A modified CRISPR system for transcriptional activation of tilapia endogenous genes


SOCIETY FOR INTEGRATIVE AND COMPARATIVE BIOLOGY
2021 VIRTUAL ANNUAL MEETING (VAM)
January 3 – Febuary 28, 2021

Meeting Abstract


P9-6  Sat Jan 2  A modified CRISPR system for transcriptional activation of tilapia endogenous genes Kim, C*; Kültz, D; Universitiy of California, Davis; UC Davis cshkim@ucdavis.edu

Recently, the CRISPR system has been repurposed for endogenous gene activation or repression by introducing a catalytically inactive form of Cas9 (dCas9) followed by fusions of the dCas9 to effector domains such as VP64 (activation), p300 (activation + epigenome editing) and KRAB (repression). This modified CRISPR system provides a simple and versatile tool for RNA-guided manipulation of endogenous gene expression. As a proof of principle for this system in a fish model we focused on developing CRISPR activation (CRISPRa) in the tilapia (Oreochromis mossambicus) OmB cell line to implement this promising approach. Building upon our previous CRISPR optimization with two different tilapia endogenous promoters used in both Cas9 and gRNA expression plasmids, the original Cas9 part in the plasmid was replaced by dCas9 (D10A, H840A) and the tilapia p300core. The p300core-mediated CRISPRa can target the specific promoter or even enhancer regions of a gene of interest due to the chromatin state modification ability of p300 which has histone acetyltransferase activity. The tilapia inositol monophosphatase 1 gene (IMPA1.1) and its promoter region were selected as target gene and gRNA target region, respectively. By targeting the IMPA1.1 promoter region (400 bp) using 6 different gRNAs with transient transfection of OmB cells, three gRNAs showed an increase in IMPA1.1. promoter-driven luciferase reporter expression and in mRNA levels of IMPA1.1 by 4- to 6-fold in regular medium. These results indicate that the p300-CRISPRa system enables efficient target gene activation with appropriate target-specific gRNAs in tilapia cells. To control for off-target effects in analyzing IMPA1.1-dependent phenotypes, non-target gRNAs will be used. This work provides the basis for quantitative proteomics to track changes in molecular phenotypes in response to targeted IMPA1.1 gene transcriptional activation and resulting cellular phenotypes.

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