High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns

Abstract

The 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 the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana , we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus .

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

“…Furthermore, sgRNAs directed against ERECTA ( ER ) or GLABROUS1 ( GL1 ) were, either in pairs or simultaneously, directly assembled in pDGE347 to generate three control constructs. Although single and double mutants could be induced with high efficiencies in the T 1 generation in previous transformations with the intron-optimized zCas9i (Grützner et al , 2020), we expected that efficiencies would decrease due to competition of many different sgRNAs for the limiting Cas9 nuclease core. Furthermore, we suspected that the 24 successive blocks of (with the exception of the 20 nt variable section of the sgRNA) identical sequence repeats within the sgRNA array might be prone to recombination events in E. coli , in Agrobacterium , during T-DNA transfer, or after T-DNA integration into the plant genome.…”

“…Recipient plasmids contain a ccdB cassette (Figure 1a), which can be excised by Bsa I/ Eco 31I and replaced by sgRNA TUs in a GoldenGate cloning reaction. For enhanced efficiency, recipient vectors were equipped with a highly intron-optimized Cas9 gene, zCas9i (Grützner et al , 2020), either under control of an Arabidopsis RIBOSOMAL PROTEIN S5a (RPS5a) promoter fragment (Tsutsui and Higashiyama, 2017, Ordon et al , 2019) or a 35S promoter fragment. Furthermore, recipient plasmids contain a positive selection marker (resistance to glufosinate (BASTA), kanamycin or hygromycin) and additional markers for positive and/or negative selection.…”

“…Empty nuclease vectors used in this work contained up to three selection markers for positive/negative selection in Arabidopsis or N. benthamiana . A highly intron-optimized Cas9 gene, zCas9i (coding for Cas9 with two nuclear localization signals), was incorportated for improved editing efficiencies (Grützner et al, 2020). A ccdB cassette can be excised by Bsa I, and replaced by an sgRNA array via GoldenGate cloning.…”

“…However, the rpp4 phenotype was detected by phenotypic analyses within family #1681 (Table S2), and one of the two segregants we analyzed indeed carried disruptive mutations within all of the 12 target genes, with edits at 40/48 target sites (Figures 5c, S12). Thus, enhanced efficiencies achieved by intron-optimization of the Cas9 sequence (Grützner et al , 2020) coupled with a high degree of multiplexing enabled us to isolate a duodecuple Arabidopsis mutant within a single generation.…”

“…In this study, we explore the efficiency and limits of multiplex-editing in the two major dicot model species A. thaliana and N. benthamiana . We first extended a previous toolkit for assembly of constructs with up to 32 sgRNAs, and also tuned the toolkit for improved efficiencies by incorporation of a highly intron-optimized Cas9 gene, zCas9i (Grützner et al , 2020). For multiplexing in N. benthamiana , we developed new markers for transgene counter-selection, and target eight genes by an array of nine different sgRNAs.…”