Engineering Plant Immunity via CRISPR/Cas13a System Fatimah Aljedaani and Magdy Mahfouz Laboratory for Genome Engineering , Division of Biological Sciences, King Abdullah University of Science & Technology
I Introduction Viruses are considered one of the major concerns that threaten the agricultural production and food security throughout the world. Genome-engineering strategies have recently emerged as promising tools to confer virus resistance in plants. Clustered regularly interspaced short palindromic repeats (CRISPR)/ and CRISPR associated genes are adaptive immunity systems found in bacteria and archaea to defend against invading phages and conjugative plasmids . Recently, CRISPR/Cas systems have been developed as the most powerful genome engineering tool. CRISPR/Cas13a (class 2 type VI-A) is a ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome.
II Aim and Objective In this work, we employed CRISPR/Cas13a to engineer plant immunity to confer resistance against an RNA virus, Turnip Mosaic Virus-GFP (TuMV-GFP). Our data indicate that CRISPR/Cas13a can be used to engineer interference against viruses, providing a potential novel mechanism for RNA-guided immunity against viruses in plants.
III Methodology
V Future Work
IV Results and Conclusions Suppression of virus accumulation in engineered Arabidopsis and N.benthamiana plants
Engineering CRISPR/Cas13 system for RNA knockdown in plants. Engineering crop plants resistance to single or multiple viruses by CRISPR/Cas13a.
Arabidopsis thaliana plants infiltrated with TuMVGFP. However, only overexpressed plants exhibited reduce virus accumulation
Engineering CRISPR/Cas13a machinery for in planta expression To engineer plants that express the CRISPR/Cas13a machinery, we codonoptimized the Leptotrichia shahii Cas13a (LshCas13a) nucleotide sequence for expression in plants
Designing crRNA against TuMV-GFP To target the TuMV-GFP virus, we designed and constructed crRNAs complementary to sequences of four different regions of TuMV-GFP genome, including two targets in GFP (GFP1 and GFP2), and the helper component proteinase silencing suppressor (HC-Pro) and coat protein (CP) sequences
Nicotiana benthamiana infiltrated with TuMV-GFP. crRNA-OE plants show less GFP signal compared to the control
TuMV-GFP express GFP protein under UV light. However, pCas13a-crRNA overexpression plants exhibit a substantial reduction of the GFP signal level under UV light compared to control, indicating viral reduction. Our data demonstrate that CRISPR/Cas13a mediate viral interference against RNA viruses.
CRISPR/pCa13a mediate interference with TuMV-GFP
Challenging pCas13a-crRNA-OE plants with TuMV-GFP RNA2 of TRV system was used to deliver the crRNA. Successful interference with the TuMV-GFP genome would result in attenuated replication and spread of the virus, which can be measured by monitoring the level and spread of the virus-mediated GFP expression to systemic leaves during the course of the infection.
A. GFP detection of T1 and T2 OE-pCas13a-crRNA Arabidopsis plants
B. GFP detection of pCas13acrRNA-OE N. benthamiana plants
To further detect the accumulation of the TuMV genome, total protein was extracted and subjected to western blot. GFP proteins were detected at 27 KD in all the samples by using mouse α-GFP All plant-OE Cas13a-crRNA samples show less GFP accumulation compared to the other samples indicating viral reduction in the OE plants. Overall, CRISPR/Cas13a mediate molecular interference against plant viruses.
VI References 1.Abudayyeh, O.O., et al., RNA targeting with CRISPR–Cas13. Nature, 2017. advance online publication. 2.East-Seletsky, A., et al., Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature, 2016. 538(7624): p. 270-273. 3.Aman, R., et al., RNA virus interference via CRISPR/Cas13a system in plants. Genome Biology, 2018. 19(1): p. 1. 4.Mahas, A., C. Neal Stewart, and M.M. Mahfouz, Harnessing CRISPR/Cas systems for programmable transcriptional and posttranscriptional regulation. Biotechnology Advances, 2017.