Development of safe harbor CRISPR inhibition and activation tools for systematic biological inquiry in zebrafish

About This Grant

Summary – We will establish and validate stable system for CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) in zebrafish, employing attP safe harbor PhiC31 integration sites. The application of CRISPRi and CRISPRa technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. It will afford the modulation of promoters and regulatory sequences alike, facilitating efficient loss of function and gain of function evaluation of biological and pathological relevance. We recently developed codon-optimized CRISPRi/a constructs for zebrafish, establishing their function in proof-of-principle experiments, using RNA injection of system components to modulate key genes in established phenotypes. We synthesized a zebrafish codon-optimized cas9 gene, harboring mutations D10A and D839A to render the protein catalytically inactive (dCas9). We then cloned codon-optimized Krüppel associated box (KRAB) and methylated CP2 (MeCP2) inactivating domains or VP64 activator domain downstream from dCas9 for CRISPRi and CRISPRa, respectively. To validate the biological function of our initial CRISPRi construct, we targeted the promoter sequences of key genes in melanocyte differentiation (sox10, mitfa, and mitfb); and melanin production (tyrosinase; tyr). Microinjection of CRISPRi mRNA with single guide RNAs (sgRNAs) targeting their promoters resulted in hypopigmented larvae (epidermal melanocytes and retinal pigmented epithelium. In addition, we evaluated CRISPRi/a modulation of mrap2a, which controls energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene (mc4r). Targeting the mrap2a promoter proximal region with CRISPRa or CRISPRi increases and decreases larval body length, respectively. However, RNA-based injections inherently display time-limited effects whose impact is unreliable beyond the development. Thus, here we propose to establish transgenic lines, stably expressing CRISPRi and CRISPRa from constructs integrated into attP safe harbor integration sites in chromosome 14 facilitated by the PhiC31 integrase (Aim 1). We will screen the efficacy of these new lines via Tol2-mediated delivery of sgRNA expression constructs directed at known genes whose modulation yield readily scored phenotypes, as above. Similarly, we will establish and validate constructs for efficient delivery of sgRNA expression to an alternate attP PhiC31 site in chromosome 24 (Aim2). The lines expressing CRISPRi/CRISPRa will be crossed with the empty alternate PhiC31 site, allowing the use of the second site for targeted integration of sgRNA expression constructs. The establishment of lines established in Aims 1 and 2 will be confirmed by the inclusion of discrete reporter cassettes expressing Cerulean [blue, Aim 1] and Venus [yellow, Aim 2] in the pineal and the lens of the eye, respectively. The efficiency of lines generated in aim 2 will be evaluated using guides designed to the promoters of genes employed in Aim 1 validation. Robust CRISPRi and CRISPRa systems in zebrafish will facilitate efficient assay of candidate gene function and disease relevance through bidirectional modulation of gene expression.