SANTON: Sequencing Analysis Toolkits for Off-target Nomination

Background Genome-wide off-target nomination and screening sequencing methods, such as synthetic oligonucleotide-based sequencing and whole genome-based sequencing, evaluate the precision and safety of gene-editing technologies. However, there remains a lack of comprehensive bioinformatics tools for analyzing the sequencing data generated by off-target nomination assays for various gene editors, including Cas9, Cas12a, and base editors.

We introduce a Sequencing Analysis Toolkits for Off-target Nomination (SANTON) for the identification and quantification of potential off-target sites using datasets generated from synthetic oligonucleotide-based sequencing and whole genome-based sequencing (e.g., Digenome-seq) methods. By applying SANTON to a Cas9 treated oligo synthesization-based sequencing data, a comprehensive set of potential off-target sites are evaluated in which the top off-target sites displayed highly constituency with a published study using in vivo method (e.g, GUIDE-seq). Utilizing SANTON to previously published Digenome-seq datasets, we identified more potential off-target regions than previous studies, including ones showing significantly higher cleavage level than the on-target site. To the best of our knowledge, SANTON is the first public package capable of analyzing synthetic oligonucleotide-based sequencing data and the first to specific optimized for the analysis of whole genome-based sequencing analysis for Cas12a and base editors. We demonstrated the capability of SANTON to effectively analyze complex cleavage patterns by a diverse editing system.

SANTON provides a powerful computational solution to support genome editing research, facilitating more reliable and comprehensive off-target profiling. Competing Interest Statement The authors have declared no competing interest. List of abbreviations - gRNA - Guide RNA - ABE - Adenine base editor - CBE - Cytosine base editor - NGS - Next generation sequencing - OT - Off-target region - PAM - Protospacer adjacent motif - SOS - Synthetic oligonucleotide-based sequencing - USER - Uracil-specific excision reagent - WGS - Whole genome-base sequencing