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US-12618098-B2 - Proximity-driven activation of CRISPR-Cas systems for detection of diverse molecular analytes

US12618098B2US 12618098 B2US12618098 B2US 12618098B2US-12618098-B2

Abstract

Provided herein are methods and compositions for rapid, highly sensitive detection of molecular analytes such as antibodies, proteins, and small molecules using protein-driven nucleic acid assemblies to activate CRISPR-Cas nucleases. Also provided herein are uses of the sensitive analyte detection methods in an analyte detection platform and in convenient low-cost diagnostic assays such as lateral flow devices for point-of-care use.

Inventors

  • Alexander Green
  • Kirstie Swingle

Assignees

  • ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY

Dates

Publication Date
20260505
Application Date
20200427

Claims (9)

  1. 1 . A method of detecting a target molecule in a sample, the method comprising the steps of: (a) contacting to the sample a first synthetic nucleotide sequence comprising a first double-stranded DNA (dsDNA) spacer sequence and a first single-stranded DNA (ssDNA) sequence that is reverse complementary to a protospacer adjacent motif (PAM) sequence, wherein the first synthetic nucleotide sequence is linked to a first antigen; and a second synthetic nucleotide sequence comprising a single-stranded sequence or a dsDNA sequence and a ssDNA PAM sequence, wherein the second synthetic nucleotide sequence is linked to a second antigen, wherein the first ssDNA and the second ssDNA PAM sequences comprise complementary sticky ends and form a double-stranded PAM (dsPAM) sequence when the first and second synthetic nucleotide sequences are brought into proximity by binding of the first and second antigens to the target molecule, and wherein the reverse complementary ssDNA PAM sequence is adjacent to the reverse complement of the spacer sequence in the first synthetic nucleotide; (b) contacting the contacted sample of (a) to: (i) a Cas nuclease that exhibits collateral ssDNase or ssRNase activity, (ii) a guide RNA (gRNA), wherein the gRNA comprises the spacer sequence, and (iii) a single-stranded DNA or RNA reporter construct; whereby, in the presence of the target molecule that binds to the first and second antigens, the gRNA forms a complex with the dsPAM sequence and the dsDNA spacer sequence and the Cas nuclease cleaves the single-stranded reporter construct; and (c) detecting cleavage of the single-stranded reporter construct, thereby detecting the presence of the target molecule in the sample.
  2. 2 . A method for producing a barcoded gRNA by: (a) contacting to a sample a first single-stranded DNA (ssDNA) sequence comprising a first gRNA barcode region and a first stem-forming region, wherein the first stem-forming region is linked to a first antigen, and a second ssDNA sequence comprising a second gRNA barcode region, a third gRNA barcode region, and a second stem-forming region having a sequence complementary to that of the first stem-forming region, wherein the second stem-forming region is linked to a second antigen, whereby, in the presence in the sample of an antibody that binds specifically to antigens of the first and second ssDNA sequences, the first and second stem-forming regions form a double-stranded DNA region; (b) contacting to the contacted sample of (a) a split DNA template that comprises (i)) a 5′ phosphorylated strand having a hairpin structure that comprises a single-stranded hairpin domain sequence, a double-stranded T7 promoter sequence, and ssDNA sequences complementary to the first and second gRNA barcode regions, and (ii) a single DNA strand comprising a sequence complementary to the third gRNA barcode region, whereby, in the presence of DNA ligase and DNA polymerase, an extended double-stranded DNA sequence is obtained that comprises a double-stranded T7 promoter sequence, a double-stranded gRNA barcode region, and a double-stranded hairpin domain that encodes a conserved gRNA hairpin; and (c) contacting the extended double-stranded DNA sequence of (b) to T7 RNA polymerase, whereby a gRNA comprising first, second, and third barcodes is produced.
  3. 3 . A method for producing a barcoded gRNA by: (a) contacting to a sample a first single-stranded DNA (ssDNA) sequence comprising a first gRNA barcode region and a first stem-forming region, wherein the first stem-forming region is linked to a first protein-binding probe, and a second ssDNA sequence comprising a second gRNA barcode region, a third gRNA barcode region, and a second stem-forming region having a sequence complementary to that of the first stem-forming region, wherein the second stem-forming region is linked to a second protein-binding probe, whereby, in the presence in the sample of one or more proteins that bind specifically to protein-binding probes of the first and second ssDNA sequences, the first and second stem-forming regions form a double-stranded DNA region; (b) contacting to the contacted sample of (a) a split DNA template that comprises (i) a 5′ phosphorylated strand having a hairpin structure that comprises a single-stranded hairpin domain sequence, a double-stranded T7 promoter sequence, and ssDNA sequences complementary to the first and second gRNA barcode regions, and (ii) a single DNA strand comprising a sequence complementary to the third barcode region, whereby, in the presence of DNA ligase and DNA polymerase, an extended double-stranded DNA sequence is obtained that comprises a double-stranded T7 promoter sequence, a double-stranded gRNA barcode region, and a double-stranded hairpin domain that encodes a conserved gRNA hairpin; and (c) contacting the extended double-stranded DNA sequence of (b) to T7 RNA polymerase, whereby a gRNA comprising the first, second, and third barcodes is produced.
  4. 4 . The method of claim 1 , wherein the target molecule is an antibody.
  5. 5 . The method of claim 1 , wherein the Cas nuclease is Cas12a, Cas13a, Cas13b, Cas13d, Cas12g1, or Cas12i1.
  6. 6 . The method of claim 1 , wherein the single-stranded reporter construct comprises a first small molecule at the 5′ end and a second small molecule at the 3′ end.
  7. 7 . The method of claim 6 , wherein the first and second small molecules are selected from biotin and FAM (fluorescein).
  8. 8 . The method of claim 1 , wherein the first ssDNA and the second ssDNA PAM sequences comprise complementary sticky ends having a length of 5 or 6 base pairs.
  9. 9 . The method of claim 1 , wherein the first ssDNA and the second ssDNA PAM sequences comprise complementary sticky ends having a length of 3 to 6 base pairs.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national stage application filed under 35 U.S.C. § 371 of International Application No. PCT/US2020/030032, filed Apr. 27, 2020, which claims the benefit of U.S. Provisional Application Nos. 62/839,050, filed Apr. 26, 2019, and 62/976,659, filed Feb. 14, 2020, each of which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT Not applicable. BACKGROUND CRISPR-Cas systems function as a prokaryotic adaptive immune defense through the recognition and degradation of invading viral RNA/DNA. These systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to transcribe guide RNAs that direct Cas proteins to cleave targeted DNA and RNA. CRISPR-Cas12a systems recognize target double-stranded DNA (dsDNA) that contains a region complementary to guide RNA along with a T-rich protospacer adjacent motif (PAM). Upon recognition, Cas12a nuclease activity is initiated and a staggered dsDNA break is fashioned after the PAM site. Once activated, Cas12a also exhibits a collateral single-stranded DNase effect to cleave surrounding single-stranded DNA (ssDNA) molecules. This effect enables Cas12a to attack viral ssDNA. Several CRISPR-Cas systems have been exploited for gene editing applications based on dsDNA cleavage capabilities that are single turnover. More recently, CRISPR-Cas systems that also display ssDNase collateral cleavage have been leveraged to develop diagnostic assays. Because this indiscriminate cleavage is multiple-turnover, these diagnostics are highly sensitive, and when coupled with an amplification reaction can reach attomolar detection limits and lower. However, the utility of CRISPR-Cas-based diagnostic tools for infections has thus far been limited to DNA/RNA detection, restricting its use to detection of infections predominately in the acute stages when viral, bacterial, fungal, and parasitic pathogens can be detected in the blood. Standard immunoassays can detect infections even after pathogens are no longer circulating in the blood since they can exploit the prevalence of antibodies and other proteins that are generated in response to infection. These assays are typically done in a capture format where a known antigen or antibody is fixed on a surface and binding of a target macromolecule is visualized using a label, enzyme, or other reporter. Common assays include Enzyme-Linked Immunosorbent Assays (ELISA) and lateral flow assays. Because of the wide range of targets and low cost for these types of assays, they are considered standard diagnostic tools and are regularly used for detection of a range of different bacterial and viral pathogens, among others. Compared to nucleic acid tests, however, immunoassays often suffer from lower specificity, making discrimination of closely related pathogens challenging, and poorer sensitivity, since they are not as amenable to amplification as nucleic acids. To circumvent the latter limitation, several labs have tried to combine protein detection/binding with nucleic acid amplification techniques. One such example uses a small-molecule ligand and target binding event to template the formation of a DNA strand that is then amplified using polymerase chain reaction (PCR). Another example is Immuno-PCR where an ELISA assay using DNA-conjugated antibodies is coupled with PCR for highly sensitive detection (Barletta et al., Am. J. Clin. Pathol. 122, 20-27 (2004)). The disadvantages of such assays is that they require multiple processing steps, are limited to fluorescent readout, or require thermal cycling. These disadvantages make them unsuitable for low-cost, point-of-care assay formats. Accordingly, there remains a need in the art for rapid, inexpensive, and highly sensitive methods for detecting diverse molecular analytes such as nucleic acid complexes formed in response to antibodies, proteins, and small-molecule analytes. SUMMARY OF THE DISCLOSURE This disclosure is related to methods and compositions for detecting diverse molecular analytes with excellent sensitivity and specificity. As described herein, the methods and compositions are useful in a platform to detect a broader range of diseases and infections, both viral and bacterial; to enable profiling of the human immune system; and to detect impurities and toxins present in drinking water, food and beverages, and other samples. In a first aspect, provided herein is a method of detecting a target molecule in a sample. The method can comprise or consist essentially of the steps of: (a) contacting to the sample a first synthetic nucleotide sequence comprising a first double-stranded DNA (dsDNA) spacer sequence and a first single-stranded DNA (ssDNA) that is reverse complementary to a protospacer adjacent motif (PAM) sequence, wherein the first synthetic nucleotide sequence is linked to a first antigen; and a second synthetic nucleotide sequence comprising a single-strande