Preliminary Study On Gene Editing Of Human Primary Lymphocytes Using CRISPR Technology

Objective:To perform double knockout of PD-1 and CTLA-4 genes in human primary lymphocytes cultured in vitro using the current efficient and rapid CRISPR technology.Method:1.Three sets of candidate sgRNA sequences were designed for each of the CTLA-4 gene and the PD-1 gene,and CRISPR-CTLA-4 and CRISPR-PD-1 knockout plasmids were constructed accordingly.The editing efficiency of each sgRNA was verified using the 293 T cell line.A CRISPR-CTLA-4-PD-1 double knockout plasmid was constructed from the two sgRNAs with the highest editing of CTLA-4 and PD-1 genes.2.Using the method of electroporation,an attempt was made to transfect the CRISPR-CTLA-4-PD-1 double knockout plasmid into human primary lymphocytes.In order to achieve the standard of electroporation transfection of primary lymphocytes,various conditions of electroporation were explored and optimized.3.Using the PCR amplification method,the CTLA-4-PD-1 fragment was amplified from the CRISPR-CTLA-4-PD-1 double knockout plasmid,and the fragment was ligated to the adenovirus expression vector to try to pass Construction of CRISPR-CTLA-4-PD-1 adenovirus infected human primary lymphocytes.4.Using the CRISPR/RNP protein complex targeting CTLA-4 and PD-1 genes,electroporation was transfected into human primary lymphocytes using optimized electroporation conditions,and observedafter transfection.The situation of gene editing and the killing effect of lymphocytes on K562 cell line after transformation.Result: The CRISPR-CTLA-4-PD-1 double knockout plasmid was successfully constructed and verified to play a gene editing role in 293 cells.The establishment of electroporation conditions for primary lymphocytes,and the first comprehensive analysis of various factors of electrotransformation,found different voltage,pulse time,electroporation buffer,cell culture state,plasmid size and purity on transfection efficiency and cells The live rate has a significant impact.Among them,the positive GFP plasmid electroporation transfected human primary lymphocytes with an efficiency of 34%.An adenovirus expression vector carrying the CRISPR-CTLA-4-PD-1 system was successfully constructed.The infection efficiency of human primary lymphocytes infected with positive adenovirus GFP reached 29.6%.The CRISPR/RNP protein complex was successfully prepared and transfected into lymphocytes,and CTLA-4 and PD-1 gene editing of lymphocytes were achieved.The modified lymphocytes were stronger against K562 cell line.Killing ability.Conclusion: The CRISPR-CTLA-4-PD-1 double knockout plasmid was constructed and the conditions of electroporation transfected human primary lymphocytes were comprehensively analyzed and optimized,which provided an important reference for solving the problem of primary lymphocytes electroporation.Significance,and also the construction of an adeno-associated plasmid with CRISPR system,laid the foundation for the subsequent double-knocking of human primary lymphocytes by adenovirus infection.Finally,transfection of CRISPR/RNP protein complexes with electroporation can effectively edit the CTLA-4 and PD-1genes of primary lymphocytes and improve their ability to kill tumor cells,thus providing new ideas for tumor immunotherapy.significance.