Single base resolution: methodologies and bioinformatic tools to treat point mutations using CRISPR-Cas systems Authors: Roy Rabinowitz, Dani Offen Affiliation: Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Israel Abstract: The CRISPR-Cas system holds a great promise in the treatment of diseases caused by genetic variations. The specificity of the system enables researchers to target a particular locus within the genome. However, the ability of the CRISPR-Cas system to distinguish between two sequences differing in a single nucleotide is limited. Therefore, Cas9 is prone to both genome-wide and allelic off-targeting. We present two bioinformatic tools to assist in designing gRNAs for treating point mutations by two distinct approaches. CrisPam identifies SNP-derived PAMs to specifically target point mutations in an allele-specific manner. The PAM sequence is essential for successful DNA binding and cleavage by the Cas enzyme. By identifying novel PAM sequences, generated by the variant nucleotide, CrisPam suggests potential Cas enzymes and their corresponding gRNAs to perform allele-specific targeting. Base editing allows precise conversion of a target nucleotide without involving DNA double strand breaks. The editing occurs within a region called the activity window in which all target nucleotides will undergo transition. The editing may result in bystander editing and cause additional unintended mutations. However, in some special cases termed synonymous corrections, bystander editing results in the proper amino acids sequence. BE-FF predicts the outcome of base editing and identifies precise corrections of the target nucleotide, as well as synonymous corrections which broaden the gene editing scope of base editors. Be-FF and CrisPam are designed to serve experimental researchers and assist in the process of gRNA design and identification of suitable CRISPR systems.