High-throughput sequencing (HTS) data of plasmid library subjected to natural and high-fidelity Cas9 cleavage

SpCas9 is an RNA-guided endonuclease in the CRISPR-Cas immune system that is used for genome editing as it can be easily programmed to generate double-stranded breaks at desired target sites. Many natural Cas9 orthologs and engineered SpCas9 variants have been used to enable high-fidelity genome editing. To understand the native cleavage specificity and cleavage activity of Cas9 variants, we employed high-throughput <i>in vitro</i> cleavage assays using a plasmid library (pLibrary). We tested the cleavage activity of two natural Cas9 orthologs – SpCas9 and SaCas9, and three engineered variants – SpCas9 HF1, HypaCas9 and HiFi Cas9. We used two different crRNA sequences and corresponding target libraries (pLibrary), referred to as pLibrary PS4 and EMX1. The data set is the high-throughput sequencing (HTS) reads of pLibrary subjected to cleavage by different Cas9 variants. The README file contains details about the content of the HTS data set, and the key to the file names and corresponding samples. This HTS data set supports the research article: "High-throughput <i>in vitro</i> specificity profiling of natural and high-fidelity CRISPR-Cas9 variants".