CriSNPr: a single interface for the curated and de novo design of gRNAs for CRISPR diagnostics using diverse Cas systems
- CSIR Institute of Genomics and Integrative Biology, India
- CSIR-Institute of Genomics and Integrative Biology, India
Abstract
CRISPR-based diagnostics (CRISPRDx) have improved clinical decision-making, especially during the COVID-19 pandemic, by detecting nucleic acids and identifying variants. This has been accelerated by the discovery of new and engineered CRISPR effectors, which have expanded the portfolio of diagnostic applications to include a broad range of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline necessitates customized detection schemes based on the fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is especially relevant for variant detection, a low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPR-based diagnostics currently exists. In this manuscript, we fill this lacuna using a unified webserver, CriSNPr (CRISPR-based SNP recognition), which provides the user with the opportunity to de-novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. Furthermore, we provide a database of curated pre-designed gRNAs as well as target/off-target for all human and SARS-CoV-2 variants reported thus far. CriSNPr has been validated on multiple Cas proteins, demonstrating its broad and immediate applicability across multiple detection platforms. CriSNPr can be found at http://crisnpr.igib.res.in/.
Data availability
The current manuscript is a computational study, so no new data has been generated for this manuscript. Experimental validation results have been presented in figures in the manuscript. The source code and related datasets have been indicated in the manuscript and also uploaded here: http://crisnpr.igib.res.in/download. All other validation data have been presented in the main manuscript itself.
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DbSNP: The NCBI database of genetic variationDbSNP: The NCBI database of genetic variation.
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Database Resources of the National Genomics Data Center, China National Center for Bioinformation in 2021.Nucleic Acids Research, doi.org/10.1093/nar/gkaa1022.
Article and author information
Author details
Debojyoti Chakraborty
Funding
CSIR (HCP23)
- Souvik Maiti
- Debojyoti Chakraborty
EMBO (GAP252)
- Debojyoti Chakraborty
Lady Tata Memorial Trust (GAP198)
- Debojyoti Chakraborty
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Tony Yuen, Icahn School of Medicine at Mount Sinai, United States
Version history
- Preprint posted: February 17, 2022 (view preprint)
- Received: February 17, 2022
- Accepted: February 7, 2023
- Accepted Manuscript published: February 8, 2023 (version 1)
- Version of Record published: February 20, 2023 (version 2)
Copyright
© 2023, Ansari et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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CriSNPr: a single interface for the curated and de novo design of gRNAs for CRISPR diagnostics using diverse Cas systems
eLife 12:e77976.
https://doi.org/10.7554/eLife.77976
Further reading
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- Computational and Systems Biology
Transcriptomic profiling became a standard approach to quantify a cell state, which led to accumulation of huge amount of public gene expression datasets. However, both reuse of these datasets or analysis of newly generated ones requires significant technical expertise. Here we present Phantasus - a user-friendly web-application for interactive gene expression analysis which provides a streamlined access to more than 96000 public gene expression datasets, as well as allows analysis of user-uploaded datasets. Phantasus integrates an intuitive and highly interactive JavaScript-based heatmap interface with an ability to run sophisticated R-based analysis methods. Overall Phantasus allows users to go all the way from loading, normalizing and filtering data to doing differential gene expression and downstream analysis. Phantasus can be accessed on-line at https://alserglab.wustl.edu/phantasus or can be installed locally from Bioconductor (https://bioconductor.org/packages/phantasus). Phantasus source code is available at https://github.com/ctlab/phantasus under MIT license.
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- Computational and Systems Biology
- Evolutionary Biology
A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.
