Objective The mouse FcγR2b, FcγR3, and FcγR4 gene clusters of about 90 kb in length wereknocked out by CRISPR/ Cas9 genome editing technique to lay the foundation for constructing an FcγR gene humanizedmouse. Methods Single guide RNAs (sgRNAs) were designed using online software to target exons of FcγR2b andFcγR3. Five candidate sgRNAs with lower off-target effects were selected for each target site. The cleavage activity ofsgRNAs was tested in vitro by the CRISPR/ Cas9 activity assay kit. The sgRNA with high cleavage activity was selected forin vitro transcription and microinjected with Cas9 mRNA into zygotes. Results A genetic modified mouse with 89,711 bpknockout fragment was confirmed by PCR detection and sequencing. The 5′ end of the FcγR2b gene, the 3′ end of theFcγR3 gene and FcγR4 gene were knocked out at the same time. Moreover, 8 sites with the highest probability of off-targetwere predicted by software, and all 8 off-target sites of the founder mouse genome were sequenced and confirmed. No smallfragment insertion or deletion was found in any predicted off-target sites. Conclusions We established a large fragmentknockout technique using CRISPR/ Cas9 genome editing technique, which may provide a new method to establish humanized mouse models by combining with BAC transgenic strategies.