Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature‐tolerant CRISPR/LbCas12a

摘要

The application of the CRISPR/Cas-system paved the way for the era of genome engineering and quickly became the standardly applied tool for targeted non-homologous end joining (NHEJ) based mutagenesis. Despite various efforts, homologous recombination (HR) based gene targeting (GT) still requires further improvement regarding efficiency for general applicability in plants (Huang and Puchta, 2019). We developed the in planta GT (ipGT) method aimed to establish a technology for crops with meagre transformation efficiencies (Fauser et al., 2012). Here, the targeting vector including the nuclease is integrated into the genome and excised at the same time as the target site is activated by double-strand break (DSB) induction (Figure 1a). We successfully adopted Cas9 for this application (Schiml et al., 2014) and improved GT efficiencies by replacing Cas9 from Streptococcus pyogenes (SpCas9) with Cas9 from Staphylococcus aureus (SaCas9), which is more efficient in DSB induction in Arabidopsis (Steinert et al., 2015). Egg-cell specific expression and screening for the most efficient transgenic lines were further key points for GT improvement (Miki et al., 2018; Wolter et al., 2018). Most recently, we tested the CRISPR/LbCas12a-system for ipGT and could demonstrate a further increase in GT efficiencies despite lower InDel rates induced by LbCas12a compared with SaCas9 at the ALS target locus (Wolter and Puchta, 2019). We speculated that the higher GT efficiency is caused by Cas12a-mediated cleavage on the PAM distal site, leaving the seed sequence unaffected by mutagenesis. Thus, further cleavage of NHEJ repaired junctions might be much more frequent with Cas12a than with Cas9, enhancing the probability for HR to take over DSB repair. This is also in line with very recently published results on enhancing GT efficiency using LbCas12a in rice and tomato (Li et al., 2019; van Vu et al., 2020). Recently, by introducing the single amino acid substitution D156R, we were able to obtain a LbCas12a nuclease variant with improved, temperature-tolerant cutting efficiency (ttLbCas12a) for plant gene editing, outperforming LbCas12a at 22°C and 28°C in mutation induction (Schindele and Puchta, 2019). Based on those findings, we were interested to test whether replacing LbCas12a by ttLbCas12a could further improve GT.