HIV or HCV detection with CRISPR-CAS13A
Inventors
Fozouni, Parinaz • Ott, Melanie • DOUDNA, Jennifer A.
Assignees
University of California • J David Gladstone Institutes • University of California San Diego UCSD
Publication Number
US-12215377-B2
Publication Date
2025-02-04
Expiration Date
2039-09-06
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Abstract
The present disclosure relates to methods using CRISPR-Cas13a enzyme, complexed with HIV or HCV crRNAs to specifically and sensitively detect and quantify the presence of HIV or HCV RNA in a sample. These methods can be used to diagnose HIV or HCV infection, quantify the concentration of HIV or HCV RNA present in a sample, identify the presence of different HIV or HCV splice variants, subtypes, or mutations, and to monitor reactivation of HIV or HCV transcription.
Core Innovation
The invention relates to methods using the CRISPR-Cas13a enzyme complexed with HIV or HCV CRISPR guide RNAs (crRNAs) to specifically and sensitively detect and quantify the presence of HIV or HCV RNA in a sample. The methods involve incubating a sample containing RNA with Cas13a protein and crRNAs to generate HIV or HCV RNA cleavage products and subsequently detecting these cleavage products without reverse transcribing the RNA prior to detection.
These methods solve the significant challenge of accurately detecting acute and chronic HIV infections, especially in the early phases where current antibody-based self-tests have a large detection window leading to false-negative results. Similarly, for HCV, early detection is crucial to prevent progression to chronic hepatitis and its complications. Current gold standard RNA detection methods require laboratory access and are not conducive to at-home frequent testing.
The invention includes quantitative detection of viral RNA concentrations, identification of HIV or HCV splice variants, subtypes, or mutations, and monitoring of reactivation of viral transcription. The method can be performed with or without RNA amplification, utilizes fluorescence detection, and may include depletion of interfering sample components or removal of nucleases. The invention also covers kits comprising Cas13a protein, crRNAs, and necessary reagents for detection.
Claims Coverage
The patent includes four independent claims covering methods for detecting HIV or HCV RNA, quantifying RNA concentration, identifying splice variants/mutations, and monitoring viral transcription reactivation. The key inventive features of these methods are the use of Cas13a protein complexed with specific crRNAs, RNA cleavage product detection without reverse transcription, and optional amplification and sample treatment steps.
RNA detection using Cas13a and crRNAs without reverse transcription
Methods comprising incubating a sample containing RNA with Cas13a protein and at least one CRISPR guide RNA (crRNA) specific to HIV or HCV to produce RNA cleavage products followed by detecting those RNA cleavage products, wherein the RNA is not reverse transcribed prior to detection.
Quantification of HIV or HCV RNA concentration via Cas13a cleavage product analysis
Methods for quantifying HIV or HCV RNA involving incubation with Cas13a and crRNAs, detection of RNA cleavage product concentration using a detector, optionally employing a standard curve, and without reverse transcription prior to analysis.
Identification of HIV or HCV splice variants and mutations by crRNA recognition and cleavage product analysis
Methods that detect the presence or absence of HIV or HCV splice variants and/or mutations by incubating RNA samples with Cas13a and crRNAs that specifically recognize such variants or mutations, followed by detection of cleavage products without reverse transcription.
Monitoring reactivation of HIV or HCV transcription using Cas13a cleavage products
Methods to monitor reactivation or rebound of HIV or HCV transcription by incubating RNA samples with Cas13a and crRNAs, detecting the amount of RNA cleavage product, with or without amplification, without reverse transcription prior to detection.
Overall, the inventive features provide methods that enable sensitive, specific, and quantitative detection of HIV or HCV RNA using Cas13a and crRNAs, without the need for reverse transcription, incorporating options for sample treatment, RNA amplification, multiplexing of crRNAs, and various detection modalities.
Stated Advantages
Allows for specific and sensitive detection and quantification of HIV or HCV RNA without the need for reverse transcription.
Enables early detection of acute HIV infections overcoming limitations of antibody-based self-tests.
Facilitates at-home testing due to elimination of laboratory-dependent RNA reverse transcription steps.
Allows identification of viral splice variants and mutations, contributing to detailed viral profiling.
Supports monitoring of viral reactivation or rebound in patients under treatment.
Adaptable to various detection methods including fluorescence-based assays.
Multiplexing of crRNAs enhances sensitivity and detection capability.
Sample treatment steps like depletion of interfering components or removal of RNase improve assay specificity and reliability.
Documented Applications
Diagnosis of HIV or HCV infection by detecting viral RNA in biological samples.
Quantification of HIV or HCV RNA viral load in samples.
Identification and differentiation of HIV or HCV splice variants, subtypes, and mutations.
Monitoring reactivation or rebound of HIV or HCV transcription in patients.
Potential use in at-home or point-of-care testing for HIV or HCV.
Use in therapeutic monitoring and treatment decision-making for HIV and HCV patients.
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