Functional study of CDH1 mutations implicated in cleft lip and palate through CRISPR-Cas9 genome editing approach in zebrafish

Abstract

CDH1 encodes the epithelial cadherin, a key molecule to establish the adherens junctions in epithelial cells and also involved in epithelial-mesenchymal transition, a mechanism that plays a central role on the embryogenesis of the lip and palate. We have recently identified, through exome analysis, two missense mutations in CDH1 (c.G760A and c.G2351A) underlying three familial cases of nonsyndromic cleft lip with or without cleft palate (NS CL/P). These mutations have never been described and were absent in 831 Brazilian controls. In silico analyses have indicated that they impair the protein function; however, their pathogenicity lacks confirmation by functional studies. Our main aim to be achieved through this collaboration with Dr. Liao, at the Massachusetts General Hospital, Harvard Medical School, is to test the relevance of CDH1 in the etiology of NS CL/P. To achieve our goals, we will analyze the functional effect of those two CDH1 mutations using zebrafish as a model. A growing number of evidences has showed that zebrafish is a very suitable model to study human genes important for craniofacial development, particularly those related to the development of the bones that constitute mandible, maxilla and palate. We are planning to introduce 2 mutations in CDH1 that completely inactivate the gene (i.e., gene knockout), as well as the 2 missense mutations previously mentioned. The mutations will be created using the CRISPR/Cas9-mediated genome editing approach. This technology has become a promising tool for studying gene functions and the effect of specific mutations on phenotypes, and it relies on the co-injection of the endonuclease Cas9 and a single-guide RNA (sgRNA), which will guide the Cas9 to a complementary target sequence. To accomplish these goals, we organized the schedule in two stages. In the first one, I will design the sgRNA constructs (at Maria Rita's lab, in São Paulo) and, as soon as I get them, I will move to Dr. Liao's lab, where I will spend 3-4 months. During this period, I will first get trained in injections on zebrafish embryos, and then introduce the CRISPR/Cas9 system targeting our candidate mutations. This will give rise to the F0 generation, which is expected to contain mosaic individuals for each of the 4 mutations we have introduced. These fish will take 3 months to grow up and breed, and during this period I will return to Brazil. Our interest is to study the F1 generation, which we expect to contain the heterozygous individuals (with mutations in all the cells of the organism). This F1 generation will be obtained by mating the F0 mosaics with wild types, just after F0 reaches sexual maturity. Once F1 generation will be obtained, they will be screened to detect the mutants. In case these are identified, we will proceed to the analysis of these mutants, which will include phenotypic characterization and morphological analysis.This BEPE application is related to the first 3-4 months of experiments. During the period of growing and breeding of F0, and growing of F1 (5 months approximately), I will return to Brazil in order to finish my other experiments and write the thesis. As soon as the F1's mutants are obtained, I will return to Dr. Liao's lab to start their characterization. For this second stage, as suggested by FAPESP, I will apply for another BEPE or use other resources to accomplish this goal. It is my goal to finish this project still during my PhD and possibly to continue it during my Post-doc training. Dr. Passos-Bueno and Dr. Liao are planning to establish a long term collaboration to functionally study candidates variants for NS CL/P in zebrafish, in order to functionally test the new variants identified by Dr. Passos-Bueno's group at Dr. Liao's lab. (AU)