Comprehensive Genome Analysis to Elucidate CRISPR-Cas Off-Target Mutation Patterns on the Basis of in vivo, in vitro, and in silico Experiments

Abstract

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Off-target mutations are one of the major concerns raised about genome editing nucle-ases, and many efforts have been made to predict them. However, the accuracy of the predictions remains unsatisfactory possibly because our knowledge of mutation pat-terns is insufficient. In this way, although this technique is already at the application stage, the basic characteristics of off-target mutations are still to be investigated. Therefore, the objective of this research is to elucidate the patterns of off-target muta-tions reported in multiple studies that utilized in vivo GUIDE-seq and in vitro Dige-nome-seq methods. The results showed that digested sites were identical or highly sim-ilar to each other in most of the cases, while they sometimes varied considerably if dif-ferent enzymes are used; 16 insignificant and 207 significant cases were found in GUIDE-seq datasets. A comparison among three independent studies for a same en-zyme and target site showed that the digested sequences patterns were similar in all eight cases. In addition, a comparative analysis between experiment-based GUIDE-seq and in silico CRISPOR methods revealed limitations in predicting off-target mutations, particularly for SpCas9 variants and alternative enzymes. While CRISPOR has shown some success in identifying off-target sequences for the WT SpCas9 enzyme, it still generates a notable number of false positives. To conclude, off-target mutations might not be really predictable, and are determined mainly by the intrinsic nature of an en-zyme, and if new variants of an enzyme is engineered, its characteristics should be re-investigated. Furthermore, we encountered problem when analyzing the Digenome-seq datasets, while Arabidopsis data could be analyzed successfully, the methodology should be further improved to analyze the human datasets.