Construction Of PLCγ2(S707Y) Point Mutation And Sp110 Knockout Mouse Model By CRISPR/Cas9 Technology And The Analysis Of PLCγ2(S707Y) Point Mutant Mouse

OBJECTIVE:Autoinflammatory diseases are genetic immune diseases caused by abnormal activation of immune cells.Phospholipase Cγ2 is an enzyme that plays a key role in BCR-mediated signaling.A variant of the PLCG2 gene c.2120C>A（p.Ser707Tyr）was found in the family suffering from autoinflammatory diseases.It was reported that mutation of PLCG2 gene c.2120C>A（p.Ser707Tyr）affected the negative regulation of PLCγ2 activation.However,the molecular mechanism behind this is still unclear.A mouse model of PLCγ2（S707Y）point mutation was constructed by CRISPR/Cas9 gene editing technology to warrant the study of the pathogenesis of related autoinflammatory diseases and the influence of B cells on autoinflammatory diseases.The sp110 gene is highly expressed in germinal center B cells,but the specific function is still unclear.The sp110 knockout mouse model was constructed to provide an experimental tool for studying the function of this gene in germinal center B cells.METHOD:The CRISPR/Cas9 technology has become an essential method in biomedical research due to its simple design and easy operation.sgRNA（single guide RNA）was designed targeting on plcg2 gene and sp110 gene specific sequence,microinjected C57BL/6 mouse fertilized eggs.Then we sequenced the region of interest to confirm the mutation of plcg2 and deletion of sp110 gene.After backcrossed for several generations,we successfully generated PLCγ2^S707Y/+homozygous mice.Six to eight weeks old PLCγ2^S707Y/+mice and PLCγ2^+/+mice were used to analyze the development of lymphocytes including T cells and B cells.Activation of T cells and B cells were evaluated by Calcium flux upon engagement of TCR and BCR.RESULTS:Sequencing of targeted region of plcg2 proved success in introducing PLCγ2（S707Y）point mutation mice.Flow cytometry analysis showed that there was no significant difference in the percentage of T、B cells and their subpopulations in lymphocytes between PLCγ2^S707Y/+mice and PLCγ2^+/+mice.There was no significant change in the calcium flux upon BCR activation in splenic B cells.Currently gRNA targeting sp110 were microinjected into C57BL/6 fertilized eggs.Further evaluation and characterization are planned after successful generation of the knockout mice.CONCLUSION:A PLCγ2（S707Y）point mutation mouse model was constructed by CRISPR/Cas9 technology.This mutation can be stably inherited in offsprings.The PLCγ2（S707Y）mutant mouse has no obvious effect on the development of T and B lymphocytes,Its role in the function and activation of B cells needs further investigation.However,this mouse provides an important experimental model for the study of human autoinflammatory diseases.