The invention relates to the field of gene editing, in particular to an sgRNA sequence and a method for preparing RH negative erythrocytes by a CRISPR-Cas9 technology. The invention provides a separable nucleotide sequence. The separable nucleotide sequence comprises an sgRNA targeting sequence and/or a complementary sequence thereof; the sgRNA targeting sequence is a nucleotide sequence shown asSEQ ID NO:1. The provided sgRNA sequence can be targeted to the separable nucleotide sequence. The nucleotide sequence provided by the invention is used for building an expression vector; the transformation from RH positive blood into RH negative blood can be realized, so that the blood transfusion requirement is met.