CRISPECTOR provides accurate estimation of genome editing translocation and off-target activity from comparative NGS data

Abstract

Controlling off-target editing activity is one of the central challenges in making CRISPR technology accurate and applicable in medical practice. Current algorithms for analyzing off-target activity do not provide statistical quantification, are not sufficiently sensitive in separating signal from noise in experiments with low editing rates, and do not address the detection of translocations. Here we present CRISPECTOR, a software tool that supports the detection and quantification of on- and off-target genome-editing activity from NGS data using paired treatment/control CRISPR experiments. In particular, CRISPECTOR facilitates the statistical analysis of NGS data from multiplex-PCR comparative experiments to detect and quantify adverse translocation events. We validate the observed results and show independent evidence of the occurrence of translocations in human cell lines, after genome editing. Our methodology is based on a statistical model comparison approach leading to better false-negative rates in sites with weak yet significant off-target activity.

CRISPR-Analytics (CRISPR-A): a platform for precise analytics and simulations for gene editing

“…This would let us know if this novel approach improved somehow the state-of-the-art. Among all the tools presented in the introduction, only ampliCan 19 and CRISPECTOR 22 apply some kind of noise correction model. To benchmark the CRISPR-A model, we compared its performance with the previously characterized data set.…”

“…Other tools are focused on the analysis of Oxford Nanopore Technologies (ONT) data, instead of Illumina-based NGS data 21 . Moreover, new analysis tools have recently been developed to cover specificity assessment related to off-targets and translocations 22 .…”

High-Throughput Imaging of CRISPR- and Recombinant Adeno-Associated Virus–Induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells

“… 26 , 29 , 47 Based on GUIDE-seq analysis, we designed a rhAmpSeq multiplex PCR panel to measure on- and off-target editing efficiencies. The NGS rhAmpSeq data were analysed with the CRISPECTOR tool 31 , 48 to contextualize our findings from the Imagestream ( Fig. 4A–C ).…”

“…Data were analysed using the CRISPECTOR software tool, as described previously, 31 to produce inferred activity rates at the analyzed sites. The rhAmpSeq data for RAG1 and RAG2 were previously published, and the FASTQ files were downloaded for additional analysis from the SRA under accession number PRJNA630002.…”

“…We recently demonstrated a combined pipline using GUIDE-seq and RNase H2-dependent multiplex assay amplification (rhAmpSeq) 30 , 31 to quantify off-target activity with next-generation sequencing (NGS). While the off-target information provided by sequencing is important, this represents an “end-product” of CRISPR-Cas activity and does not provide detailed genotoxicity charecterization based on DDR induction.…”