JACKIE: Fast Enumeration of Genome-Wide Single- and Multicopy CRISPR Target Sites and Their Off-Target Numbers

Abstract

Zinc finger protein-, transcription activator like effector-, and CRISPR-based methods for genome and epigenome editing and imaging have provided powerful tools to investigate functions of genomes. Targeting sequence design is vital to the success of these experiments. Although existing design software mainly focus on designing target sequence for specific elements, we report here the implementation of Jackie and Albert's Comprehensive K-mer Instances Enumerator (JACKIE), a suite of software for enumerating all single- and multicopy sites in the genome that can be incorporated for genome-scale designs as well as loaded onto genome browsers alongside other tracks for convenient web-based graphic-user-interface-enabled design. We also implement fast algorithms to identify sequence neighborhoods or off-target counts of targeting sequences so that designs with low probability of off-target can be identified among millions of design sequences in reasonable time. We demonstrate the application of JACKIE-designed CRISPR site clusters for genome imaging.

kmerDB: A database encompassing the set of genomic and proteomic sequence information for each species

“…Kmer data from kmerDB can find applications in comparative genomics and evolutionary studies [42] , [50] , aiding sequence specification like identifying highly-specific CRISPR target sites [60] . Prime sequences can serve as genetic barcodes or targetable landing sites in biotechnological applications, facilitating tracking of cells or organisms through genetic tagging.…”

kmerDB: A Database Encompassing the Set of Genomic and Proteomic Sequence Information for Each Species

“…Third, the identification of nullomers can be used for studies in evolution as a marker of negative selection (Koulouras and Frith 2021; Georgakopoulos-Soares, Yizhar-Barnea, et al 2021), for pathogen detection or therapeutics (Silva et al 2015; Pratas and Silva 2021), as anti-cancer agents (Alileche et al 2012; Alileche and Hampikian 2017), in cancer detection (Georgakopoulos-Soares, Barnea, et al 2021; Montgomery et al 2023), as vaccine adjuvants (Patel et al 2012) or in forensic applications (Goswami et al 2013). Fourth, kmer information derived from kmerDB can be used for comparative genomics and evolutionary studies (Perry and Beiko 2010; Sims et al 2009) and for sequence specification such as the identification of CRISPR target sites that are highly specific (Zhu and Cheng 2022). Fifth, prime sequences can be used as genetic barcodes or targetable landing pads in biotechnological applications.…”

Multifaceted roles of cohesin in regulating transcriptional loops

“…To avoid interference of dCas9 binding on loop formation, we selected probes at least 5kb away from the ChIA-PET shores. A design window of 2kb was selected and overlapped with unique sites using the JACKIE pipeline v1.0 (Zhu et al, 2022) and off-target prediction software Cas-OFFinder v2.4 (Bae et al, 2014). The gRNAs were selected per design window minimizing, in order, 1-mismatch, 2-mismatch and 3-mismatch predicted off-target sites and the activity of gRNA is further optimized by selecting within 40~60 GC%.…”