Knock Out PPARγ Gene Of Chicken Cells By CRISPR/Cas9

PPARγ has been proved to control many genes involved in fat cell differentiation and the lipid metabolism, and is widely involved in fat absorption, transport, generation and decomposition in the study of mammal, However, it is not be deeply studied in chicken.CRISPR/Cas9 system can target to a specific genome locus by a Cas9 protein and a guide RNA(gRNA) including 20 nt sequence that binds to the target DNA sequence adjacent to the PAM. Then it can connect nicks effectively with intracellular DNA repair mechanisms so as to edit the endogenous gene efficiently. This study targeted the PPARγ gene in the genome of chicken DF-1 and preadipocytes using CRISPR/Cas9 system, to lay the foundation for studying the function of PPARγ in the fat cell differentiation and production of transgenic chicken.1. In order to target the PPARγ gene in chicken DF-1 and preadipocytes using CRISPR/Cas9 system, this study designed a sgRNA via http://crispr.Mit.edu of MIT by taking PPARγ exon4 as template and constructed pll3.7-U6-sgPPARγ-CMV-spCas9(sgPPARγ-Cas9) expression vector and surrogate reporter system of pB-CMV-DsRed-CAG-PPARγ.200 bp repeat.Puro-T2A-GFP(RPG-PPARγ) successfully to target PPARγ gene.2. In order to verify the target efficiency of the CRISPR/Cas9 system in DF-1 cellline,we con-transfected the sgPPARγ-Cas9 expression vector and RPG-PPARγ reporter vector to the DF-1 cell line, At 48 h after transfection, a considerable fraction of cells expressed Ds-Red,and a small portion of cells expressed eGFP in the experimental group, whereas the cells only expressed Ds-Red in the control group. This indicated that the CRISPR/Cas9 system worked in DF-1 cells in reporter vector level; the subsequent T7E1 assay demonstrated the mutation rate was only 0.75%. This indicated that the CRISPR/Cas9 system worked in DF-1 cells in genome level; At the same time, the transfected cells were selected by 3μg/m L puromycin for4 days to get the positive cells, the target sequence of the selected cells were subjected to T7E1 assay and the mutation increased to 60.7%; and subsequent sequence analysis showed that the mutation efficiency increased to 95%. And there was no detectable off-target mutation of three potential off-target sites according to T7E1 analysis in our study, suggesting the CRISPR/Cas9 system is a robust tool for chicken genome editing.3. With the purpose of studying the function of PPARγ gene, we targeted PPARγ gene with CRISPR/Cas9 system in chicken preadipocytes. Using opti-pulsing buffer, the sgPPARγ-Cas9 expression vector and RPG-PPARγ reporter vector were con-transfected to the chicken preadipocytes with the condition of 280 V 1mS 1time, At 48 h after transfection, a considerable fraction of cells expressed Ds-Red, and a small portion of cells expressed eGFP in the experimental group. And then selected by 1.5μg/mL puromycin for 3 days, The mutation rate of the selected preadipocytes is 33.1% via T7E1 assay and 45% by sequencing,indicating that this system work in the chicken fat cell.Thus, this research contributes to building CRISPR/Cas9 system to edit the chicken gnome, to study the role of PPARγ in the formation and differentiation of chicken fat cells via PPARγ gene knockout individual, the results can provide theoretical basis for breeding to improve poultry meat quality traits.