CRISPR-RNP Technology-mediated Knock-in Of Egfp Into β-actin Site Of Chicken DF-1 Cells

The Genomic Safe Harbors（GSHs）means some sites into which foreign genes are integrated to maintain stable expression without abnormal regulation on the structure and function of endogenous genes.So far,CRISPR/Cas9 knock-in technology has been successfully applied in the transgenic research of many kinds of mammals.Although CRISPR/HDR（Homology Directed Repair）solves the problem of random integration of foreign genes,it also results in lower expression level of transgenes because of position effect and changes of gene copy number.Some GSHs,such as Rosa26,β-actin and so on,have been found in the mammalian genome,and these harbors have been used for the precise knock-in of transgenes.However,the application of CRISPR/Cas9 technology is restricted in the field of transgenic poultry because of the little knowledge about poultry GSHs.As to knock-in efficiency of a transgene and host safety,CRISPR/Cas9 knock-in mediated by ribonucleoprotein（RNP）,formed by single guide RNA（sg RNA）and Cas9protein,is much better than that mediated by plasmids or virus vectors.The purpose of this study was to have enhanced green fluorescent protein（EGFP）knocked into the intron region ofβ-actin gene in chicken fibroblasts（DF-1）by CRISPR-RNP technique,so that the EGFP could be expressed under the regulation of endogenousβ-actin promoter,thus providing basic data for verification of chickenβ-actin gene as a GSH,and laying a technical foundation for transgenic chicken research.1.Prokaryotic expression and purification of His-Cas9 fusion protein and its application in the verification of chickenβ-actin sg RNA activityThe recombinant plasmid p ET-NLS-Cas9-6×His was transformed into Escherichia coli BL21.The expression of His-Cas9 fusion protein was induced by IPTG,and the concentration of IPTG,induction temperature and induction time were optimized.Soluble expression of His-Cas9 fusion protein was determined firstly,then His-Cas9 protein was purified by Ni-NTA argose medium under natural conditions.Based on the DNA sequence in intron region of chickenβ-actin,4 sg RNAs were designed by online tool（https://zlab.bio/guide-design-resources）.And double-stranded DNA（ds DNA）was cleaved by RNP to verify the cleavage activity of His-Cas9 protein,and the suitable sg RNA was screened.The results showed that:（1）The best induction condition is 0.5 m M IPTG,at 18℃for 16 h.（2）The His-Cas9fusion protein without unspecific protein can be obtained by elution of 250m M imidazole.（3）The activity of the purified His-Cas9 fusion protein was similar to that of a commercial Cas9 protein.（4）Four sg RNAs were obtained by in vitro transcription to recognize the upstream 20～200 bp intron region of chickenβ-actin initiation code,and all of them could specifically guide the His-Cas9 fusion protein to cleave the corresponding ds DNA.（5）The activity of sg RNA3 was the highest（99.7%）with the strongest specificity.2.CRISPR-RNP technology mediated knock-in of EGFP into chickenβ-acin site in vitroThe left and right homologous arms（LHA,RHA）and EGFP fragments were cloned by PCR respectively.The recombinant plasmid p MD^TM-18T-LHA-EGFP-RHA was constructed by overlapping PCR and TA cloning techniques,and the ds DNA donor template was amplified using the plasmid as a template.Single-Stranded DNA（ss DNA）donor was prepared by phosphatase digestion.The CRISPR-RNP element was transfected into chicken DF-1 cells by the transfection reagent CRISPR-Fectin^TM and electroporation respectively,and the transfection conditions were optimized.The average fluorescence intensity was calculated by Image J,the positive rate of EGFP was analyzed by flow cytometry,the EGFP integration was analyzed by genotyping,and the expression of EGFP was verified by Western Blot.Finally,five potential off-target sites were analyzed by T7E1 enzyme.The results show that:（1）The ss DNA donor template of EGFP reporter gene was successfully prepared.（2）The transfection reagent CRISPR-Fectin^TM did not get satisfactory results.（3）Pulsing 1 time at 170V for 3ms is a suitable electroporation condition for chicken DF-1 cells,which brought about 11.8%of positive rate and 50.963 of average fluorescence intensity;the positive cell rate increased with the raising amount of ss DNA donor.（4）Genotyping analysis indicated that EGFP was accurately knocked into the target harbor.（5）Western Blot detection showed that EGFP was expressed in chicken DF-1.（6）No off-target phenomenon was found.In brief,the His-Cas9 fusion protein with the same activity as commercial Cas9 protein was obtained in this study,and EGFP was accurately knocked into theβ-actin intron of chicken DF-1 cells by CRISPR-RNP gene editing technique,which drives the expression of EGFP reporter gene under the action of endogenous promoter.