Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in Plants

Abstract

Running title: High efficiency site-targeted mutagenesis with an intronized Cas9 gene AbstractThe recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself.To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

Continual improvement of CRISPR‐induced multiplex mutagenesis in Arabidopsis

“…While we have shifted the distribution to 4-to 6-plex mutants, even higher levels will be needed to rapidly mutate genomes and large gene families. It would be interesting to combine the RPS5A::BP-BP vector with different strategies to express gRNAs and/or by including introns in the Cas9-coding sequence (Gr€ utzner et al, 2020). Experimenting with the different gRNA expression strategies would be especially interesting, given that the gRNAs are the limiting factor in the RPS5A::BP-BP vector.…”

“…In Arabidopsis, the removal of the C‐terminal NLP NLS from the SV40‐NLP configuration reduced mutagenesis below the background level of the assay (Osakabe et al., 2016). It was shown that an SV40 NLS on both ends of a Cas9 containing 13 introns compared to a single C‐terminal SV40 NLS increased the number of T1 plants with a KO phenotype from 58 to 72% (Grützner et al., 2020). In wheat and maize protoplasts, the double‐BP architecture improved the Cas9 and Cas12a adenine base editor (ABE) editing rates compared to a C‐terminal triple SV40 NLS (Gaillochet et al., 2023).…”

Continual improvement of multiplex mutagenesis in Arabidopsis

“…While we have shifted the distribution to 4-6-plex mutants, even higher levels will be needed to rapidly mutate genomes and large gene families. It would be interesting to combine the RPS5A::BP-BP vector with different strategies to express gRNAs and/or by including introns in the Cas9 coding sequencing 32 . Experimenting with the different gRNA expression strategies would be especially interesting given that the gRNAs are the limiting factor in the RPS5A::BP-BP vector.…”

“…In Arabidopsis, the removal of the C-terminal NLP NLS from the SV40-NLP configuration reduced mutagenesis below the background level of the assay 31 . It was shown that an SV40 NLS on both ends of a Cas9 containing 13 introns, instead of a single C-terminal SV40 NLS, increased the number of T1 plants with a KO phenotype from 58% to 72% 32 . In wheat and maize protoplasts, the double-BP architecture improved Cas9 and Cas12a adenine base editor (ABE) editing rates compared to a C-terminal triple SV40 NLS 33 .…”

Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing

“…Examples of 5'UTR intron mediated expression improvements have led to inclusion of 5'UTR intron in many transgenes' designs (11). Benefits from adding multiple introns into transgenes are however controversial, while some studies describe benefits achieved with constructs containing multiple endogenous introns (12) and heterologous introns (13)(14)(15) other studies report no added benefit achieved by more than a single intron (16).…”