A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants

Abstract

Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase (PDS) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

Fig. 1. Agrobacterium-mediated transient expression of GUS and CRISPR/Cas9 genes in tobacco.

a, b T-DNA constructs for (a) GUS and (b) CRISPR/Cas9 (Cas9 and a PDS-targeted sgRNA) expression. The GUS construct consists of a GUSPlus gene interrupted by a cas1 intron (in), to prevent bacterial expression, under the control of a CaMV 35S promoter, and a kanamycin resistance gene (Kan^R). b The CRISPR construct (hCas9-NtPDS) consists of a Cas9 gene interrupted with an IV2 intron (in), an sgRNA targeting the tobacco PDS gene under the control of a U6 promoter, and a kanamycin (Kan^R) resistance gene. Primer sets 1, 2, and 3 were used for PCR analysis to determine the presence of T-DNA fragments stably integrated into the tobacco genome. c The fourth exon of the tobacco PDS gene was selected as the target site for the sgRNA. d, e Histochemical staining of GUS activity in tobacco leaf discs (d) without or (e) with timentin to suppress Agrobacterium growth. d GUS activity in tobacco leaf discs 2–6 days following infection by Agrobacterium. e Reduction in GUS activity in tobacco explants transferred to timentin-containing media, to suppress Agrobacterium growth, for an additional 5 days following the initial 2–6 day co-incubation. The difference in GUS expression activities between explants in d and those in e indicated transient GUS expression demonstrating that transient GUS expression peaked at 3–4 days post infection

Fig. 2. CRISPR/Cas9-mediated mutations were identified with high-throughput sequencing analysis of the PCR-amplified sgRNA-target region of the pds-12 mutant.

The red-marked TGG sequence on the x axis represents the three PAM nucleotides. Black circles represent nucleotide variant frequencies (NVF) of PCR product amplified from wild-type (WT) DNA. Red triangles represent the NVF of PCR product amplified from the genomic DNA of a 42-plant pool containing the pds-12 mutant line, displaying much higher NVF at positions 45–51. Blue squares represent the NVF of the same PCR products as the red triangles, but diluted six times with the WT PCR product, demonstrating that the NVF at nucleotide positions 45–51 are significantly reduced following the dilution. Using the WT PCR product dilution method, mutations at positions 45–51 were identified, consistent with the DNA sequencing results of the pds-12 mutant plant (Table 2)

Fig. 3. Identification of mutant plants using high-resolution melt (HRM) analysis of PCR products derived from pooled plant samples.

a HRM analysis of DNA from pooled samples containing pds-12 with various ratios of mutant (MT) to wild-type (WT) plant tissue. The pooled samples were as follows: a 100% WT plant pool, a 2-plant pool (1MT + 1WT), a 7-plant pool (1MT + 6WT), a 20-plant pool (1MT + 19WT), and a 30-plant pool (1MT + 29WT). Melt curves demonstrate that the 20-plant pool sample can be clearly distinguished from those of the 100% WT pool. b HRM analysis of the 7-plant pool containing different pds mutants. HRM curves of the 100% WT pool (negative control) or 7-plant pools containing a single pds-9, pds-10, pds-11, pds-12, pds-13, or pds-14 mutant demonstrate that HRM analysis is effective to identify single mutants from pooled samples of 7-plants. The WT curve is representative of the three negative control pools used in this single-blind screen. Difference RFU values in b represent the average of three replicates at each temperature point. Error bars show variance for each temperature point for each mutant line

Fig. 4. PCR verification of non-transgenic pds mutant lines using three primer sets (Fig. 1b) targeted to the three different regions of the T-DNA insert.

Primer set 1 was used to amplify the T-DNA region between the left T-DNA border and the 5′ end of the CaMV 35S promoter. Primer set 2 was used to amplify the 3′ end of the Cas9 coding sequence. Primer set 3 was used to amplify a region of the kanamycin resistance gene (Kan^R). The absence of all three T-DNA PCR fragments was indicative of non-transgenic plants. WT wild type