Search

EP-4041912-B1 - A MOLECULAR SENSING PLATFORM AND METHODS OF USE

EP4041912B1EP 4041912 B1EP4041912 B1EP 4041912B1EP-4041912-B1

Inventors

  • KARLIKOW, Margot
  • PARDEE, Keith
  • MOUSAVI, Peivand Sadat

Dates

Publication Date
20260513
Application Date
20201009

Claims (15)

  1. An oligonucleotide primer pair comprising: (a) a promoter primer comprising, from 5' to 3', a transcriptional promoter, and a proximal detection target segment that has, or is complementary to, the sequence of a proximal portion of a detection target nucleic acid; and (b) a crRNA primer comprising, from 5' to 3', a crRNA encoding segment that is a sequence encoding a crRNA or the reverse complement of a sequence encoding a crRNA, and a distal detection target segment that has, or is complementary to, the sequence of a distal portion of the detection target nucleic acid, wherein the target segments in each primer permit amplification from the detection target nucleic acid.
  2. The primer pair of claim 1, wherein the transcriptional promoter is a T7 promoter, T3 promoter, or SP6 promoter, optionally wherein the crRNA primer is between 30 and 200 base pairs in length.
  3. The primer pair of any one of claims 1 or 2, wherein the crRNA primer is comprised in an array, the array comprising a nucleic acid of up to 8000 base pairs in length.
  4. A system for target nucleic acid-specific generation of a crRNA-encoding nucleic acid, the system comprising: (a) at least one primer pair of any one of claims 1-3; (b) a polymerase, optionally a DNA polymerase, optionally a RNA polymerase, optionally wherein the DNA polymerase is suitable for use in isothermal amplification method selected from Lamp, NASBA, RPA, NEAR, and/ or the polymerase is selected from AMV-RT, Bsu, IsoPol, and HDA; or a polymerase suitable for use in PCR optionally Q5; and (c) components for nucleic acid amplification.
  5. The system of claim 4, wherein if the target nucleic acid is RNA, the system comprises a reverse transcriptase.
  6. A kit for detecting a target nucleic acid, the kit comprising a primer pair of any one of claims 1 to 3, and optionally one or more of: (a) a DNA polymerase, and optionally components for nucleic acid amplification; (b) an RNA polymerase, and optionally components for transcription; (c) a Cas enzyme; and (d) at least one component selected from: a signal-generating CRISPR-sensitive reporter; a function restoring nucleic acid; and a DNAcrRNA.
  7. The kit of claim 6, wherein the signal-generating CRISPR-sensitive reporter is a molecular beacon or the signal-generating CRISPR-sensitive reporter is a CRISPR-sensitive DNA sensor and the kit further comprises a DNA ligase, optionally wherein the CRISPR-sensitive DNA sensor comprises: (a) a non-functional CRISPR-sensitive DNA reporter construct comprising a non-functional expression cassette with at least one CRISPR target site inserted or naturally present in the expression cassette, the non-functional expression cassette having a reporter construct upstream end upstream of the CRISPR target site and a reporter construct downstream end downstream of the CRISPR target site, and (b) at least one function-restoring nucleic acid, the function-restoring nucleic acid comprising a downstream flanking end and a function restoring repair insert, optionally an upstream flanking end, wherein the upstream flanking end interfaces with the reporter construct upstream end and/or the downstream flanking end interfaces with the reporter construct downstream end and one or both of the flanking ends permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal; or wherein the CRISPR-sensitive DNA sensor comprises: (a)a non-functional DNA reporter construct comprising a non-functional expression cassette, the non-functional expression cassette having a single stranded part; (b)at least one function-restoring nucleic acid (e.g. supplemented dsDNA), the function-restoring nucleic acid comprising a CRISPR target site inserted or naturally present in the function restoring nucleic acid, and a function restoring repair insert complementary to the single stranded part of the non-functional DNA reporter construct, the function restoring insert being releasable upon CRISPR mediated cleavage of the function restoring nucleic acid; wherein the function restoring repair insert interfaces (hybridizes) with the reporter construct single stranded part permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal; or wherein the CRISPR-sensitive DNA sensor comprises a non-functional CRISPR-sensitive DNA reporter construct, the reporter construct comprising: (a)a promoter, (b)a reporter cassette; (c)a function-blocking region, optionally in the promoter, or within a transcription start site or in a coding region of the reporter cassette; (d)CRISPR-Cas target sites that flank the function blocking region; (e) a reporter construct upstream end upstream of the function-blocking region; and (f) a reporter construct downstream end downstream of the function-blocking region; wherein the upstream end is capable of interfacing with the downstream end to permit function-restoring repair of the reporter construct when the CRISPR target sites are actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal.
  8. A method of target-nucleic acid-specific generation of a crRNA-encoding nucleic acid in a sample putatively containing the target nucleic acid, the method comprising: (a) contacting the sample with a system, the system comprising: (i) a primer pair of any one of claims 1 to 3; (ii) a polymerase; and (iii) components for nucleic acid amplification; and (b) incubating the sample and the system of step a. under conditions for target-specific amplification of the target sequence to generate a crRNA-encoding nucleic acid.
  9. The method of claim 8 for detecting a target nucleic acid in a sample, the method further comprising: (c) optionally, separating the crRNA-encoding nucleic acid, optionally wherein separating the crRNA-encoding nucleic acid comprises i) isolating the crRNA-encoding nucleic acid, from the system; or ii) inactivating the primers; (d)contacting the crRNA-encoding nucleic acid with an RNA polymerase and components for transcription; (e) incubating the crRNA-encoding nucleic acid, RNA polymerase and components for transcription under conditions for the generation of a crRNA; (f) contacting the crRNA with a CRISPR-Cas protein, optionally the CRISPR-Cas protein Cas12a; (g)incubating the crRNA and CRISPR-Cas protein under conditions to allow the binding of the crRNA to the CRISPR-Cas protein to generate an active CRISPR-Cas effector protein; (h)contacting the active CRISPR-Cas effector protein with a signal-generating CRISPR-sensitive reporter; (i) incubating the system under conditions to allow the generation of signal from the signal-generating CRISPR-sensitive reporter; and (j) detecting the presence or absence of the signal.
  10. The method of claim 8 or 9, wherein the sample is (a) a biological sample, optionally the biological sample is obtained from a tissue sample, saliva, blood, plasma, sera, stool, urine, semen, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, skin swab, or mucosal membrane surface, or (b) an environmental sample, optionally the environmental sample is or is obtained from a food sample, a beverage sample, a surface, a soil sample, a water sample, exposure to atmospheric air or other gas sample, or (c) a sample comprising a barcode, or a combination thereof and/or wherein the target nucleic acid is purified or amplified from the sample prior to the application of the method.
  11. The method of any one of claims 8-10, wherein the polymerase is a DNA polymerase, optionally selected from Bsu, IsoPol, AMV-RT or Q5, or any DNA polymerase, suitable for use in isothermal amplification, optionally selected from HDA, Lamp, NASBA, RPA, NEAR, or suitable for PCR or combinations thereof, or wherein target nucleic acid is an RNA and the polymerase is a reverse transcriptase optionally AMV-RT.
  12. The method of any one of claims 9-11, wherein (a)the promoter primer comprises a T7 promoter and the RNA polymerase is T7 polymerase; (b)the promoter primer comprises a T3 promoter and the RNA polymerase is T3 polymerase; or (c)the promoter primer comprises a SP6 promoter and the RNA polymerase is SP6 polymerase.
  13. The method of any one of claims 9-12, wherein the signal-generating CRISPR-sensitive reporter is a molecular beacon (MB) optionally wherein the molecular beacon which comprises a CRISPR sensitive nucleic acid linker, a fluorophore and a quencher, wherein the CRISPR sensitive nucleic acid linker is double stranded and optionally wherein the fluorophore and the quencher are opposite.
  14. The method of claim 9, wherein the signal-generating CRISPR-sensitive reporter is a CRISPR-sensitive DNA sensor, and the method further comprises in step h. contacting the active CRISPR-Cas effector protein with components for function-restoring repair of the signal-generating reporter and incubating the active CRISPR-Cas effector protein, signal-generating CRISPR-sensitive reporter, and components under conditions to allow a function restoring repair of the signal generating reporter optionally wherein the CRISPR-sensitive DNA sensor comprises: (a)a non-functional CRISPR-sensitive DNA reporter construct comprising a non-functional expression cassette with at least one CRISPR target site inserted or naturally present in the expression cassette, the non-functional expression cassette having a reporter construct upstream end upstream of the CRISPR target site and a reporter construct downstream end downstream of the CRISPR target site, and (b)at least one function-restoring nucleic acid, the function-restoring nucleic acid comprising a downstream flanking end and a function restoring repair insert, optionally an upstream flanking end optionally comprising a promoter, wherein the upstream flanking end interfaces with the reporter construct upstream end and/or the downstream flanking end interfaces with the reporter construct downstream end and one or both of the flanking ends permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal; or wherein the CRISPR-sensitive DNA sensor comprises: (a)a non-functional DNA reporter construct comprising a non-functional expression cassette, the non-functional expression cassette having a single stranded part; (b)at least one function-restoring nucleic acid optionally supplemented dsDNA, the function-restoring nucleic acid comprising (i) a CRISPR target site inserted or naturally present in the function restoring nucleic acid, and (ii) a function restoring repair insert complementary to the single stranded part of the non-functional DNA reporter construct, the function restoring insert being releasable upon CRISPR mediated cleavage of the function restoring nucleic acid; wherein the function restoring repair insert interfaces (hybridizes) with the reporter construct single stranded part permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal; or wherein the CRISPR-sensitive DNA sensor comprises a non-functional CRISPR-sensitive DNA reporter construct, the reporter construct comprising: (a)a promoter, (b)a reporter cassette, (c)a function-blocking region optionally in the promoter, or within a transcription start site or in a coding region of the reporter cassette, (d)CRISPR-Cas target sites that flank the function blocking region; (e) a reporter construct upstream end upstream of the function-blocking region; and (f) a reporter construct downstream end downstream of the function-blocking region wherein the upstream end is capable of interfacing with the downstream end to permit function-restoring repair of the reporter construct when the CRISPR target sites are actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal.
  15. A method of detecting a target nucleic acid in a sample putatively containing the target nucleic acid of claim 9, wherein the signal-generating CRISPR-sensitive reporter in step (h) is a CRISPR-sensitive DNA sensor comprising: (i) a non-functional CRISPR-sensitive DNA reporter construct comprising a non-functional expression cassette with at least one CRISPR target site inserted or naturally present in the expression cassette, the non-functional expression cassette having a reporter construct upstream end upstream of the CRISPR target site and a reporter construct downstream end downstream of the CRISPR target site, and (ii) at least one function-restoring nucleic acid, the function-restoring nucleic acid comprising a downstream flanking end and a function restoring repair insert, optionally an upstream flanking end, wherein the upstream flanking end interfaces with the reporter construct upstream end and/or the downstream flanking end interfaces with the reporter construct downstream end and one or both of the flanking ends permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal; the method further comprising incubating the system under conditions to allow the generation of signal from the signal-generating CRISPR-sensitive reporter; and detecting the presence or absence of the signal.

Description

FIELD The present invention relates to an oligonucleotide primer or primer pair, a system for target nucleic acid-specific generation of a crRNA-encoding nucleic acid, a kit for detecting a target nucleic acid, a method of target-nucleic acid-specific generation of a crRNA-encoding nucleic acid in a sample putatively containing the target nucleic acid and a method of detecting a target nucleic acid in a sample putatively containing the target nucleic acid. INTRODUCTION Sensors such as nucleic acid sensors have multiple applications. Diagnostics Molecular diagnostics use pathogen genomes, or the sequences of disease (e.g. cancer-related mutations), as a biomarker or molecular barcode for detection. Such molecular technologies compare favorably to traditional antibody-based diagnostics, which are expensive to develop and generally only probe for surface markers on pathogens. Molecular diagnostics, can be developed relatively inexpensively and may probe not only for the presence of disease, but also for other relevant clinical features, such as drug resistance. Molecular technologies also offer an incredible level of signal amplification. Taken together, these advantages have resulted in molecular-based methods becoming the gold standard for diagnostics. Polymerase Chain Reaction (PCR) is by far the most common mode of detection for molecular diagnostics. It is a powerful technique that allows for the detection of minute amounts of specific nucleic acids through a series of amplification reactions that require thermal cycling. In the clinic, PCR has been embedded into dozens of benchtop diagnostic instruments and is the process that underlies almost all modern diagnostics done in the clinic. These systems, however, have been largely confined to use in laboratory settings because of their costly and bulky hardware, and the requirement for specialized personnel. Molecular barcoding Molecular barcoding, is the use of molecular technologies to detect synthetic or natural DNA sequences from commodities for the purpose of identifying the product or other related information such as manufacturing origin. The concept is analogous to optical barcoding systems, such as Universal Product Code (UPC) or Quick Response Codes (QR codes), but with the advantage that the "barcode" can be embedded throughout the product, making tampering or fraud more difficult. There have been previous commercial efforts using DNA labels, but these have required samples to be sent away for sequencing, making the system impractical for most applications. De-centralized molecular capabilities The rising cost of health care and the need for de-centralized technologies for maintaining public health have led to a significant effort toward building low cost and portable diagnostics. While it would be ideal to deploy PCR to point-of-care (POC) settings, cost and technical requirements have largely restricted PCR to the lab. One important area of focus has been the development of isothermal nucleic acid amplification methods. As the name suggests, these amplification reactions operate at a single temperature, rather than thermal cycling, and as such do not require sophisticated equipment. In fact, heating for these reactions can even be provided using a chemical heater (e.g. calcium oxide and water reaction) (Curtis et al., 2012). Other benefits of isothermal methods include a simplified workflow, meaning that work can be done outside of the lab by individuals with little to no training (Yan et al., 2014). Commercial isothermal reactions have recently become available and with these, practical applications of isothermal amplification as diagnostics (Gan et al., 2014; Linnes et al., 2014). Unfortunately, however, these diagnostics suffer from significant rates of false positives because of off-target amplification. A method that uses sensors downstream of isothermal amplification to provide a second sequence-specific step, can improve detection specificity and performance (Pardee et. al. 2016). Toehold switch-based RNA sensors have the advantage of adding an extra sequence-specific check point to isothermal amplification. This additional step significantly improves detection specificity. Unfortunately, toehold switches do not appear to be as sensitive as CRISPR-based approaches. Other de-centralized sensor platforms use Cas12 and Cas13 (SHERLOCK, DETECTR). These efforts have demonstrated quantification down to attomolar concentrations, detection down to zeptomolar concentrations and sensor multiplexing. While exciting, challenges limit the practical implementation of these most recent technologies. For example, these methods rely on the storage and deployment of RNA-based reagents. RNA is unstable, and SHERLOCK requires that pre-packaged RNAs be used to both guide the specificity of the technology (e.g. crRNAs) and create the signal for positive results (e.g. RNA-based reporters). Not only is RNA prone to physical/chemical deterioration, but upon expos