Large Genome Segment Deletion in Oryza sativa Protoplasts Utilizing CRISPR Cas9 Synchronized Cuts

The newly-discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has enabled rapid genome editing in plants. Moreover, CRISPR/Cas9-based editing opens up the possibility of functional characterization of large chromosomal segments, since CRISPR/Cas9 allows for large deletions targeted to specific chromosome regions. Current techniques for plant transformation, however, involve a lengthy tissue culture process for regeneration of edited plants, which creates a bottleneck when testing new targets for gene editing. This study explores the use of plant protoplasts as a testbed for rapid testing of single guide RNA (gRNA) designs for CRISPR/Cas9 gene editing in rice, using a region on rice chromosome 10 containing a nuclear-plastid DNA (NUPT) segment as a target for making a large deletion. Plant protoplasts, which are plant cells with the cell wall removed, offer a rapid method to validate the effectiveness of CRISPR/Cas9 constructs prior to their full implementation in tissue culture. This study first optimized a plant protoplast isolation technique in rice using the Texas rice variety Presidio. Next, four different gRNAs were designed as two nested pairs, one flanking a smaller deletion (12.1 kb) and another flanking a larger deletion segment (107 kb) containing a large chloroplastic insertion. To design the gRNAs, the sequence of the Presidio genome at these locations was first obtained by sequencing PCR amplicons from the target loci. Then a ribonucleoprotein complex of Cas9 plus the gRNA for each target was used to test the gRNA efficiency in vitro by cutting PCR amplicons flanking each target cut site, which showed successful editing at all four cut sites individually. Next, plasmids containing Cas9 and each pair of gRNAs were transformed into rice protoplasts, and Cas9 expression was detected after mRNA analysis, demonstrating successful expression of the plasmids within the protoplasts. Lastly, in vivo activity of the CRISPR system was validated for at least one of the cut sites, although the two large segment deletions were not detected in subsequent analysis of the edited protoplasts. Future efforts will be needed to further test and improve the frequency of making large chromosomal deletions before it can be widely used