The Effect of Transcriptional Activators via a Blue Light-Inducible CRISPR-Cas9 System

Abstract

Increasing interest in genetic engineering has warranted the need for highly dynamic control of gene upregulation, which has resulted in improvements to the conventional CRISPR activation system (CRISPRa). One of these improvements, the light-inducible CRISPR activation system (LICAS) utilizes light-inducible heterodimers CIBN and CRY2FL fused to catalytically deactivated Cas9 (dCas9) and a transcriptional activator VP64. LICAS dynamically regulates endogenous gene expression in the presence of blue light. Recently, researchers developed the tripartite transcriptional activator VPR (VP64-p65-Rta), which showed higher levels of transcriptional gene activation than VP64 when utilized for the conventional CRISPRa system. However, VPRs activation levels for LICAS are yet to be determined. Therefore, in this study, we investigated 2 VPR and VP64 in CRISPRa and LICAS to compare their gene activation levels. First, we investigated dCas9-VPR and dCas9-VP64 in CRISPRa by targeting the Interleukin 1 Receptor Antagonist (IL1RN) gene. We confirmed previous studies contending that VPR induced higher gene activation than VP64 for IL1RN. In LICAS, when we compared VPR with VP64 by fusing each transcriptional activator to a full-length cryptochrome (CRY2FL), higher gene activation was observed via CRY2FL-VP64. Nevertheless, we demonstrated that our novel CRY2FL-VPR construct succeeded in activating the IL1RN in LICAS. However, its activation efficiency was relatively weak compared to CRY2FL-VP64. Despite this fact, we proved that VPR activator domains can be fused to CRY2FL without deactivating its ability to complex and function with sgRNAs in human cells. With further investigation, the CRY2FL-VPR design may propel improvements to dynamic endogenous gene activation methods for genetic research and therapy. Keywords: CRISPR-Cas9, CRISPR