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Loss-of-function study is an effective approach to research gene functions.However, currently most of such studies have ignored an important problem (in this paper, we call it "off-target" problem), that is, if the target gene is an overlapping gene (A gene whose expressible nucleotides overlaps with that of another one), loss-of-function mutation by deleting the complete open reading frame (ORF) may also cause the gene it overlaps partially (or totally) lose functions, resulting a phenotype which is rather different from that of single gene mutation.