Creation of Advance Construction of CRISPR/Cas9 via using Multi Round Gateway Cloning

Abstract

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated system) is used to edit specific genomic sequences with precision and efficacy. CRISPR/Cas9 system mediated double strand binding site through error-prone non homologous end joining repair in targeted sequences, which the deletion or insertion can be achieved by knock out gene. Creation advance construct of CRISPR/Cas9 of soluble starch synthase II-1, 2 and 3 (SSSII) genes in single destination binary vector through multi round LR reaction of Gateway technology. Gateway cloning system developed the suitable for the site-specific DNA recombination of lambda-bacteriophage and ccdB the cytotoxic protein, which is poisonous to most E. coli strains. A genetic transformation vector designed with appropriate gRNAs, Cas9, and antibiotic resistance was used to create SSSII knockout mutants.