Screening Essential Genes For The Proliferation Of Cashmere Goat Dermal Papilla Cells By CRISPR Library

Dermal papilla cells(DPCs)are special fibroblasts,which exist at the bottom of hair follicles.The mechanism of cashmere growth and periodic development in cashmere goats is highly related to the proliferation of DPCs,and this process is regulated by several genes.Studies have shown that Hoxc13 gene has an important role in controlling hair formation,and it is mainly expressed in DPCs.Mice treated with Hoxc13 overexpression or defect show abnormal hair growth.At present,a large number of candidate genes related to cashmere fiber growth and periodic development have been identified by transcriptomic or other methods,however,the number of gene list is large,bringing inconvenience for the selection key regulatory genes for the following functional studies.Therefore,it is necessary to innovate in the screening method.CRISPR-based screening system can achieve multi-target genome editing,and has been widely used in functional genomics,including the screening of genes related to cell resistance and cell proliferation.Therefore,in this study,we used CRISPR focused library to achieve gene knockout in Shanbei White Cashmere goat,and then identified the essential genes for the proliferation of DPCs.The test process and specific results are as follows:(1)Design and construction of a focused CRISPR library.A total of 822 differentially expressed genes in cashmere goat skin and hair follicles identified in our laboratory and published on NCBI were used to construct the library.The library contains 4,910 sg RNA,of which 40 are negative control sg RNA.and the other 4,870 sg RNA target 822 protein-coding genes respectively.The quality of the library was evaluated by high-throughput sequencing technology,and it was found that 85% of the sg RNA in the library had the best GC content;97.85% of sg RNA targeted exons common to all Ref Seq isotypes;86% of sg RNA targeted the 5 ’mRNA.These results show that the CRISPR focused library has high editing activity and can be used for genetic screening.(2)Identification of DPCs derived from cashmere goats.The cells,observed under the microscope showed that the cells had the characteristics of agglutinative growth,flattening and polygonal;immunofluorescence staining showed that α-SMA and Vimentin dermal papilla cell molecular markers were positive,indicating the DPCs can be used for CRISPR screening.(3)Screening of essential genes for proliferation of DPCs.DPCs was infected with lentivirus library under the condition of MOI 0.5,after 48 hours,puromycin was used for screening.The surviving cells were collected on the 0th day(D0),9th day(D9)and 15 th day(D15)for high-throughput sequencing.The essential genes for DPCs proliferation were screened by high-throughput sequencing and bioinformatics techniques,and a total of 57 candidate genes were obtained.Combined with GO analysis and KEGG analysis,it was found that these genes were mainly enriched to cell growth,cell cycle and apoptosis.(4)Functional verification of essential genes.Among the top 10 essential genes,SBDS,HJURP and FNDC5,were selected as candidate genes for functional identification.siRNA was used to interfere the expression of the SBDS,HJURP and FNDC5 genes in DPCs,and the effect on the proliferation of DPCs was detected by CCK8 proliferation test,EDU and flow cytometry.The results showed that interfering with the expression of SBDS gene and HJURP gene inhibited the proliferation of DPCs,while interfering with the expression of FNDC5 gene promoted the proliferation of DPCs.In this study,DPCs was isolated and purified from the back skin of Shanbei White Cashmere goat.A total of 822 differentially expressed genes in cashmere goat skin and hair follicles identified in our laboratory and published data on NCBI were used to construct the library.we used this library to achieve gene knockout in Shanbei White Cashmere goat,and then identified the essential genes for the proliferation of DPCs.Based on the negative screening strategy,high-throughput sequencing and bioinformatics techniques were used to screen and identify the essential genes underlying proliferation in DPCs.This study provides a new method for further mining and identification of key genes,which have important research and breeding value in cashmere goats,and further provides a reference approach for the study of functional genomics in livestock.