Abstract
Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.
Highlights
Introduction The bacterial defense system CRISPRCas has been engineered to be a versatile genome editing tool[1,2,3,4,5,6]
Besides 20-bp gRNAcomplementary sequence, CRISPR/Cas[9] requires extra universal nucleotides NGG adjacent to the target site, termed as protospacer adjacent motif (PAM), to initiate DNA editing, which limits the choice of targeting site but helps to improve the specificity[7]
Translocation requires the joining of two separate DSBs, so placing a locus-specific primer at induced DSBs helps to identify other unknown DSBs, as showed by LAMHTGTS17
Summary
Introduction The bacterial defense system CRISPRCas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) has been engineered to be a versatile genome editing tool[1,2,3,4,5,6]. Neither of the abovementioned methods is capable of determining in vivo editing efficiency of CRISPR/Cas enzymes In this regard, targeted sequencing is an alternative high-throughput sequencing way to roughly determine the editing efficiency of CRISPR/Cas[9] through counting minor mutations generated at the Cas[9] cleavage sites[2], but PCR amplification bias leads to quantification inaccuracy. Different with targeted deep sequencing, tracking of indels by decomposition (TIDE)[23] utilizes Sanger sequencing and a specific algorithm to evaluate insertions/deletions (indels) amplified by PCR Both T7 endonuclease I (T7EI) assay[3] and restriction fragment length polymorphism (RFLP)[24] amplify indels via PCR to omit deep sequencing, but T7EI tends to miss tiny indels and RFLP relies on an appropriate restriction enzyme cutting site spanning the Cas[9] target site
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