Conferring resistance to geminiviruses with the CRISPR-Cas prokaryotic immune system

Abstract

To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR-Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR-Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses.

Figure 1 |. Testing Cas9-sgRNA activity at targets within the BeYDV.

a, Illustration of the wild type BeYDV genome (reverse complemented) and sgRNA target sequences. Red nucleotides, sgRNA sequence; black nucleotides, BeYDV sequence; blue nucleotides, protospacer adjacent motif; lightning bolts, predicted sites for Cas9 cleavage; IR, inverted repeat; 9 nt, nanonucleotide sequence; M1, motif 1; M2, motif 2; M3, motif 3; +/−, DNA strand that pairs with the sgRNA; NOS, nopaline synthase. b, Approach to assess Cas9 and sgRNA activity within N. benthamiana leaves by measuring, c, GFP expression and d, CFUs. n, number of biological replicates; error bars represent standard deviation; *P < 0.05.

Figure 2 |. Restricting BeYDV infection with the CRISPR–Cas system.

a, Illumina next-generation sequencing of four sgRNA targets. Dark bars, sgRNA gTM3⁺ (negative control). b, PCR detection of deletions within the genome of mobile BeYDV. The ∼657 bp band indicates unmodified sequences or sequences with small INDELs. The ∼349 bp band indicates sequences containing deletions; sequences of three clones for each biological replicate are shown. c, Representative images of transgenic plants challenged with BeYDV 35 days after inoculation. d, Quantitative PCR of virus levels within transgenic plants 37 days after inoculation.