Application Of CRISPR/Cas9 System For Chicken And Pig Genome Editing And The Development Of A Novel Seamless Gene Editing Technology

Genome editing, as a technology for editing the biological genome, can delete, insert or modify the specific DNA sequence. However, the spontaneous homologous recombination efficiency is extremely low, which limited the wide application of genome engineering technology. Recently, researchers found that the homologous recombination could improve rapidly when the DNA double strand break was specifically introduced in the target site. The site specific DNA double strand break can be introduced by customer designed nucleases, such as Zinc finger nuclease(ZFN) and transcription activator-like effector nuclease(TALNE). Although they can improve gene editing efficiency significantly, it is a technical challenge to design and construct an active ZFN. TALEN is also tedious to assemble as same as ZFN.CRISPR/Cas9 recently emerges as a new technology of artificial nuclease. It was originated from a immune system of bacteria and archaebacteria, which was composed of cas9 protein and gRNA that recognized the target site. The CRISPR/Cas9 system is extensively used in animals and plants after it was proved to introduce the double strand break in site-specific genome in vitro by Jinek M et al.Successfully, we knocked out genes in chicken DF-1 cell line and pig primary kidney fibroblast by the CRISPR/Cas9 system originated from the Streptococcus thermophilus and coupled with SSA-RPG reporter system, which were all constructed by our team. In addition, we developed a novel seamless genome editing system using a two-step CRISPR/Cas9 system based on HR and SSA repair mechanisms.1. We knocked out EDN3, ATP5 E and OVA genes in DF-1 cell line by the CRISPR/Cas9 system coupled with SSA-RPG reporter vector. We used the SSA-RPG reporter to test the CRISPR/Cas9 activity intuitionisticly and easily in chicken DF-1 cell line. CRISPR/Cas9 and the corresponding SSA-RPG were co-transfected into HEK293 T cells. After transfected 48 h, a considerable fraction of cells expressed Ds-Red and eGFP. This indicated that the CRISPR/Cas9 system can work in both HEK293 T and DF-1 cells. The subsequent T7E1 assay demonstrated that the mutation frequencies within the EDN3, ATP5 E and OVA loci were 0.7%, 0.5% and 3.0% respectively. In order to improve the mutation efficiency, we used the puroR gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The sensitivity of DF-1 cells to puromycin was tested in advance to determine the appropriate concentration of puromycin screening on DF-1 cells. The result showed that the optimal concentration is 3 ?g/mL puromycin and the drug selection lasted for 4 days. After 4 days selection for enriching positive cells, the mutation rate increased to 60.7%, 61.3% and 47.3% via the T7E1 assay, and subsequent sequence analysis showed that the mutation efficiency increased to 95%. In addition, there were no detectable off-target mutations in three potential off-target sites using T7E1 assay. It is suggested the CRISPR/Cas9 combined with SSA-RPG reporter system is a robust tool for chicken genome editing.2. We successfully knocked out RELA gene in pig primary kidney fibroblast by the CRISPR/Cas9 system and SSA-RPG reporter system. Three gRNAs were designed to screen the highest gRNA in the CRISPR/Cas9 system. The result showed that the activity of RELA2.CRISPR/Cas9 is higest in the three sgRNA by comparing the ration of green fluorescence to red fluorescence in HEK293 T cells. Subsequently, the RELA2.CRISPR/Cas9 was used to target the RELA gene in pig primary kidney fibroblast. In order to improve the electroporation efficiency, the Poly-L-glutamic acid or Poloxamer 188 was added in the pulsing buffer. The result showed the electroporation efficiency improved obviously when 4% Poloxamer 188 was added. Using this pulsing buffer, the pig primary kidney fibroblast was transfected with CRISPR/Cas9 and SSA-RPG vectors. At 48 h after transfection, a considerable fraction of cells expressed Ds-Red and eGFP. This indicated that the CRISPR/Cas9 system also worked in pig primary kidney fibroblast. After puromycin screening, the puromycin resistant clones were selected. Sequence analysis showed all the clones were mutant in RELA gene and the biallelic mutant efficiency was 80%. It is suggested the CRISPR/Cas9 combined with SSA-RPG reporter system is a robust tool for pig genome editing and the screening of positive clones. It will lay a foundation for genome editing and breeding.3. Based on the HR and SSA-mediated DNA repair mechanism, we designed a novel seamless gene editing system. We first tried and optimized this novel system with CCR5 gene of the HEK293 T cells. We contructed 2 CIRSPR/Cas9 vectors to target CCR5 and eGFP gene respectively and a HR/SSA-Donor vector that include the TK-PuroR-eGFP cassette with positive and negative marker. The CCR5.CRISPR/Cas9 and Donor was transfected into HEK293 T for the first target. By CRISPR/Cas9 mediated homologous recombination, the donor vector with TK-PuroR-GFP cassette can be integrated exactly into the HEK293 T genome and the SalⅠwas introduced into the target site by HR. The positive clones were screened by puromycin and green fluorescence observation. The subsequent detection showed 100% of the clones were targeted in one allele. Five potential off-target sites were selected for off-target detection. There haven’t any off-target cleavage by sequencing analysis. The positive clones were amplified and transfected with eGFP.CRISPR-Cas9 vector for the second target. The TK- Puro R-GFP cassette was remove seamlessly by SSA mechanism and GCV screening. The 32 bp mutant to ?32/SalⅠin HEK293 T. Sequence analysis showed the mutation rate is 2.08%. Five potential off-target sites were sequenced and no off-target cleavage was observed. It is suggested that it is an effective method for positive clone screening and marker cassette deletion without scar by using a two-step CRISPR/Cas9 system combined with the HR and SSA repair pathway.