针对HLA-I类分子高效基因编辑方法的建立及其应用

Establishment and Application of Efficient Gene Editing Method for Classical HLA-I Molecules

目的:建立一种HLA-I类分子高效基因编辑的方法,以制备编辑HLA-I类通用型造血干细胞。方法:针对β2微球蛋白基因设计并合成sgRNA,将NLS-Cas9-NLS核酸酶与sgRNA按照不同的摩尔比(1∶1-1∶4)形成RNP复合物,分别设置对照组和四个转染组,通过核转染方式转入HEK-293细胞或者CD34+造血干细胞。培养72 h后,应用流式细胞术检测转染细胞表面HLA-I类分子表达情况,T7E1酶切反应鉴定切割作用,DNA直接测序方法鉴定序列套峰出现情况。结果:流式细胞术检测发现转染后HEK-293细胞或者CD34+造血干细胞有不同程度的HLA-I类分子阴性表达细胞群,直接测序不同转染组在sgRNA PAM序列附近均有明显套峰出现。在HEK-293细胞转染中,NLS-Cas9-NLS核酸酶与sgRNA摩尔比为1∶4时HLA-I类分子阴性表达细胞群比例最高(87.69±0.83)%,摩尔比为1∶3时T7E1酶切割效率最高(38±2.0)%。在CD34+造血干细胞转染中,NLS-Cas9-NLS核酸酶与Easyedit sgRNA摩尔比为1∶2时,HLA-I类分子阴性表达细胞群比例为(91.56±3.39)%,摩尔比为1∶1时T7E1酶切割效率为64±8.45%。结论:本研究提供了一种针对HLA-I类分子进行高效基因编辑的方法,该方法可以有效地将细胞表面的HLA-I类分子进行沉默表达,并成功应用于编辑CD34+造血干细胞。

Objective:To establish an efficient gene editing method of HLA-I gene to prepare HLA-I universal hematopoietic stem cells.Methods:The easyedit small guide RNA(sgRNA)was designed according to the sequences ofβ2 microglobulin gene and synthesized by GenScript company.RNP complexes were formed by NLS-Cas9-NLS nuclease and Easyedit sgRNA according to different molar ratios(1∶1~1∶4).Control group and four transfection groups were performed respectively.HEK-293 cells and CD34+hematopoietic stem cells were nucleotransfected with RNP complex by Lonza 4D Nucleofector system.The expression of HLA-I on the surface of HEK-293 cells was detected by flow cytometry after transfection for 72 hours,the cleavage effect was determined by T7E1 enzyme digestion reaction and the presence of nested peak in the DNA sequence was identified by direct sequencing.Results:The transfection groups had different levels of HLA-I negative expression cell populations by flow cytometry after transient transfection of HEK-293 cells and CD34+hematopoietic stem cells with different molar concentrations of RNP complex for 72 hours.There were nested peaks proximal to the sgRNA PAM sequence in the transfection groups by direct DNA sequencing,indicating that sgRNA had obvious editing effect.In the transfection of HEK-293 cells,the highest proportion of HLA-I negative expression cells was(87.69±0.83)%when the molar ratio of NLS-Cas9-NLS nuclease to Easyedit sgRNA was 1∶4.The cutting efficiency of T7E1 was the highest up to(38±2.0)%when the molar ratio was 1∶3.In the transfection of CD34+hematopoietic stem cells,the proportion of HLA-I negative expression cells was(91.56±3.39)%when the molar ratio was 1∶2,and the cutting efficiency of T7E1 was(64±8.45)%when the molar ratio was 1∶1.Conclusion:This study provides an efficient gene editing method for classical HLA-I molecules,which can effectively silence the expression of class HLA-I molecules on the cell surface,and is suitable for stem cell system with difficult transfection.