The authors describe the development of dimeric RNA-guided FokI nucleases (RFNs) for highly specific genome editing in human cells. RFNs, which require two guide RNAs (gRNAs) to function, show improved specificity compared to monomeric Cas9 nuclease systems, which can induce unwanted off-target mutations. The study introduces a method for expressing multiple gRNAs with any 5' end nucleotide, broadening the targeting range of RFNs. RFNs were shown to reduce off-target mutations and induce fewer indels and point mutations compared to monomeric Cas9 nickases. The authors also developed software tools to aid in identifying potential target sites and off-target sites for RFNs. Overall, the dimeric RFN system offers enhanced specificity and precision for genome editing applications.The authors describe the development of dimeric RNA-guided FokI nucleases (RFNs) for highly specific genome editing in human cells. RFNs, which require two guide RNAs (gRNAs) to function, show improved specificity compared to monomeric Cas9 nuclease systems, which can induce unwanted off-target mutations. The study introduces a method for expressing multiple gRNAs with any 5' end nucleotide, broadening the targeting range of RFNs. RFNs were shown to reduce off-target mutations and induce fewer indels and point mutations compared to monomeric Cas9 nickases. The authors also developed software tools to aid in identifying potential target sites and off-target sites for RFNs. Overall, the dimeric RFN system offers enhanced specificity and precision for genome editing applications.