In-silico Analysis for Prediction of Mutational Position and Designing of sgRNA for HDT701 Gene in Indian Rice cv. RPBio-226

Abstract

Background: In eukaryotes, Histone acetylation and deacetylation plays a prominent role in controlling gene expression and chromatin structure modifications. Both Histone acetyltransferases (HAT) and Histone deacetylases (HDACs) work in opposition to regulate chromatin acetylation. Reduced levels of histone H4 acetylation and increased susceptibility to the rice disease M. oryzae result from overexpression of HDT701 in rice. By changing the levels of histone H4 acetylation in defense-related and pattern recognition receptor (PRR) genes in rice, HDT701 reduces the activity of innate immunity which promotes resistance to M. oryzae. Crispr cas9 technology was created to change genes and modify characteristics, as well as to produce resistance to a variety of infections by focusing on possible biomolecules involved in plant defense mechanisms. Therefore the present study was aimed to design single guided RNA (sgRNA) and predict the gene mutational position in the HDT gene in RPBio-226 rice cv. Methods: To begin with, the DNA was isolated using CTAB method. Specific pair of primers was designed from the reference gene for amplification of HDT gene. In addition to, the PCR product was sequenced and the resulting sequence was applied to the creation of sgRNA. Furthermore, CHOPCHOP is a Bioinformatic search tool used to identify CRISPR–Cas single guide RNA (sgRNA) targets. Result: The isolated genomic DNA was quantified using nanodrop and found that the concentration of the DNA was 800-1000ng/ul with the purity of 1.8. The full gene was amplified with OsHDT701 gene primers and sequenced. Based on the OsHDT701 gene sequence, oligo single guide RNA (sgRNA) was generated by using the http://chopchop.cbu.uib programme. The target site for designing sgRNA was found from 168 basepair to 190 basepair with the deletion of a nucleotide at 174th position.