CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum

Abstract

Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

“…Generating larger deletions by simultaneous expression of two gRNA targeting sequences in close proximity makes such screens more feasible. In fact, simultaneous targeting of genes using multiple gRNAs has been demonstrated repeatedly in plants Mao et al, 2013;Upadhyay et al, 2013;Brooks et al, 2014;Gao et al, 2015). In our study, we achieved highfrequency deletions of approximately 200 bp in rice (Fig.…”

Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

“…All genes were targeted with 100% success rate, and more than 80% of the regenerated T0 plants contained biallelic mutations. These results, along with previous publications, illustrate that the Cas9-gRNA technology can be used to efficiently promote the alteration of individual or multiple gene sequences (for review, see Kumar and Jain, 2015), gene families, or homologous genes in polyploid species and delete chromosomal segments of various sizes (Brooks et al, 2014;Zhou et al, 2014;Gao et al, 2015). In this report, analysis of T0 plants and T1 segregating populations indicated that stably integrated Cas9 and gRNA expression cassettes remain active, often resulting in chimeric T0 plants and segregation distortions.…”

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

“…One major challenge is the ability of CRISPR‐Cas9 to induce mutations in polyploid genomes, in particular those with closely related subgenomes. CRISPR‐Cas9 was effective in diploid and tetraploid potato, tetraploid tobacco and hexaploid wheat (Butler et al ., 2015; Gao et al ., 2014; Wang et al ., 2014). The presence of mutations in the different homeologous genes was only assessed in wheat, but the low number of regenerated mutants prevented accurate estimation of the type of alleles obtained as well as their frequency (Wang et al ., 2014).…”