JPWO2020006036A5 - - Google Patents

Claims (61)

A method for amplifying and / or detecting a target double-stranded nucleic acid, wherein:
(A) The sample containing the target double-stranded nucleic acid is mixed with the amplification reaction mixture, and the amplification reaction mixture is as follows:
(I) Amplified CRISPR system, the amplified CRISPR system includes a first CRISPR / Cas complex and a second CRISPR / Cas complex, and the first CRISPR / Cas complex is the first CRISPR / Cas enzyme and the above. It comprises a first guide molecule that induces the first CRISPR / Cas complex to the first strand of the target nucleic acid, and the second CRISPR / Cas complex is the second CRISPR / Cas enzyme and the second CRISPR /. Includes a second guide molecule that directs the Cas complex to the second strand of said target nucleic acid;
(Ii) Helicase;
(Iii) A primer pair comprising a first primer and a second primer, where the first primer comprises a portion complementary to the first strand of the target nucleic acid, and the second primer is of the target nucleic acid. Containing moieties complementary to said second strand; as well as (iv) polymerase;
(B) The target nucleic acid is amplified by opening the R-loop of the target nucleic acid using the first CRISPR / Cas complex and the second CRISPR / Cas complex, and the helicase is used to obtain the target nucleic acid. Unraveling the first and second strands, annealing the strands with the primer pair, and extending the strands with the polymerase; and (c) opening, unraveling and annealing under isothermal conditions. And, by repeating the extension, the target nucleic acid is further amplified; and (d) optionally, the amplified target nucleic acid is detected.
The method described above. The method of claim 1, wherein the CRISPR / Cas enzyme is an inactivated CRISPR / Cas enzyme selected from the group consisting of inactivated Cas9, inactivated Cas12a, inactivated Cas12b, and inactivated Cas12c. The inactivated CRISPR / Cas enzyme is an inactivated Cas9 protein with mutations in the HNH and RuvC domains, or the inactivated CRISPR / Cas enzyme is a mutation corresponding to SpCas9 D10A and N863A or SpCas9 D10A and H840A. The method of claim 2, wherein the inactivated Cas9 protein has. The inactivated CRISPR / Cas enzyme is described in Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, S. et al. Mutans, S.M. agalactiae, S.A. equimimilis, S. sanguinis, S.A. pneumonia; C.I. jejuni, C.I. colli; N. salsuginis, N. et al. tergarcus; S. auricalis, S.A. carnosus; N. Mentingitides, N.M. gonorroea; L. gonorrhoeae. monocytogenes, L. ivanovii; C.I. Botulinum, C.I. Difficile, C.I. tetany, C.I. ｓｏｒｄｅｌｌｉｉ、Ｆｒａｎｃｉｓｅｌｌａ ｔｕｌａｒｅｎｓｉｓ １、Ｐｒｅｖｏｔｅｌｌａ ａｌｂｅｎｓｉｓ、Ｌａｃｈｎｏｓｐｉｒａｃｅａｅ科菌ＭＣ２０１７ １、Ｂｕｔｙｒｉｖｉｂｒｉｏ ｐｒｏｔｅｏｃｌａｓｔｉｃｕｓ、Ｐｅｒｅｇｒｉｎｉｂａｃｔｅｒｉａ属菌ＧＷ２０１１＿ＧＷＡ２＿３３＿１０、Ｐａｒｃｕｂａｃｔｅｒｉａ属菌ＧＷ２０１１＿ＧＷＣ２＿４４＿１７、Ｓｍｉｔｈｅｌｌａ種ＳＣＡＤＣ、Ａｃｉｄａｍｉｎｏｃｏｃｃｕｓ種ＢＶ３Ｌ６、Ｌａｃｈｎｏｓｐｉｒａｃｅａｅ科菌ＭＡ２０２０、Ｃａｎｄｉｄａｔｕｓ Ｍｅｔｈａｎｏｐｌａｓｍａ ｔｅｒｍｉｔｕｍ、Ｅｕｂａｃｔｅｒｉｕｍ ｅｌｉｇｅｎｓ、Ｍｏｒａｘｅｌｌａ ｂｏｖｏｃｕｌｉ 237, Leptospira indai, Lachnospiraceae ND2006, Porphyromonas creviolicans 3, Prevotella disiens, and Porphyromonas macacae. The method of claim 2, wherein the inactivated CRISPR / Cas enzyme is an inactivated Cas12 protein. The method of claim 5, wherein the inactivated Cas12 protein has a mutation in the RuvC domain, or the inactivated Cas12 enzyme is inactivated Cas12a, Cas12b, Cas12c. The method of claim 6, wherein the deactivated Cas12a has a mutation corresponding to D908A or E993A of AsCpf1. 前記失活Ｃａｓ１２タンパク質は、Ｆｒａｎｃｉｓｅｌｌａ ｔｕｌａｒｅｎｓｉｓ、Ｐｒｅｖｏｔｅｌｌａ ａｌｂｅｎｓｉｓ、Ｌａｃｈｎｏｓｐｉｒａｃｅａｅ科菌、Ｂｕｔｙｒｉｖｉｂｒｉｏ ｐｒｏｔｅｏｃｌａｓｔｉｃｕｓ、Ｐｅｒｅｇｒｉｎｉｂａｃｔｅｒｉａ属菌、Ｐａｒｃｕｂａｃｔｅｒｉａ属菌、Ｓｍｉｔｈｅｌｌａ種、Ａｃｉｄａｍｉｎｏｃｏｃｃｕｓ種、Ｌａｃｈｎｏｓｐｉｒａｃｅａｅ科菌、Ｃａｎｄｉｄａｔｕｓ Ｍｅｔｈａｎｏｐｌａｓｍａ ｔｅｒｍｉｔｕｍ、Ｅｕｂａｃｔｅｒｉｕｍ ｅｌｉｇｅｎｓ、Ｍｏｒａｘｅｌｌａ ｂｏｖｏｃｕｌｉ、Ｌｅｐｔｏｓｐｉｒａ ｉｎａｄａｉ 、Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｃｒｅｖｉｏｒｉｃａｎｉｓ、Ｐｒｅｖｏｔｅｌｌａ ｄｉｓｉｅｎｓ及びＰｏｒｐｈｙｒｏｍｏｎａｓ ｍａｃａｃａｅ、Ｓｕｃｃｉｎｉｖｉｂｒｉｏ ｄｅｘｔｒｉｎｏｓｏｌｖｅｎｓ、Ｐｒｅｖｏｔｅｌｌａ ｄｉｓｉｅｎｓ、Ｆｌａｖｏｂａｃｔｅｒｉｕｍ ｂｒａｎｃｈｉｏｐｈｉｌｕｍ、Ｈｅｌｃｏｃｏｃｃｕｓ ｋｕｎｚｉｉ、Ｅｕｂａｃｔｅｒｉｕｍ種、Ｍｉｃｒｏｇｅｎｏｍａｔｅｓ（Ｒｏｉｚｍａｎｂａｃｔｅｒｉａ）群菌、Ｆｌａｖｏｂａｃｔｅｒｉｕｍ種、Ｐｒｅｖｏｔｅｌｌａ ｂｒｅｖｉｓ、Ｍｏｒａｘｅｌｌａ ｃａｐｒａｅ、Ｂａｃｔｅｒｏｉｄｅｔｅｓ ｏｒａｌ、Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｃａｎｓｕｌｃｉ、Ｓｙｎｅｒｇｉｓｔｅｓ ｊｏｎｅｓｉｉ、 Prevotella bryantii, Anaerovibrio species, Butyrivivrio fibrisolvens, Candidatus Methanomethylophilus, Bacteroidota species, Oribacteria species, Oribacteria species, Pseudibisu. The method of claim 5, wherein the inactivated Cas12 is an inactivated Cas12b protein having a mutation in the Nuc domain. The method of claim 9, wherein the deactivated Cas12b has a mutation corresponding to D570A, E848A, or D977A of AacC2c1. 前記失活Ｃａｓ１２ｂタンパク質は、Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ａｃｉｄｏｔｅｒｒｅｓｔｒｉｓ、Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ｃｏｎｔａｍｉｎａｎｓ、Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ｍａｃｒｏｓｐｏｒａｎｇｉｉｄｕｓ、Ｂａｃｉｌｌｕｓ ｈｉｓａｓｈｉｉ、Ｃａｎｄｉｄａｔｕｓ Ｌｉｎｄｏｗｂａｃｔｅｒｉａ、Ｄｅｓｕｌｆｏｖｉｂｒｉｏ ｉｎｏｐｉｎａｔｕｓ、Ｄｅｓｕｌｆｏｎａｔｒｏｎｕｍ ｔｈｉｏｄｉｓｍｕｔａｎｓ、Ｅｌｕｓｉｍｉｃｒｏｂｉａ門菌ＲＩＦＯＸＹＡ１２、Ｏｍｎｉｔｒｏｐｈｉｃａ ＷＯＲ＿２細菌ＲＩＦＣＳＰＨＩＧＨＯ２、Ｏｐｉｔｕｔａｃｅａｅ科菌ＴＡＶ５、Ｐｈｙｃｉｓｐｈａｅｒａｅ綱菌ＳＴ－ＮＡＧＡＢ－ Ｄ１、Ｐｌａｎｃｔｏｍｙｃｅｔｅｓ門菌ＲＢＧ＿１３＿４６＿１０、Ｓｐｉｒｏｃｈａｅｔｅｓ門菌ＧＷＢ１＿２７＿１３、Ｖｅｒｒｕｃｏｍｉｃｒｏｂｉａｃｅａｅ科菌ＵＢＡ２４２９、Ｔｕｂｅｒｉｂａｃｉｌｌｕｓ ｃａｌｉｄｕｓ、Ｂａｃｉｌｌｕｓ ｔｈｅｒｍｏａｍｙｌｏｖｏｒａｎｓ、Ｂｒｅｖｉｂａｃｉｌｌｕｓ種ＣＦ１１２、Ｂａｃｉｌｌｕｓ種ＮＳＰ２．１、Ｄｅｓｕｌｆａｔｉｒｈａｂｄｉｕｍ ｂｕｔｙｒａｔｉｖｏｒａｎｓ、Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ｈｅｒｂａｒｉｕｓ、Ｃｉｔｒｏｂａｃｔｅｒ ｆｒｅｕｎｄｉｉ、Ｂｒｅｖｉｂａｃｉｌｌｕｓ ａｇｒｉ（例えば、ＢＡＢ－２５００ ), And the method according to claim 9 or 10, derived from a bacterial species selected from the group consisting of Alicyclobacillium nodulans. The first inactivated CRISPR / Cas enzyme and the second inactivated CRISPR / Cas enzyme are the same, or the first inactivated CRISPR / Cas enzyme and the second inactivated CRISPR / Cas enzyme are different. The method according to any one of claims 1 to 11. 12. The method of claim 12, wherein the inactivated CRISPR / Cas enzyme is dCas9, dCas12a, dCas12b, dCas12c, or dCas14. The polymerases include Bst 2.0 DNA polymerase, Bst 2.0 WarmStart DNA polymerase, Bst 3.0 DNA polymerase, full length Bst DNA polymerase, giant fragment Bst DNA polymerase, giant fragment Bsu DNA polymerase, phi29 DNA polymerase, T7 DNA polymerase. , Gst polymerase, Taq polymerase, E.I. The method according to any one of claims 1 to 13, selected from the group consisting of a cleno fragment of colli DNA polymerase I, a KlenTaq DNA polymerase, a Pol III DNA polymerase, a T5 DNA polymerase, and a sequencenase DNA polymerase. The helicase was UvrD helicase, CRISPR-Cas3 helicase, E.I. colli helicase I, E.I. colli helicase II, E.I. colli helicase III, E.I. coli helicase IV, Rep helicase, DnaB helicase, PriA helicase, PcrA helicase, T4 Gp41 helicase, T4 Dda helicase, SV40LargeT antigen, yeast RAD helicase, RecD helicase, RecQ helicase, heat resistant T.I. tengcongensis UvrD helicase, heat resistant T.I. thermophilus UvrD helicase, heat resistant T.I. Selected from the group consisting of aquaticus DnaB helicase, Dda helicase, papillomavirus E1 helicase, paleobacillus MCM helicase, eukaryotic MCM helicase, and T7 Gp4 helicase, or said helicase from Thermoanaerobutter with mutant D403A / D404A. PcrA helicase, PcrA helicase from Bacillus species FJAT-27231 with D407A / D408A mutation, PcrA helicase from Bacillus megaterium with D415A / D416A mutation, Bacillus helicase from D407A / D408A mutation, Bacillus silk A PcrA helicase from the Paeniclostridium sordellii with a variant,
The method according to any one of claims 1 to 14. Amplification of the target nucleic acid is performed at about 37 ° C. to 65 ° C., amplification of the target nucleic acid is performed at about 50 ° C. to 59 ° C., or amplification of the target nucleic acid is performed at about 60 ° C. to 72 ° C. The method according to any one of claims 1 to 15, wherein the amplification of the target nucleic acid is performed at room temperature. The method according to claim 1, wherein the method is performed under mesothermal and isothermal conditions. 17. The method of claim 17, wherein the polymerase is a mesophilic polymerase. The method of claim 18, wherein the mesophilic polymerase is a Sau LF polymerase. 19. The method of claim 19, wherein the amplification of the target nucleic acid is performed at about 37 ° C. The helicase is E.I. E. coli UvrD helicase, optionally having two or more substitutions from D to A, such as D403 and D404, or said helicase is one or more variants corresponding to D409A and D410A. 17. The method of claim 17, wherein the helicase is a pcrA (UvrD) type helicase, or the helicase is Tte-UvrD D409A / D410A. The target nucleic acid sequence is about 20-30, about 30-40, about 40-50, or about 50-100 nucleotides in length, or the target nucleic acid sequence is about 100-200, about 100 in length. 1. 500, or about 100-1000 nucleotides, or the target nucleic acid sequence is about 1000-2000, about 2000-3000, about 3000-4000, or about 4000-5000 nucleotides in length. To the method according to any one of 21. The method according to any one of claims 1 to 22, wherein the first primer or the second primer comprises an RNA polymerase promoter. The method according to any one of claims 1 to 23, comprising one or more additives selected from clauding agents, cofactors, single-stranded binding proteins, and / or assisted proteins. The crowding agent is polyethylene glycol, the cofactor is ATP, the single-stranded binding protein is selected from T4 gp32 or thermophilic SSB (ET-SSB), and / or. 24. The method of claim 24, wherein the assist protein is dCpf1 and / or UvsX recombinase. In addition, a method selected from the group consisting of gel electrophoresis, insertion dye detection, PCR, real-time PCR, fluorescence, fluorescence resonance energy transfer (FRET), mass analysis, lateral flow assay, colorimetric assay, and CRISPR-based detection system. The method of any one of claims 1 to 25, comprising detecting the amplified nucleic acid. The method according to any one of claims 1 to 26, wherein the amplified target nucleic acid is detected by a system based on CRISPR Cas13, a system based on CRISPR Cas12, or a combination thereof. 27. The method of claim 27, wherein the amplified target nucleic acid is detected by a CRISPR Cas-13 based system comprising the LwaCas13 enzyme. The method according to any one of claims 1 to 28, wherein the target nucleic acid is detected with attomol sensitivity, or the target nucleic acid is detected with femtomole sensitivity. The target nucleic acid is selected from the group consisting of genomic DNA, mitochondrial DNA, viral DNA, plasmid DNA, circulating acellular DNA, environmental DNA, synthetic double-stranded DNA, and RNA, or the target nucleic acid is RNA. And the RNA is reverse transcribed into the cDNA prior to amplification,
The method according to any one of claims 1 to 29. 30. The method of claim 30, wherein the amplification is recombinase polymerase amplification (RPA). The method according to any one of claims 1 to 31, wherein the reaction is carried out in less than about 2 hours, less than about 90 minutes, less than about 60 minutes, less than about 30 minutes, or less than about 15 minutes. The method according to any one of claims 1 to 32, wherein the sample is a biological sample or an environmental sample. The biological sample may be blood, plasma, serum, urine, feces, sputum, mucus, lymph, lubricant, bile, ascites, pleural effusion, serous tumor, saliva, cerebrospinal fluid, atrioventricular fluid or vitreous fluid, or any body. 33. The method of claim 33, which is a secretion, a leak, an exudate, or a fluid obtained from a joint, or a swab on the surface of the skin or mucous membrane. The sample is blood, plasma, or serum obtained from a human patient, or the sample is a plant sample, the sample is an unpurified sample, or the sample is a purified sample. The method according to any one of claims 1 to 34. A method for amplifying and / or detecting a target single-stranded nucleic acid, wherein:
(A) Converting the single-stranded nucleic acid in a sample into a target double-stranded nucleic acid; and (b) performing the step according to claim 1.
The method described above. 36. The method of claim 36, wherein the target single-stranded nucleic acid is an RNA molecule. 37. The method of claim 37, wherein the RNA molecule is converted to the double-stranded nucleic acid by a reverse transcription and amplification step. The target single-stranded nucleic acid is single-stranded viral DNA, viral RNA, messenger RNA, ribosome RNA, translocated RNA, microRNA, small-molecular-weight interfering RNA, nuclear small-molecular-weight RNA, synthetic RNA, synthetic single-stranded DNA, long. The method according to any one of claims 36 to 38, which is selected from the group consisting of strand untranslated RNA, premicroRNA, viral dsRNA, and nonviral dsRNA. Further included is an amplifier primer containing a portion of the primer pair that is contained in the first primer; and the first primer of the primer pair intervenes in the first step of amplification, and the amplifier primer is the primer. The method of claim 1, comprising a step intervening in a subsequent step of amplification with the pair of said second primers. 40. The method of claim 40, wherein the amplifier primer and the first primer of the primer pair each contain a T7 promoter sequence. 40. The method of claim 40, wherein the amplifier primer has a length of about 23 to about 35 nucleotides. A system that amplifies and / or detects a target double-stranded nucleic acid in a sample, and:
a) Amplified CRISPR system, the amplified CRISPR system includes a first CRISPR / Cas complex and a second CRISPR / Cas complex, and the first CRISPR / Cas complex is the first CRISPR / Cas enzyme and the first. It comprises a first guide molecule that induces one CRISPR / Cas complex to the first strand of the target nucleic acid, and the second CRISPR / Cas complex is a second CRISPR / Cas enzyme and the second CRISPR / Cas. Includes a second guide molecule that directs the complex to the second strand of said target nucleic acid;
b) Helicase;
c) A primer pair containing a first primer and a second primer, where the first primer comprises a portion complementary to the first strand of the target nucleic acid, and the second primer is said to the target nucleic acid. Includes a part complementary to the second strand;
d) Polymerase; and optionally e) said system comprising a detection system to detect amplification of said target nucleic acid. 43. The system of claim 43, wherein the CRISPR / Cas enzyme is an inactivated CRISPR / Cas enzyme selected from the inactivated Cas9 enzyme or the inactivated Cas12 enzyme. 44. The system of claim 44, wherein the inactivated Cas12 enzyme is an inactivated Cas12a, Cas12b, or Cas12c enzyme. 43. The system of claim 43, wherein the CRISPR / Cas enzyme is a Cas9 or Cas12 enzyme. 46. The system of claim 46, wherein the Cas12 enzyme is a Cas12a, Cas12b, or Cas12c enzyme. The polymerases include Bst 2.0 DNA polymerase, Bst 2.0 WarmStart DNA polymerase, Bst 3.0 DNA polymerase, full length Bst DNA polymerase, giant fragment Bst DNA polymerase, giant fragment Bsu DNA polymerase, phi29 DNA polymerase, T7 DNA polymerase. , Gst polymerase, Taq polymerase, E.I. The item according to any one of claims 43 to 47, which is selected from the group consisting of a cleno fragment of colli DNA polymerase I, a KlenTaq DNA polymerase, a Pol III DNA polymerase, a T5 DNA polymerase, and a sequencenase DNA polymerase, and a Sau LF DNA polymerase. The system described. The helicase is UvrD helicase, CRISPR-Cas3 helicase, Rep helicase, PcrA helicase, E.I. colli helicase I, E.I. colli helicase II, E.I. colli helicase III, E.I. coli helicase IV, Rep helicase, DnaB helicase, PriA helicase, PcrA helicase, T4 Gp41 helicase, T4 Dda helicase, SV40LargeT antigen, yeast RAD helicase, RecD helicase, RecQ helicase, heat resistant T.I. tengcongensis UvrD helicase, heat resistant T.I. thermophilus UvrD helicase, heat resistant T.I. Selected from the group consisting of aquaticus DnaB helicase, Dda helicase, papillomavirus E1 helicase, paleobacillus MCM helicase, eukaryotic MCM helicase, and T7 Gp4 helicase, or said helicase from Thermoanaerobutter with mutant D403A / D404A. PcrA helicase, PcrA helicase from Bacillus species FJAT-27231 with D407A / D408A mutation, PcrA helicase from Bacillus megaterium with D415A / D416A mutation, Bacillus helicase from D407A / D408A mutation, Bacillus silk A PcrA helicase from the Paeniclostridium sordellii with a variant,
The system according to any one of claims 43 to 48. 49. The system of claim 49, wherein the helicase is Tte-UvrD D409A / D410A. The system of any one of claims 43-50, wherein the CRISPR / Cas enzyme, the helicase, and the polymerase function at the same temperature, and in some cases at about 37 ° C. A system for amplifying and / or detecting a target single-stranded nucleic acid in a sample, and the following:
a) A reagent that converts the target single-stranded nucleic acid into a double-stranded nucleic acid:
b) The system comprising the components of claim 43. 13. System. 53. The system of claim 53, wherein the detection system comprises the Cas13 enzyme LwaCas13. 43. The system of claim 43, further comprising an additional amplifier primer having promoter sequence identity with the first primer of the primer pair. A kit for amplifying and / or detecting a target single-stranded nucleic acid or a target double-stranded nucleic acid in a sample according to any one of claims 43 to 51 or 54, and an instruction manual for use. The kit, including a set of. The kit according to claim 56, further comprising a reagent for purifying the target single-stranded nucleic acid or the double-stranded nucleic acid in the sample. 28. The system of claim 48, wherein the helicase is Tte-UvrD D409A / D410A, or the helicase has one or more D to A substitutions in the enzyme. 40. The system of claim 48, further comprising one or more additives selected from clouding agents, cofactors, single-stranded binding proteins, and / or assisted proteins. The crowding agent is polyethylene glycol having a molecular weight of 20 K and is optionally provided at about 2.5% to about 6.5%, or the cofactor is ATP and optionally 0. Provided at 75-about 5 mM, the single-stranded binding protein is selected from T4 gp32 or thermophilic SSB (ET-SSB), and / or the assist protein is dCpf1 and / or UvsX recombinase. The system according to claim 59. 56. The system of claim 56, comprising mesophilic DNA polymerase, specifically Sau LF polymerase.