Characterizing the in vivo functions of Cyfip1 in the development of cardiovascular, hematopoietic, and nervous systems by precise targeted genome editing in zebrafish
Actin cytoskeleton is the most abundant and crucial protein in most eukaryotic cells, involving a broad range of essential cellular processes. The major regulator for actin dynamics is the WAVE regulatory complex (WRC). The activation and function of the WRC is governed through its own subunit called Cyfip. In vertebrates, there are two Cyfip isoforms, Cyfip1 and Cyfip2, which have been shown to have different expression patterns and distinct functions in various biological systems. However, there is a limited understanding of the in vivo functions of Cyfip proteins in animals, especially in vertebrates. With the development of CRISPR/Cas9-based gene editing technologies and zebrafish as a vertebrate model organism, we have established cyfip1 and cyfip2 knockout mutant in zebrafish using a newly developed and efficient CRISPR/Cas9-based short homology targeted integration strategy named GeneWeld. Together with a novel gene inactivation method called pPRISM-Stop vector, we explored the in vivo functions of each Cyfip isoform in various biological systems, including cardiovascular, hematopoietic, retinotectal, and spinal motor nervous systems. With the high efficiency of the GeneWeld method for precise targeted integration of pPRISM-Stop cassette into each cyfip locus, we were able to recover cyfip1 and cyfip2 germline transmitting adults with on-target integration with frequencies at 13% for both cyfip loci (3/24 for cyfip1 and 2/16 for cyfip2). Despite an unexpected integration of the vector backbone into cyfip2 locus uncovered later, we were able to successfully establish stable lines of true cyfip1 knockout mutant to investigate the phenotypes from its homozygous deletion. Intriguingly, we discovered that cyfip1 abolishment during early stage of development led to mismigration or stalled development of endothelial cells and stenotic vessels accompanied by disrupted blood circulation, substantial reduction of the HSPCs in various hematopoietic tissues, as well as aberrant axon branching and abnormal axon terminals. Taken together, our study demonstrated efficient targeted integration at zebrafish cyfip1 locus using CRISPR/Cas9 short homology targeted GeneWeld strategy and pPRISM-Stop-mediated gene inactivation method to establish, for the first time, stable cyfip1 knockout mutant zebrafish lines to analyze cyfip1 knockout phenotypes and characterize the in vivo functions of cyfip1 in cardiovascular, hematopoietic, retinotectal and spinal motor nervous systems. Although additional samples and further analysis is necessary to make final conclusions, the pronounced morphological and microscopic phenotypes discovered in this study suggested the promising essential in vivo functions of cyfip1 in the development of cardiovascular, hematopoietic, retinotectal, and spinal motor nervous systems, which worth investigating more profoundly to fully characterize the in vivo functions and identify molecular mechanisms of cyfip1, and the WRC-mediated actin remodeling, in these physiological systems
