CRISPR/CAS9 Technology Inhibited The Expression Of BCL11A Gene And The Construction Of Its DNA-binding Region SgRNAs Library

Objective:CRISPR/Cas9 technology was used to inhibit the expression of BCL11 A,and explore the effect of BCL11 A on the expression of g-amma globin gene.The sgRNAs lentivirus library in the DNA binding region of γ-globin gene transcription repressor BCL11 A was constructed,laying the experimental foundation for further screening the accurate DNA binding sites of BCL11 A.Methods:1.Apply online design software(http://chopchop.cbu.uib.no),design two sgRNAs of BCL11 A enhancer+58.2.To construct lentiviral recombinant plasmid vector,which was identified by enzyme digestion and verified by Sanger sequencing;The experiment was divided into three groups: sgRNA1,sgRNA2 and blank.3.Packaged lentiviral plasmid vector: the lentiviral recombinant plasmid vector and the packaged plasmid were transfected into HEK293 T cells to concentrate the virus.4.Three groups of K562 cells were infected with lentivirus,positive infected cells were screened,and DNA was extracted for PCR amplification.The amplification product was sent to Sanger sequencing,the m-utant activity was determined by T7E1 enzyme,and TA vector was connected for monoclonal sequencing.5.Positive infected cells were induced by hemin for 0,2,4 and 6 days,and the expression of BCL11 A and γ-globin gene in each group was detected by real-time fluorescence quantitative PCR.6.Use CRISPR gRNA-design software(CRISPRwiz)to design all possible sgRNAs within a 3.5kb region at the 5’ upstream of human delta globin gene as well as synthesize sgRNAs library.7.LentiCRISPRv2 vector was recombined with the enriched sgRNAs library to construct the sgRNAs plasmid library.Sequencing quality control of the plasmid library through NGS(" next-generation " sequencing technology).8.Qualified plasmid library was tested and packaged into lentivirus.Results:1.Two lentiviral recombinant plasmid vectors were verified by enzyme dig-estion and identified by sequencing,and the recombinant vector was constructe-d successfully.2.A set of peaks arise in the positive infected cells of SgRNA2 suggest that they were edited.The results of T7E1 enzyme digestion showed that the infected cells of SgRNA2 has mutated activity,monoclonal sequencing linked to TA vector showed that gene editing produced a 463 bp deletion.3.SgRNA1 group showed no mutation activity.4.Positive infected cells in sgRNA2 were induced by hemin for 6 days,and the real-time fluorescence quantitative PCR results suggested that reduce the level of BCL11 A expression in positive infected cells resulted in a increase of γ-globin gene expression.5.The lentiviral library of sgRNAs within a 3.5kb region at the 5’ upstream of human delta globin gene was constructed.Conclusion: 1.The BCL11 A enhancer+58 of K562 cells was successfully edited and mutated.2.After editing BCL11 A gene,the expression of BCL11 A was inhibited in K562 cells after erythroid induction,and the expression of γ-globin gene was increased.3.The lentiviral library of sgRNAs within a 3.5kb region at the 5’ upstream of human delta globin gene was obtained.