Optimizing genome editing strategy by primer-extension-mediated sequencing

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.

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

“…CRISPR/Cas systems show great potential in GE, but their off‐targeting may cause severe problems for the host organisms. Off‐targeting can lead to chromosomal rearrangements, causing damage at imperfectly matched genomic loci and limiting GE application for therapeutic purposes . As well as interfering with chromosome stability, off‐target effects may cause loss of functional‐gene activity that causes diverse physiological or signaling abnormalities ( Figure ).…”

“…PEM‐seq has been developed and widely used to detect off‐target effects and determination of specificity and editing efficiency of CRISPR/Cas9 . LAM‐HTGTS has been combined with targeted sequencing by PEM‐seq for the effective analysis of CRISPR/Cas9 induced off‐target mutations via translocation capture.…”

“…Furthermore, PEM‐seq has been employed for testing a variety of extensively used methods which were developed for reducing Cas9 off‐target activity, whereby PEM‐seq can assess comprehensively the specificity and editing efficiency of CRISPR/Cas9. This could greatly help in choosing an appropriate GE strategy for given loci …”

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

“…25 Interestingly, this mutant has also been shown to reduce off-target activity. 25,28 We hypothesized this increased specificity is due to reduced binding to all PAMs, thereby limiting the affinity for all sites without complete gRNA matches. This would effectively reduce the potency of the inhibition of the non-target pool by reducing the affinity for non-targets with NGG PAMs as well as non-targets with non-canonical PAMs.…”

The clinical potential of gene editing as a tool to engineer cell‐based therapeutics

“…They also benefit from accelerated nuclease kinetic activity, potentially reducing the activity window of the nuclease and thus opportunities for off‐target effects [27–29]. Whilst some report that CRISPR can lead to increased DNA cleavage at off‐target sites compared to the paired binding approaches of ZFNs and TALENs, strategies to reduce off‐target activity are underway [30]. These include the quantification of imperfect Cas9‐induced DSB repair products through primer‐extension‐mediated sequencing (PEM‐seq) to improve determination of editing specificity and efficiency; as well as the use of high‐fidelity Cas9 variants.…”