et al 2017;Yin et al 2017;Malzahn et al 2019;Lee et al 2019;Bernabé-Orts et al 2019;Jia et al 2019). Generally, LbCas12a and FnCas12a were shown to be more reliable in plant genome editing than AsCas12a.…”