| The acquired immune system and the innate immune system are two major branches of the body’s immune system.The innate immune system is a non-specific and rapid defense mechanism,while the acquired immune system can bring to the body a long-term protection mechanism which is specified to the antigen.Acquired immune system is classified into humoral immunity and cellular immunity.Follicular helper T cells(TFH)play a important role in humoral immunity.However,the molecular mechanisms underlying differentiation of TFH remains unresolved,so it will be of great significance to find out novel critical regulators in differentiation of follicular helper T cells.Early gene screening techniques mostly relied on the use of shRNA for genetic interference at the RNA level.RNA interference had problems of low efficiency and high frequency of off-target.Therefore,in this study,we generated a small sgRNA library transduced into CD4+T cells from Cas9-SM-EIIA mice.The edited T cells were adoptively introduced into recipient mice followed by LCMV infection and TFH differentiation of donor cells was analyzed by flow cytometry and next-generation sequencing.The results show that the sgRNA of two candidate genes were significantly enriched in TFH cells compared to the sgRNA of other genes,suggesting that these two candidate genes may play a negative regulatory role in the differentiation of TFH cells.Subsequently,we individually transfected the candidate sgRNA in Cas9-SM-EIIA CD4+T cells,and then adoptively introduced it back to the recipient mice.After LCMV infection,the percentage of donor cells transfected with sgRNA differentiated into TFH cells was significantly greater than that of uninfected T cells.These results indicate that these two candidate genes may play a negative regulatory role in the differentiation of TFH cells.The CRISPR-Cas9 screening system provides strong technical support for further TFH research. |
