| β-thalassemia is one of the common single-gene hereditary diseases worldwide.It is hemolytic anemia caused by the reduction or deletion ofβ-globin synthesis caused by the mutation ofβ-globin gene.Children with severe thalassemia begin to develop symptoms about half a year after birth,showing progressive aggravation of anemia,hepatosplenomegaly,infection,developmental delay,cardiac failure,liver failure and other symptoms.Without treatment,most patients die before the age of five.Human hemoglobin is a tetramer composed of twoα-globin like chains and twoβ-globin like chains.It mainly expresses fetal hemoglobin Hb F（α2γ2）during embryonic stage and gradually changes to adult hemoglobin Hb A（α2β2）after birth.Reactivation of gamma-globin expression that has been turned off in patients with beta-thalassaemia may lead to treatment of this disease.BCL11A,as an important transcriptional repressor regulatingγ-toβ-globin switch,is considered to be a target for activatingγ-globin in the treatment ofβ-thalassemia.This paper mainly carried out two parts,one part was based on the reports in previous literature that BCL11A may bind to 3.5kb upstream region of HBD.We used CRISPR/Cas9 lentivirus library to carry out saturation mutation in this region,and screened out sg RNA sites related to high Hb F expression through high-throughput sequencing.Some sg RNAs were selected for functional verification alone.The other part is to edit the BCL11A erythroid specific enhancer and its binding site on theγ-globin gene promoter by single-and double-gene editing,and select the best combination according to the expression level of Hb F for optimized experiment and off-target detection of gene editing.The main results are as follows:1.A CRISPR lentivirus library targeting 3.5kb upstream region of HBD was constructed.After HUDEP-2 cells were infected,high-throughput sequencing was performed on cells with high/low Hb F expression,and Hb F scores related to all sg RNA were obtained.For the 3.5kb upstream region of HBD gene,we designed a total of 1,084 sg RNAs s in all possible directions.The synthesized g RNA was cloned into CRISPR lentivirus vector and packaged into lentivirus after quality control.HUDEP-2 cells were infected with a MOI of 0.4.After proliferation,drug screeing and erythroid induced differentiation,the cells with the highest and lowest expression of Hb F were separated by flow cytometry.After high-throughput sequencing and evaluation,Hb F socre corresponding to each sg RNA was calculated and mapped.The complexity of the 3.5kb region upstream of HBD on globin switch regulation was demonstrated.2.Seven sg RNAs were screened for phenotypic verification in HUDEP-2 cells and CD34+cells,and a large number of HUDEP-2 single-clonal mutant cell lines were constructed and their functions were verified.According to the location of sg RNA on the genome and Hb F score,four sg RNAs were selected for validation in the first batch,among which sg0347 and g0349 were located near the potential BCL11A protein binding motif 2.5kb upstream of HBD gene,and g0801 and sg0877 were located on PYR sequence.We plotted the proliferation curve of lentivirus-infected cells and analyzed the apoptosis rate（for HUDEP-2 cells only）by flow cytometry.sg0347 and sg0349 significantly inhibited the proliferation of both CD34+and HUDEP-2 cells,and almost no on-target mutation was detected.After sequence analysis,it was found that these two sg RNA targeted sites were exactly in the Alu sequence,which resulted in severe off-target and cell apoptosis.The other two sg0801 and sg0877 targeted to the PYR region had a slight inhibitory effect on CD34+and HUDEP-2 cell growth and with low editing efficiency.In edited HUDEP-2 cells,the expression of HBG was elevated by q PCR.For the second batch,we selected 3 sg RNAs according to reads count of sg RNA,namely sg0127,sg0596 and sg0657.After lentivirus-mediated gene editing in CD34+and HUDEP-2 cells,the three sg RNAs had no effect on cell proliferation,and the editing efficiency was high,especially in HUDEP-2 cells,which reached about 70%on-target mutation rate.The erythroid surface markers and apoptosis rate of HUDEP-2cells were detected by flow cytometry,and the m RNA and protein expression levels of globin gene in CD34+and HUDEP-2 cells were detected by q PCR and flow cytometry,respectively.The m RNA and protein levels ofγ-globin gene increased slightly but not steadily without affecting erythroid differentiation.We constructed a large number of HUDEP-2 single-clonal cell lines from the second batch of 3 sg RNAs,and found that some of the single-clonal cells showed a sharp increase in the expression ofγ-globin gene and Hb F protein.Among them,the-6 bp homozygous mutation caused by sg0127 showed high Hb F expression,while the+1 bp homozygous mutation showed low Hb F expression.After transcription factor prediction,we speculated that 6 bp deletion might lead to the binding failure of transcription factor NF-Y,thereby promotingγ-globin expression.3.In two cell models,the promoting effect of double-gene editing on Hb F was better than that of single-gene editing,and no off-target event was detected.We selected sg E1,sg E2 targeting the erythroid specific enhancer h+58 of the trans-transcription factor BCL11A and sg P1,sg P2 targeting BCL11A binding motif on HBG promoter region.After the single-gene editing of HUDEP-2,we selected sg P1and sg E1 with better effect as the combination of double-gene editing,and verified it in HUDEP-2 and CD34+cell models.The results showed that double-gene editing sg P1+E1 had better promotion effect onγ-globin than single-editing sg P1 and sg E1group.After optimizing the editing efficiency of HUDEP-2,the proportion of Hb F expressing cells could reach more than 50%.We performed amplicon deep sequencing and whole genome sequencing on CD34+cells,and no off-target was detected,indicating that the double-gene editing has good safety.In conclusion,on the one hand,this study demonstrated the complex role of this region in globin switch regulation through CRISPR library high-throughput screening and independent validation of 3.5kb upstream of HBD,and found a regulatory site of globin switch that may be involved in transcription factor NF-Y.On the other hand,it was verified that double-gene editing had better promotion effect onγ-globin than single-gene editing,which provided a new strategy for gene therapy ofβ-thalassaemia. |
