This study presents a method to enhance the specificity of CRISPR-Cas9 nucleases by using truncated guide RNAs (gRNAs). Traditional gRNAs, which are 20 nucleotides long, can cause off-target mutations. However, shorter gRNAs with less than 20 nucleotides of complementarity to the target DNA sequence significantly reduce off-target mutations without affecting on-target editing efficiency. Combining truncated gRNAs with paired Cas9 nickases further reduces off-target mutations. The researchers found that gRNAs with 17 or 18 nucleotides of complementarity, termed "trugRNAs," are effective and more specific than longer gRNAs. These trugRNAs were tested on multiple human genes and showed high efficiency in inducing on-target mutations and HDR-mediated genome editing. They also demonstrated reduced mutagenesis at off-target sites, with some sites showing decreases of up to 5000-fold. TrugRNAs were also effective in reducing off-target effects when used with paired nickases. The study highlights that trugRNAs provide a simple and effective strategy to improve the specificity of CRISPR-based systems. The findings suggest that trugRNAs can be used to enhance the specificity of both Cas9 nucleases and dCas9 fusion proteins. The study also discusses the implications of these findings for future research and therapeutic applications of CRISPR technology.This study presents a method to enhance the specificity of CRISPR-Cas9 nucleases by using truncated guide RNAs (gRNAs). Traditional gRNAs, which are 20 nucleotides long, can cause off-target mutations. However, shorter gRNAs with less than 20 nucleotides of complementarity to the target DNA sequence significantly reduce off-target mutations without affecting on-target editing efficiency. Combining truncated gRNAs with paired Cas9 nickases further reduces off-target mutations. The researchers found that gRNAs with 17 or 18 nucleotides of complementarity, termed "trugRNAs," are effective and more specific than longer gRNAs. These trugRNAs were tested on multiple human genes and showed high efficiency in inducing on-target mutations and HDR-mediated genome editing. They also demonstrated reduced mutagenesis at off-target sites, with some sites showing decreases of up to 5000-fold. TrugRNAs were also effective in reducing off-target effects when used with paired nickases. The study highlights that trugRNAs provide a simple and effective strategy to improve the specificity of CRISPR-based systems. The findings suggest that trugRNAs can be used to enhance the specificity of both Cas9 nucleases and dCas9 fusion proteins. The study also discusses the implications of these findings for future research and therapeutic applications of CRISPR technology.