The invention relates to a design method of an sgRNA and a lentivirus carrier formed by the sgRNA and plasmids. In the method, the design length of the sgRNA is 17bp or 18bp, and the tail end of the sgRNA is provided with a G or A; the target sequence is (N16)GNGG, (N16)ANGG, (N16)CNCC or (N16)TNCC; when a first basic group is A, C or T, g is additionally arranged in front of the first basic group. Whether a similar sequence exists in a gene group is searched in a TagScan database; when the sgRNA with the length of 17bp is additionally provided with a completely-matched sequence, the sgRNA is abandoned, and other appropriate sequences are tested; for the sgRNA which is 18bp long, when only one non-matched nucleotide is provided, the sgRNA is also abandoned, and other sequences with the designed sgRNA are continuously tested. The method for designing the sgRNA is optimized, the high knock-out efficiency and low target missing or no target missing can be realized.