Methods for detecting and sequencing a target nucleic acid
Inventors
Chiu, Charles • Granados, Andrea • DOUDNA, Jennifer A. • Harrington, Lucas B. • Chen, Janice S. • Deng, Xianding
Assignees
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Publication Number
US-12644145-B2
Publication Date
2026-06-02
Expiration Date
Abstract
The present disclosure provides methods for characterizing a target DNA present in a sample. The methods involve contacting the sample with a type V CRISPR/Cas effector protein and one or more guide RNAs, where the contacting generates a cleavage product comprising a 5′ overhang; and ligating a double-stranded nucleic acid adapter to the cleavage product, to generate a ligation product. The ligation product includes the target DNA, which can be sequenced. The sample can be subjected to one or more amplification steps prior to the contacting step, with primers that provide for amplification of nucleic acids of, e.g., specific pathogens, categories of pathogens, two or more different pathogens, or two or more different categories of pathogens.
Core Innovation
The disclosure characterizes a target DNA present in a sample by amplifying nucleic acids, generating a PAM-distal cleavage product using a type V CRISPR/Cas effector protein with guide RNAs, and ligating a double-stranded nucleic acid adapter to the cleavage product. The contacting generates a cleavage product comprising a 5′ overhang, which becomes the substrate for adapter ligation.
The adapter comprises a 3′ deoxyadenosine overhang and a 5′ overhang that comprises a stretch of from 3 to 15 contiguous nucleotides complementary to a contiguous stretch of nucleotides of the same length in the 5′ overhang of the PAM-distal cleavage product. Ligation generates a ligation product comprising the adapter and the PAM-distal cleavage product, after which the nucleotide sequence of the PAM-distal cleavage product present in the ligation product is determined.
The disclosed system is extended with pre-amplification and sequencing modalities such as next generation sequencing including nanopore sequencing, to enable determination of the PAM-distal cleavage product sequence. The disclosure also describes a Type V CRISPR/Cas-based detection rationale in which target-DNA guide RNA activation of a Type V effector triggers promiscuous cleavage of non-target single-stranded DNA to produce a measurable signal.
Claims Coverage
The consolidated claim coverage presents one independent inventive workflow with 7 inventive features. It centers on recombinase polymerase amplification, Type V CRISPR/Cas guided PAM-distal cleavage producing a 5′ overhang, adapter ligation using specified 3′ and 5′ overhang features, and determining the nucleotide sequence of the PAM-distal cleavage product.
Amplification followed by Type V CRISPR/Cas guided PAM-distal 5′ cleavage
Amplifying nucleic acids in the sample using recombinase polymerase amplification, and contacting the sample with a type V CRISPR/Cas effector protein and one or more guide RNAs comprising a region that binds the type V CRISPR/Cas effector protein and a guide sequence that hybridizes with the target DNA, wherein the contacting generates a PAM-distal cleavage product comprising a 5′ overhang.
Adapter ligation using complementary 5′ overhang and 3′ deoxyadenosine overhang
Ligation of a double-stranded nucleic acid adapter to the cleavage product, wherein the adapter comprises a 3′ deoxyadenosine overhang and a 5′ overhang comprising a stretch of from 3 to 15 contiguous nucleotides complementary to a contiguous stretch of nucleotides of the same length in the 5′ overhang of the PAM-distal cleavage product, generating a ligation product comprising the adapter and the PAM-distal cleavage product.
Sequencing of the PAM-distal cleavage product in the ligation product
Determining the nucleotide sequence of the PAM-distal cleavage product present in the ligation product.
Type V CRISPR/Cas effector protein selection within Cas12 family
The type V CRISPR/Cas effector protein is selected from Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12d, or Cas14a proteins.
Nanopore sequencing for sequence determination
Sequence determination is performed using nanopore sequencing.
Non-hybridizing single-stranded detector DNA with detectable signal measurement
The method further contacts a sample with a non-hybridizing, single-stranded detector DNA and measures a detectable signal generated by type V CRISPR/Cas effector-mediated cleavage of the detector DNA.
Low concentration detection threshold
The method detects target DNA in a sample at a concentration as low as 200 fM.
The consolidated inventive coverage focuses on Type V CRISPR/Cas-driven PAM-distal cleavage producing a 5′ overhang, attachment of a double-stranded nucleic acid adapter with specified 3′/5′ overhang complementarity, and sequence determination of the PAM-distal cleavage product within the adapter-containing ligation product. Coverage is further refined by specific Type V/Cas protein selections, optional detector DNA signal measurement, nanopore sequencing, and an explicit low detection threshold.
Stated Advantages
The problem being solved is the ability to characterize and sequence target DNA present in a sample using Type V CRISPR/Cas guided cleavage, in a manner compatible with sequencing workflows.
Documented Applications
Characterizing a target DNA present in a sample.
Sequencing workflows.
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