| Objective:CRISPR/Cas9 is a commonly used gene editing technology that has been widely used in the study of gene editing in mouse and human cells.Primary hematopoietic stem cells(HSCs)are difficult to be infected by exogenous genes carrying large fragments due to their scarce cell number,so gene editing in primary HSCs is very difficult.In this topic,we mainly use CRISPR/Cas9 technology to establish the methodology of gene editing at the HSC level.Methods:Cas9-EGFP/Cre-ER transgenic mouse models with inducible expression of Cas9 protein were first generated by crossing Cas9-EGFP knockout mice with Cre-ER mice.Primary HSCl as well as HSC2 cell cells from this mouse were sorted using flow cytometry and infected using sgPax5-Crimson lentivirus to knock out the Pax5 gene,and the results showed that the infection efficiency of the lentivirus was able to reach 95%.The infected cellular DNA was subjected to targeted sanger sequencing,which showed a knockout efficiency of 97.85%.The infected cells were then transplanted into lethally irradiated recipients to examine the effect of HSC knockout of the Pax5 gene on reconstitution and lineage differentiationResults:1)Cas9-EGFP knock-in mice were first backcrossed with C57BL/6J(CD45.2)for ten generations to ensure a pure CD45.2 genetic background,and then crossed with Cre-ERT2(CD45.2)mice to finally obtain Cas9fl-Cre+/-positive offspring mice.2)Two weeks after Tamoxifen injection,only myeloid cells in peripheral blood could express EGFP;at four weeks after induction,lymphoid cells,myeloid cells,and platelets in peripheral blood could express a certain proportion of EGFP in addition to mature erythrocytes;at the 8th week after induction,the expression of EGFP in peripheral blood lineage cells was in a stable state.3)According to the results of single-cell RNA sequencing and single-cell transplantation,it was found that HSC2 is a group of hematopoietic stem cells with multi-lineage differentiation ability but lymphoid-bias,which plays an important role in the generation and development of lymphoid cells.4)HSCs(HSC1 and HSC2)were infected in vitro with sgPax5/sgScramble-Crimson lentivirus for 72 h.The infection efficiency of HSC1 Pax5 knockout and control group was 69.0%±21.0 vs.74.8%± 13.6,respectively;the infection efficiency of Pax5 knockout control group of HSC2 was 51.9%± 33.2 vs.61.4%± 17.7,respectively.5)After 48 hours of in vitro infection of HSCs with sgPax5/sgScramble-Crimson lentivirus,they were transferred to co-cultured with OP-9 mouse stromal cells for 7 days.Whole genomic DNA of infection-positive cells was extracted,and Pax5-targeted sequencing was performed.The results showed that the editing efficiency of CRISPR/Cas9 was about 92%.6)The total reconstitution rate in the peripheral blood of Pax5 knockout recipient mice was lower than that of the control group,in which myelocytes and T cells were able to reconstitute normally,while mature B cells were lacked in the peripheral blood.There is no big difference between HSC1 and HSC2 group.7)After Pax5 knockout,the proportion of B cells in the bone marrow of recipient mice was significantly increased,of which pro-B(45.3%±15.7 vs.7.0%± 12.6,P<0.01)was predominant;the proportion of T lymphocytes and myeloid cells did not change greatly compared with the control group;in the level of hematopoietic progenitor cells,a significant increase in the proportion of MPP3(0.49%± 8.2 vs.2.8%± 25.7,P<0.01)occurred;there was no significant effect at the level of hematopoietic stem cells.There is no significant difference between HSC1 and HSC2 group.Conclusions:1)An efficient and convenient gene knockout method at the level of a small number of highly purified HSCs was established by optimizing the in vitro culture system of HSCs and simplifying the exogenous plasmid size.2)Based on this approach,this study successfully knocked out Pax5 at the level of mouse bone marrow primary HSCs,resulting in the arrest of Pro-B in the bone marrow and the absence of mature B cells in the peripheral blood,consistent with the results of previous studies. |
