Optimization Of Gene Knock-in Editing System By MMEJ And Investigation Of The Expression Patterns Of Connexins In Zebrafish

Connexins are structurally related transmembrane proteins that assemble to form vertebrate gap junctions.Connexins mediate the exchange of materials between adjacent cells.They deliver diverse chemical messages into and out of a cell,including ions,metabolites,hormones and other small signaling molecules less than about 1.2 kD in size.To date,the connexins gene family comprise 33 members in the zebrafish.One of the challenge of gap junction in the zebrafish is to study connexins in a physiological environment.The goals of our project are to understand expression patterns and regulatory mechanisms of connexins in vivo.We insert the fluorescent protein into the C-terminal of the connexin to form a fusion protein,and then use a light-sheet microscope to describe the expression profile of the connexin in the body to understand the communication between cells in the body and its related functions.Therefore,it is necessary to establish an efficient,precise and stable gene knock-in editing system.We optimized the microhomology-mediated end joining(MMEJ),our donor vector uses short homology of 25-40 bp and contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms.We succeeded to drive EGFP into targeted genes(Cx43 and Cx43.4)in frame,by MMEJ at 25 bp homology arm and a single strand break in the donor vector(25-single).We observed high rates of germline transmission(50%-57.14%).The 25-single donor vector promotes the precise integration of targeted sites in the genome at high frequency.We successfully constructed konck-in at four zebrafish,Cx45.6-EGFP,Cx47.1-EGFP,Cx28.9-EGFP and Cx39.9-EGFP.Furthermore,we confirmed that this precise genome modification was heritable.We observed high rates of germline transmission(18.8%-77.8%)for targeted knock-in at six zebrafish loci and efficient integration at high frequency.Cx43-EGFP,Cx43.4-EGFP and Cx39.9-EGFP observed fluorescence in the F1 progeny of among these six genes,while the expression of EGFP was not observed in Cx45.6-EGFP,Cx28.9-EGFP and Cx47.1-EGFP.Cx43-EGFP is specifically expressed on notochord and the cornea of the eyes.Cx43.4-EGFP is expressed on the eye retina,notochord and hatching glands.Cx39.9-EGFP is specifically expressed on the muscle cell membrane,and it is significantly enhanced in the sarcomere area.Cx45.6-EGFP,Cx28.9-EGFP and Cx47.1-EGFP were expressed in the juvenile stage by the mRNA expression level.The EGFP inserted the C-terminus didn’t affect the expression of the target gene,and the expression of fluorescent mRNA was about nonfluorescent ten times.It was preliminarily proved that fluorescence was not observed due to low background expression.In summary,we designed a 25-single donor plasmid based on MMEJ.This simple method enables precise targeted gene knock-in in zebrafish with a germline transmission rate(19-78%).We successfully constructed 6 Cx gene knock-in zebrafish,and collected 3 Cx expression profiles.