CRISPR

1. CRISPR overview and the application of CRISPR in Tragopogon

CRISPR/Cas9 is a powerful tool for genome editing. There are two components of the CRISPR/Cas9 system: the Cas9 endonuclease and the single-guide RNA (sgRNA) (Figure 1). The ribonucleoprotein Cas9-sgRNA complex recognizes and binds any genomic regions that contain a protospacer adjacent motif (PAM) sequence, which is NGG (where N represents any nucleotide) for SpCas9. If the spacer sequence of the sgRNA (i.e., the first 20 nucleotides at its 5′ end; Figure 1) matches the genomic sequence immediately upstream of the PAM sequence, Cas9 will cleave both strands of the genomic DNA, leaving blunt ends at the position between the third and fourth nucleotides upstream of PAM (Figure 1). The double-stranded DNA break (DSB) will be repaired by one of the two innate DNA repair systems: the non-homologous end-joining (NHEJ) pathway or homology-directed repair (HDR) pathway. The error-prone NHEJ pathway is efficient and could introduce a small insertion or deletion (indel) at the DSB point (Figure 1). The HDR pathway is less efficient but more accurate. In the presence of a DNA template, either single- or double-stranded, the resultant DNA sequence will be the same as the template, which has been used for gene replacement or targeted insertion (Figure 1).

Tragopogon is a model system for studies of recent and recurrent polyploidy. Application of a robust CRISPR/Cas9 system in Tragopogon permits unique studies of biased fractionation, the gene-balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae.

2. Plasmid construction

In a proof-of-principle study, we designed sgRNAs that target the phytoene desaturase gene in Tragopogon (TraPDS). PDS encodes an essential enzyme that participates in the carotenoid biosynthesis pathway, and the loss-of-function mutant of PDS has an albino phenotype. In addition, PDS is typically a single-copy gene in plant genomes. For these reasons, a pds mutant can be efficiently generated and visually identified.

CRISPR/Cas9 target sites are located within the second and third exon of the TraPDS gene (Figure 2a). sgRNA gTraPDS1 and gTraPDS2 are designed based on sequences of the target sites (TraPDS1 and TraPDS2, respectively). Various plasmids have been designed for CRISPR/Cas9-mediated edition of TraPDS gene (Figure 2b). Different versions of Arabidopsis U6 promoters (AtU6–1, AtU6–26 and AtU6–29) are used to drive the expression of sgRNAs; Glycine ubiquitin promoter (GmUbi) drives the expression of the GFP gene; Arabidopsis ubiquitin promoter (AtUbi) drives the expression of the Cas9 gene; the expression of hygromycin resistance gene (HygR) is driven by the cauliflower mosaic virus 35S promoter (35S).

3. CRISPR-mediated Tragopogon mutant

Albino shoots were regenerated and TraPDS has been edited by CRISPR. For shoot #1–1, based on the DNA sequencing chromatogram, at least two mutation types were detected at the TraPDS1 CRISPR/Cas9 target site: one allele had a 2-bp deletion and one nucleotide substitution, and one allele showed a 7-bp deletion (Figure 3a). For shoot #1–2, at least three mutation types were identified at the TraPDS1 CRISPR/Cas9 target site based on the chromatogram: one allele had a 3-bp deletion and one nucleotide insertion; one allele showed an 11-bp deletion; and one allele had a 7-bp deletion and one nucleotide substitution (Figure 3b).

References:

Shan, S., Mavrodiev, E. V., Li, R., Zhang, Z., Hauser, B. A., Soltis, P. S., … & Yang, B. (2018). Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy. Molecular ecology resources, 18(6), 1427-1443.

Shan, S., Soltis, P. S., Soltis, D. E., & Yang, B. (2020). Considerations in adapting CRISPR/Cas9 in nongenetic model plant systems. Applications in plant sciences, 8(1), e11314.