Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene

Abstract

Background: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9 (WT Cas9 nuclease, HiFi Cas9 nuclease, Cas9 D10A nickase) and two Cas12a nucleases (AsCas12a and LbCas12a), using the rice phytoene desaturase (PDS) gene as a target site.

Findings: Delivery of two Cas9 nucleases (WT Cas9, and HiFi Cas9) and one Cas12a nuclease (LbCas12a) resulted in targeted mutagenesis of the PDS gene. LbCas12a had a higher editing efficiency than that of WT Cas9 and HiFi Cas9. Editing by Cas9 enzymes resulted in indels (1-2 bp) or larger deletions between 20-bp to 30-bp, which included the loss of the PAM site; whereas LbCas12a editing resulted in deletions ranging between 2 bp to 20 bp without the loss of the PAM site.

Conclusions: In this work, when a single target site of the rice gene OsPDS was evaluated, the LbCas12a RNP complex achieved a higher targeted mutagenesis frequency than the AsCas12a or Cas9 RNPs.

Keywords: CRISPR; Cas12a; Cas9; Ribonucleoproteins; Rice; Synthetic guide RNAs.

Fig. 1

Choice of Carotenoid biosynthesis pathway to evaluate CRISPR-Cas nucleases. a Carotenoid biosynthesis pathway in rice. Phytoene desaturase (PDS) is a single copy gene involved in the synthesis of zeta-carotene from phytoene. GGPS, geranylgeranyl pyrophosphate synthase; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, zeta-carotene desaturase. b Schematic diagram showing OsPDS gene structure, OsPDS-Exon1 and relative position of crRNA1/2 for CRISPR-Cas9 and crRNA3 for CRISPR-Cas12a. PDS-F and PDS-R, forward and reverse primer pair for PCR and NGS analysis. c Flowchart showing RNP complex delivery and editing efficiency comparison analysis. d Regenerating green and albino rice plantlets on hygromycin containing rooting medium

Fig. 2

NGS analysis of rice lines generated from two Cas9 (WT Cas9 and HiFi Cas9) and one Cas12a (LbCas12a) RNP complex/selectable marker plasmid co-delivery. Total reads do not always add to 100% because small percentages of low frequency reads were excluded. These low frequency events are likely due to sequencing or alignment errors. Blue letters, target sequences in PDS exon 1; Red letters, PAM sequences; White letter in black box, substitution; Green letter with underscore, insertions; WT, wild type; SNP, single nucleotide polymorphism