Provided is a method for knocking out target genes in T cells in vitro based on the CRISPR-Cas9 system. Also provided are crRNA targeting target genes TRAC, B2M and PD1, and a kit comprising sgRNA formed by linking the crRNA and tracrRNA corresponding to a Cas9 protein, the Cas9 protein, an oligodeoxyribonucleic acid (N-oligo) or a milt DNA fragment. The kit is used to knock out the TCR, B2M and/or PD1 genes in T cells. Also provided are T cells with gene knock out obtained according to the method of the present invention and the use thereof.