Biomedical Engineering and Bioengineering Commons^™

Guide Rnas Preparation For In-Vitro Crispr-Cas9 Complex Delivery Targeting Genes That Affect Wound Healing., Prashant Khatiwada

Biomedical Engineering Undergraduate Honors Theses

CRISPR-Cas9 technology has widely been used as a viable genome engineering platform to make site-specific insertion, deletion, and breaks. The nuclease dead version of Cas9 or dCas9 can be used for the activation and repression of target gene sites using specific activation or repression domains. In this study, CRISPR guide RNAs were designed for a CRISPR inhibition approach to repress the transcriptional activity of the target genes. An expression plasmid vector composed of a U6 promoter sequence, BbsI restriction sites, and a chimeric gRNA sequence was digested, and the phosphorylated forward and reverse gRNAs were ligated with the plasmid vector. …