Highly efficient CRISPR systems for loss-of-function and gain-of-function research in pear calli

Abstract CRISPR/Cas systems have been widely used for genome engineering in many plant species. However, their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only a few reports on the application of the CRISPR/Cas9 system in pear have been documented, and have shown very low editing efficiency. Here we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand the targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taking these results together, we have built a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.