Genome editing is valuable for understanding gene function and for gene therapy.But the rate of traditionally homologous recombination is very low and the Cre/loxP leaves behind a 34 bp sequence together with the targeted mutation and the transposase can reintegrate the released piggyback into other chromosomal sites.Here we present a seamless strategy for editing CCR5 gene using the CRISPR-Cas9 system twice based on the HR and SSA repair mechanism respectively.In the first targeting, up to 100% clones were targeted in CCR5 gene on one allele.In the second targeting, 2.08% clones were targeted and repaired by SSA repair mechanism resulting in a 32 bp fragment replaced by SalI enzyme site seamlessly.This study provided a novel method for seamless genome editing and would promote the production for transgenic animals.