Abstract
BackgroundDelivery 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.FindingsDelivery 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.ConclusionsIn 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.
Highlights
Delivery of clustered regularly interspaced short palindromic repeats (CRISPR) reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes
Clustered regularly interspaced short palindromic repeatsCRISPR associated (CRISPR-Cas) is an adaptive immune system in prokaryotes that protects against invading bacteriophages by performing cleavage of their DNA (Horvath and Barrangou 2010; Garneau et al 2010; Gasiunas et al 2012)
The CRISPR-Cas9 system from Streptococcus pyogenes recognizes an NGG protospacer adjacent motif (PAM) to create double strand breaks upstream of the PAM site, whereas the CRISPR-Cas12a system recognizes the TTTV PAM to create double strand breaks downstream of the PAM recognition site (Svitashev et al 2015; Kim et al 2017). These two proteins are of use for gene editing in different genomic contexts as Cas9 can be used for editing GC-rich regions and Cas12a can be used for editing AT-rich regions
Summary
Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. CRISPR-Cas9 and CRISPR-Cas12a from the Type II CRISPR systems are two major nucleases that have been exploited to edit plant genomes (Nekrasov et al 2013; Svitashev et al 2015; Kim et al 2017). Because the purpose of this experiment was to compare the efficacies of Cas9 and Cas12a nucleases for editing genomes, we identified DNA sequences that could be targeted by both Cas9 and Cas12a.
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