CRISPRitz: rapid, high-throughput and variant-aware in silico off-target site identification for CRISPR genome editing

Abstract

Motivation Clustered regularly interspaced short palindromic repeats (CRISPR) technologies allow for facile genomic modification in a site-specific manner. A key step in this process is the in silico design of single guide RNAs to efficiently and specifically target a site of interest. To this end, it is necessary to enumerate all potential off-target sites within a given genome that could be inadvertently altered by nuclease-mediated cleavage. Currently available software for this task is limited by computational efficiency, variant support or annotation, and assessment of the functional impact of potential off-target effects. Results To overcome these limitations, we have developed CRISPRitz, a suite of software tools to support the design and analysis of CRISPR/CRISPR-associated (Cas) experiments. Using efficient data structures combined with parallel computation, we offer a rapid, reliable, and exhaustive search mechanism to enumerate a comprehensive list of putative off-target sites. As proof-of-principle, we performed a head-to-head comparison with other available tools on several datasets. This analysis highlighted the unique features and superior computational performance of CRISPRitz including support for genomic searching with DNA/RNA bulges and mismatches of arbitrary size as specified by the user as well as consideration of genetic variants (variant-aware). In addition, graphical reports are offered for coding and non-coding regions that annotate the potential impact of putative off-target sites that lie within regions of functional genomic annotation (e.g. insulator and chromatin accessible sites from the ENCyclopedia Of DNA Elements [ENCODE] project). Availability and implementation The software is freely available at: https://github.com/pinellolab/CRISPRitzhttps://github.com/InfOmics/CRISPRitz. Supplementary information Supplementary data are available at Bioinformatics online.

Delivery Approaches for Therapeutic Genome Editing and Challenges

“…There are multiple in silico tools available for predicting putative off-target sites. The recently developed CRISPRitz platform is the only bioinformatics tool that simultaneously accounts for mismatches, bulges, and genetic variants while distinguishing between coding and noncoding off-target sites [ 48 ]. However, in silico CRISPR design tools cannot replace the need for additional validation of off-target gene editing using targeted deep sequencing or, ideally, whole-genome sequencing to accurately quantify the Cas9 specificity.…”

Faster and better CRISPR guide RNA design with the Crackling method

“…Filtering based on features such as: GC content, presence of TTTT, gRNA secondary structure [18] sgRNA Scorer 2.0 Scoring using machine learning model, trained on [6] [7] CHOPCHOP Flag indicating position 20 containing guanine [17] A B (3,5,12,20) s (1,9,16,20) s (1,2,8,9) s (1,2,3,4) s (1,2,19,20)…”

“…Other tools such as CRISPRseek [21], CasOT [20] and CRISPRitz [4] exist. However, we do not consider them here as already published results show that they perform poorly compared to others which we have included [11,4].…”

kRISP-meR: A Reference-free Guide-RNA Design Tool for CRISPR/Cas9

“…Recent studies have showed that considerable variation is present across human populations and individuals contain large scale variations such as duplications not present in the reference [15]. The issue of individual specific genomic variations has been addressed in tools that are capable of designing personalized sgRNAs, namely AlleleAnalyzer [16], CrisFlash [17] and CRISPRitz [18] while Sun et al presented a tool to design sgRNAs for non-reference plant genomes [19]. However, these tools use reference genomes and sequencing reads to detect variations and may lack specificity if the individual contains duplications or regions missing from the reference.…”