The study introduces dimeric RNA-guided FokI nucleases (RFNs) for highly specific genome editing. RFNs require two guide RNAs (gRNAs) for activity, enhancing specificity compared to monomeric Cas9 nucleases. They are more efficient and specific than paired Cas9 nickases, reducing off-target mutations. RFNs can target a broader range of sequences due to a novel method for expressing gRNAs with any 5' end nucleotide. The study demonstrates that RFNs induce fewer unwanted mutations than monomeric Cas9 nickases and are more precise in genome editing. RFNs are effective in human cells and can reduce known off-target mutations to undetectable levels. The study also identifies a previously unknown side-effect of monomeric Cas9 nickases: the introduction of point mutations at specific positions in target sites. RFNs are more specific and efficient, making them a valuable tool for genome editing applications requiring high precision. The study provides a robust, user-friendly platform for CRISPR-based genome editing with improved specificity and mutagenesis profiles.The study introduces dimeric RNA-guided FokI nucleases (RFNs) for highly specific genome editing. RFNs require two guide RNAs (gRNAs) for activity, enhancing specificity compared to monomeric Cas9 nucleases. They are more efficient and specific than paired Cas9 nickases, reducing off-target mutations. RFNs can target a broader range of sequences due to a novel method for expressing gRNAs with any 5' end nucleotide. The study demonstrates that RFNs induce fewer unwanted mutations than monomeric Cas9 nickases and are more precise in genome editing. RFNs are effective in human cells and can reduce known off-target mutations to undetectable levels. The study also identifies a previously unknown side-effect of monomeric Cas9 nickases: the introduction of point mutations at specific positions in target sites. RFNs are more specific and efficient, making them a valuable tool for genome editing applications requiring high precision. The study provides a robust, user-friendly platform for CRISPR-based genome editing with improved specificity and mutagenesis profiles.