577. Empower Multiplex CRISPR-Mediated Gene Manipulation with Self-Cleaving Ribozymes and tRNA

Abstract

Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has been recently introduced as an efficient tool to edit the genome. Moreover, it can be targeted to specific gene loci by using single guide RNA (sgRNAs) for genetic manipulation and potential therapy. However, its targeting capability is often restricted by (gRNA) and generation of large deletions in genome by CRISPR requires expression of two distinct guide RNAs simultaneously. Here we report an innovative modification to increase the CRISPR/cas9 editing efficiency in the form of Ribozyme-flanked guide RNA/CRISPR system, which expresses two gRNA in equal molar amount. Our results show that this approach can be used to generate large DNA deletion with transfection of only one plasmid in human and mouse cells. Furthermore, we also used this system to target multiple transcriptional activators to a single promoter and the results showed that RNA level of targeting gene was statistically significantly up-regulated via Ribozyme gRNA/hCas9. Taken together our data, we prove that this strategy can be used in gene editing study broadly to enhance the targeting and the editing efficiency of CRISPR system.