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.
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
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Accumulating evidence to make decisions is a core cognitive function. Previous studies have tended to estimate accumulation using either neural or behavioral data alone. Here, we develop a unified framework for modeling stimulus-driven behavior and multi-neuron activity simultaneously. We applied our method to choices and neural recordings from three rat brain regions—the posterior parietal cortex (PPC), the frontal orienting fields (FOF), and the anterior-dorsal striatum (ADS)—while subjects performed a pulse-based accumulation task. Each region was best described by a distinct accumulation model, which all differed from the model that best described the animal’s choices. FOF activity was consistent with an accumulator where early evidence was favored while the ADS reflected near perfect accumulation. Neural responses within an accumulation framework unveiled a distinct association between each brain region and choice. Choices were better predicted from all regions using a comprehensive, accumulation-based framework and different brain regions were found to differentially reflect choice-related accumulation signals: FOF and ADS both reflected choice but ADS showed more instances of decision vacillation. Previous studies relating neural data to behaviorally inferred accumulation dynamics have implicitly assumed that individual brain regions reflect the whole-animal level accumulator. Our results suggest that different brain regions represent accumulated evidence in dramatically different ways and that accumulation at the whole-animal level may be constructed from a variety of neural-level accumulators.
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- Biochemistry and Chemical Biology
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The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.
