Xiang Ji, Xiaomin Si, Yi Zhang, Huawei Zhang, Feng Zhang and Caixia Gao

Genome Biology201819:197 https://doi.org/10.1186/s13059-018-1580-4

Published: 15 November 2018

ABSTRACT

The CRISPR/Cas9 system has recently been engineered to confer resistance to geminiviruses in plants. However, we show here that the usefulness of this antiviral strategy is undermined by off-target effects identified by deep sequencing in Arabidopsis. We construct two virus-inducible CRISPR/Cas9 vectors that efficiently inhibit beet severe curly top virus (BSCTV) accumulation in both transient assays (Nicotiana benthamiana) and transgenic lines (Arabidopsis). Deep sequencing detects no off-target effect in candidate sites of the transgenic Arabidopsis. This kind of virus-inducible genome-editing system should be widely applicable for generating virus-resistant plants without off-target costs.

See https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1580-4

Figure 1: Off-target effects generated by overexpression of the CRISPR/Cas9 system, and validation of the VIGE system in transient assays based on Nicotiana benthamiana. a Off-target frequencies detected by deep sequencing in transgenic C3 and non-transgenic plants (n = 3). The genomic DNA of leaves was extracted for analysis. For each site, mismatches relative to the on-target site are shown by colored boxes, and bases in the spacer sequence are numbered from 1 (most PAM-proximal) to 20 (most PAM-distal). b Diagram of the virus inducible system that confers geminivirus resistance with high specificity. c Expression of the pV86-GUS and pC86-GUS reporter constructs. Leaves were stained with X-gluc 4, 5, and 6 days after virus infection. Scale bar, 1 cm. d Detection of BSCTV DNA accumulation level of individually expressed pCambia–BSCTV vector (left panel) and the Cas9 transcription level of inducible vectors (pV86-401 or pC86-401) co-expressed with pCambia–BSCTV (right panel) from 0 dpi to 4 dpi (n = 3). e Phenotypes reflecting the activities of the inducible systems (pV86-401 and pC86-401) containing B7, B15, C3, and C11 sgRNAs targeting the BSCTV genome in tobacco plants. White arrows indicate systemic leaves with altered phenotypes after BSCTV infection. Scale bar, 2 cm. f Virus loads in local and systemic leaves (n = 3) analyzed by quantitative PCR. Values are means ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference by two-tailed Student’s t test.