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Screening of CRISPR/Cas9 gRNA for mimicking Powdery Mildew resistant MLO ol-2 mutant



Archana Prajapati & Vikrant Nain*



School of Biotechnology, Gautam Buddha University, Greater Noida 201312, India;



Email: vikrant@gbu.ac.in; Fax: +91-1202344205; Phone: +91-1202344283; *Corresponding author; Archana Prajapati - E-mail: btphd2018003@gbu.ac.in


Article Type

Research Article



January 14, 2021; Revised June 9, 2021; Accepted June 9, 2021, Published June 30, 2021



Powdery Mildew (PM) caused by fungal pathogen Oidium neolycopersici (O. neolycopersici) affects both greenhouse and field-grown tomato production. Resistance to PM disease can be achieved by selective inactivation of Mildew Resistance Locus O (MLO) genes encoding heptahelical transmembrane domains, which confer susceptibility to fungal pathogens. Natural loss-of-function mutation is a 19 base pair (bp) deletion in the SlMLO1 gene locus responsible for fungal resistance in S. lycopersicum var. cerasiforme. Introgression of these resistance alleles through breeding into elite varieties is possible. However, this is a long and labour-intensive process and has limitations due to linkage drag. Nonetheless, recent developments in the field of genome editing technology particularly CRISPR/Cas9 systems allows quick, effective and accurate genome modification at the target gene locus. Therefore, it is of interest to determine the efficacy and exact deletion that mimics the natural ol-2 (Slmlo1) mutation present in wild tomatoes using CRISPR/Cas9. 947 putative guide RNAs (gRNAs) were designed using Cas9 variants to broaden Protospacer Adjacent Motif (PAM) compatibility and to enhance DNA specificity against the SlMLO1 locus. 60 out of 947 gRNAs were selected based on the recognition of the PAM sequence, the MIT specificity ranking, the off-target sites, their distance from the 19bp natural ol-2 mutation, the secondary structure of the gRNAs, and their minimum free energy. In depth analysis of these 60 gRNAs helped in the selection of the top five gRNAs based on the above-mentioned criteria. These gRNAs are useful for introducing deletions identical to natural ol-2 mutants and impart resistance against fungal pathogen O. neolycopersici in cultivated tomato crops.



Genome editing, Powdery mildew resistance, CRISPR/Cas9, SlMLO1 locus, Oidium neolycopersici



Prajapati & Nain, Bioinformation 17(6): 637-645 (2021)


Edited by

P Kangueane






Biomedical Informatics



This is an Open Access article which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. This is distributed under the terms of the Creative Commons Attribution License.