Patent Application
20250019753
The disclosure relates to methods for nucleic acid detection.
Sensitive, accurate and efficient detection of nucleic acid sequence variants is essential for precision medicine, where the individualized treatment is provided based on the unique genetic profile of each patient. However, profiling rare DNA or RNA variants with low allele frequencies in cancer samples has challenged current molecular diagnostic technologies for a long time (Khodakov D, et al. Adv Drug Deliv Rev 105, 3-19 (2016). [PubMed: 27089811]). The first-generation sequencing (FGS) approaches are not sensitive enough to detect a mutation rate under 10%. The next-generation sequencing (NGS) approaches are time-consuming and not economical. Allele-specific PCR methods are prone to artificially introduced mutations, while the specificity of qPCR-based methods depends excessively on the primers and the probes. Detection sensitivity of PCR-based methods can be enhanced by restriction digestion of wild-type (WT) sequences in the sample to enrich target sequences, but only at a cost of much complicated workflow (Zhao A H, et al. J Hematol Oncol 4, 40 (2011). [PubMed: 21985400]).
CRISPR-based gene editing systems have shown great potential for rapid and sensitive nucleic acid detection, including those based on Cas9, Cas12, and Cas13. Recently, Cas12-or Cas13-based detection has been applied to SARS-COV-2 diagnosis in coordination with isothermal amplification, which proved highly effective because the samples have little contaminants from host nucleic acid. In sharp contrast, for detection of rare genetic variants and mutations, the majority of the DNA or RNA are WT sequences, which significantly hampers the analysis.
Xianfeng, WANG et al. (Dual Methylation-Sensitive Restriction Endonucleases Coupling with an RPA-Assisted CRISPR/Cas13a System (DESCS) for Highly Sensitive Analysis of DNA Methylation and Its Application for Point-of-Care Detection, ACS Sens. 2021, 6, 2419-2428) disclosed dual methylation-sensitive restriction endonucleases (BstUI/HhaI) coupling with an RPA-assisted CRISPR/Cas 13a system for site-specific methylation detection of the SEPT9 gene. However, only the SEPT9 gene was tested in this article. It is unable to conclude that this method is applicable to detect any methylation(s) in any sequence of interest. Further, BstUI/HhaI used in this article are only sensitive to DNA CpG methylation in genome of a mammal, but not to Dam methylation or Dem methylation. And only Cas13a was used in the detection. Methods for sensitively, and simply detecting mutations, such as deletion, insertion and/or substituent in a nucleic acid of interest, were not disclosed in this article and remains elusive.
In general, provided are methods, kits, and materials for amplifying, enriching or detecting a target nucleic acid, especially in a small amount, in a sample by digesting or degrading non-target nucleic acids in the sample before or during amplification and may detect the amplified target nucleic acids.
In some embodiments, the disclosure provides a method for amplifying or enriching target nucleic acids with alternation(s) of interest at a specified site in a sample, including digesting or degrading non-target nucleic acids without the alternation(s) of interest at the specified site in the sample by exposing the non-target nucleic acids to one or more proteins having an activity of cleaving nucleic acid that recognizes the base(s) at the specified site before or during amplification.
In some embodiments, the disclosure provides a method for detecting a target nucleic acid with alternation(s) of interest at a specified site in a sample, including digesting or degrading non-target nucleic acids without the alternation(s) of interest at the specified site in the sample by exposing the non-target nucleic acids to one or more proteins having an activity of cleaving nucleic acid that recognize the specified site before or during amplification, and detecting the amplified target nucleic acid.
In one or more embodiments, the protein having an activity of cleaving nucleic acid is selected from the group consisting of: a restriction endonuclease, a Cas enzyme, an Ago enzyme, a ZFN enzyme, a TALEN enzyme, and a functional complex thereof, such as Cas enzyme/sgRNA complex, Ago/gDNA complex, or Cas12a/crRNA complex. Preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex. Preferably, the Ago/gDNA complex is a pfAgo/gDNA complex. Preferably, the Cas12a/crRNA complex is a LbCas 12a/crRNA complex.
In one or more embodiments, the proteins having an activity of cleaving nucleic acid is a restriction endonuclease, and the non-target nucleic acid contains the recognizing site of the restriction endonuclease at the specified site.
In one or more embodiments, the restriction endonuclease is selected from the group consisting of BclI, BsaBI, AclWI, Bst4CI, Xmil, BlsI, PspFI, CviKI-1, CviJI, MscI, EcoP15I, BspACI, BseLI, BstKTI, PspN4I, BspLI, NlaIV, Bmil, MaiI, RsnAI, BspEI, HpaII, Mrol, Kpn2I, BcoDI, BstDEI, BpulOI, BtsIMutI, BasBI, BtsCI, NspI, Fail, EcoRV and Btsal.
In one or more embodiments, the Ago enzyme is selected from a group consisting of pfAgo, cbAgo, LrAgo, pfAgo-mut, Apol, pfAgo, TtAgo and MjAgo.
In one or more embodiments, the protein having an activity of cleaving nucleic acid is a Cas enzyme, and a gRNA having a targeting region which binds to the non-target nucleic acid sequence at the specified site is used to direct the Cas enzyme cleavage of the nucleic acid sequence.
In one or more embodiments, the Cas enzyme is selected from the group consisting of Cas 9, Cas 12, Cas 13 and Cas 14, especially SpCas9, SaCas9, HypaCas9, StlCas9, spCas9-NG, LbCas12a, spCas9-mut, and ScCas9.
In one or more embodiments, exposing the non-target nucleic acids to the one or more proteins having an activity of cleaving nucleic acid is performed by adding the protein(s) in an amplification mixture for amplifying the target nucleic acids.
In one or more embodiments, the amplification is selected from the group consisting of: helicase-dependent amplification (HAD), polymerase chain reaction (PCR), DNA ligase chain reaction (LCR), isothermal DNA amplification, QBeta RNA replicase, RNA transcription-based amplification reactions, loop-mediated isothermal amplification (LAMP), RT-LAMP, recombinase polymerase amplification (RPA), reverse transcription-recombinase polymerase amplification (RT-RPA), helicase-dependent amplification (HDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification
(SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA).
In one or more embodiments, the amplification mixture is an isothermal nucleic acid amplification mixture.
In one or more embodiments, the amplification is RPA.
In one or more embodiments, the alternation includes deletion, substitution and insertion of one or more base(s) at the specified site as compared to the non-target nucleic acid.
In one or more embodiments, the alternation is alternation of two or more continuous bases as compared to the non-target nucleic acid.
In one or more embodiments, detection of the amplified target nucleic acid indicates the presence of the alternation in the subject.
In one or more embodiments, the target nucleic acid is detected by DNA staining, nucleic acid amplification, spectrophotometry, sequencing, fluorescent probe hybridization, fluorescence resonance energy transfer, optical microscopy, electron microscopy, CRISPR-based detection, visual based detection, sensor-based detection, color detection, gold nanoparticle based detection, electrochemical detection, semiconductor-based sensing or combinations thereof.
In one or more embodiments, the amplified target nucleic acid is detected with one or more protein(s) capable of recognizing a specific nucleic acid sequence, or a functional complex thereof.
In one or more embodiments, the protein(s) capable of recognizing a specific nucleic acid sequence include Cas enzyme, Ago enzyme, ZFN enzyme, TALEN enzyme, and functional complexes thereof.
In one or more embodiments, the Cas enzyme is selected from the group consisting of Cas 9, Cas 12, Cas 13 and Cas 14, especially includes but is not limited to SpCas9, SaCas9,HypaCas9, StlCas9, spCas9-NG, LbCas 12a, spCas9-mut, and ScCas9.
In one or more embodiments, the Ago enzyme is selected from the group consisting of pfAgo, cbAgo, LrAgo, pfAgo-mut, Apol, pfAgo, TtAgo and MjAgo.
In one or more embodiments, the functional complex is selected from the group consisting of Cas enzyme/sgRNA complex, Ago/gDNA complex and Cas12a/crRNA complex: more preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex: the Ago/gDNA complex is a pfAgo/gDNA complex: the Cas12a/crRNA complex is a LbCas12a/crRNA complex.
In one or more embodiments, the protein capable of recognizing a specific nucleic acid sequence is selected from the group consisting of LbCas12a, FnCas 12a, Lb5Cas12a, HkCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, LbuCas13a, LwaCas13a, LbaCas13a, PprCas13a, HheCas13a, EreCas13a, AsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, spCas9, pfAgo, cbAgo, LrAgo, Cas12b, Cas12a-mut, Cas 12b-mut, AapCas 12b, BrCas12b, CcaCas13b, PsmCas13b and AacCas12b, or functional complexes thereof.
In one or more embodiments, digesting or degrading and detecting are carried out sequentially or simultaneously.
In one or more embodiments, digesting or degrading and detecting are carried out in the same reaction system.
In yet another embodiment, the disclosure provides a kit for amplifying or enriching and may detect target nucleic acids with alternation(s) of interest at a specified site in a sample, including reagents for amplification of target nucleic acids with alternation(s) of interest at the specified site in the sample, and reagents for digesting non-target nucleic acids without the alternation(s) of interest at the specified site in the sample.
In one or more embodiments, the alternation includes deletion, substitution and/or insertion of one or more base(s) at the specified site as compared to the non-target nucleic acid.
In one or more embodiments, the alternation is alternation of two or more continuous bases as compared to the non-target nucleic acid.
In one or more embodiments, the reagents for digesting non-target nucleic acids include a protein having an activity of cleaving nucleic acid.
In one or more embodiments, the protein having an activity of cleaving nucleic acid is selected from the group consisting of: a restriction endonuclease, a Cas enzyme, an Ago enzyme, a ZFN enzyme, a TALEN enzyme, and a functional complex thereof, such as Cas enzyme/sgRNA complex, Ago/gDNA complex, or Cas 12a/crRNA complex. Preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex. Preferably, the Ago/gDNA complex is a pfAgo/gDNA complex. Preferably, the Cas12a/crRNA complex is a LbCas 12a/crRNA complex.
In one or more embodiments, the proteins having an activity of cleaving nucleic acid is a restriction endonuclease, preferably is selected from the group consisting of BclI, BsaBI, AclWI, Bst4CI, Xmil, BlsI, PspFI, CviKI-1, CviJI, Mscl, EcoP151, BspACI, BseLI, BstKTI, PspN4I, BspLI, NlaIV, Bmil, MaiI, RsnAI, BspEI, HpaII, Mrol, Kpn2I, BcoDI, BstDEI, Bpu1OI, BtsIMutI, BasBI, BtsCI, NspI, Fail, EcoRV and Btsal.
In one or more embodiments, the proteins having an activity of cleaving nucleic acid is a Ago enzyme, preferably selected from a group consisting of pfAgo, cbAgo, LrAgo, pfAgo-mut, Apol, pfAgo, TtAgo and Mj Ago.
In one or more embodiments, the protein having an activity of cleaving nucleic acid is a Cas enzyme, preferably is selected from the group consisting of Cas 9, Cas 12, Cas 13 and Cas 14, especially SpCas9, SaCas9, HypaCas9, StlCas9, spCas9-NG, LbCas12a, spCas9-mut, and ScCas9.
In one or more embodiments, the reagents for digesting non-target nucleic acids include restriction endonucleases, or Cas enzymes and a guide RNA having targeting region which binds to the non-target nucleic acid.
In one or more embodiments, the reagents for amplification of target nucleic acids with alternation(s) of interest at the specified site in the sample include reagent(s) used to perform any of helicase-dependent amplification (HAD), polymerase chain reaction (PCR), DNA ligase chain reaction (LCR), isothermal DNA amplification, QBeta RNA replicase, RNA transcription-based amplification reactions, loop-mediated isothermal amplification (LAMP), RT-LAMP, recombinase polymerase amplification (RPA), reverse transcription-recombinase polymerase amplification (RT-RPA), helicase-dependent amplification (HDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA).
In one or more embodiments, the reagents for amplification include reagents for PCR or isothermal amplification reaction. More preferably, the reagents for amplification include reagents used for RPA.
In one or more embodiments, the kit further includes reagents used for detecting the target nucleic acids, such as reagents used for DNA staining, nucleic acid amplification, spectrophotometry, sequencing, fluorescent probe hybridization, fluorescence resonance energy transfer, optical microscopy, electron microscopy, CRISPR-based detection, visual based detection, sensor based detection, color detection, gold nanoparticle based detection, electrochemical detection, semiconductor-based sensing or combinations thereof.
In one or more embodiments, the reagent(s) used for detecting the target nucleic acids include one or more protein(s) capable of recognizing a specific nucleic acid sequence, or a functional complex thereof.
In one or more embodiments, the protein(s) capable of recognizing a specific nucleic acid sequence include Cas enzyme, Ago enzyme, ZFN enzyme, TALEN enzyme, and functional complexes thereof.
In one or more embodiments, the Cas enzyme is selected from the group consisting of Cas 9. Cas 12, Cas 13 and Cas 14, especially includes but is not limited to SpCas9, SaCas9, HypaCas9, StlCas9, spCas9-NG, LbCas 12a, spCas9-mut, and ScCas9.
In one or more embodiments, the Ago enzyme is selected from the group consisting of pfAgo, cbAgo, LrAgo, pfAgo-mut, Apol, pfAgo. TtAgo and MjAgo.
In one or more embodiments, the functional complex is selected from the group consisting of Cas enzyme/sgRNA complex, Ago/gDNA complex and Cas12a/crRNA complex; more preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex; the Ago/gDNA complex is a pfAgo/gDNA complex; the Cas12a/crRNA complex is a LbCas12a/crRNA complex.
In one or more embodiments, the protein capable of recognizing a specific nucleic acid sequence is selected from the group consisting of LbCas12a, FnCas12a, Lb5Cas12a, HkCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, LbuCas13a, LwaCas13a, LbaCas13a, PprCas13a, HheCas13a, EreCas13a, AsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, spCas9, pfAgo, cbAgo, LrAgo, Cas12b, Cas12a-mut, Cas12b-mut, AapCas12b, BrCas12b, CcaCas13b, PsmCas13b and AacCas12b, or functional complexes thereof.
In one or more preferred embodiments, the kit contains a mixture including reagents for RPA and the reagents for digesting the nucleic acid that do not contain the alteration. In some embodiments, the mixture also includes reagents used for detecting the target nucleic acids.
In one or more embodiments, the kit includes the kit includes the protein for cleavage listed in any one of the ID No. in Table A and reagent(s) for the amplification method listed in the same ID No., and includes the protein for detection listed in the same ID No. for detection.
As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
The term “endonuclease activity” refers to an enzyme activity of cleaving a polynucleotide chain by separating nucleotides other than the two end ones.
“The protein having an activity of cleaving nucleic acid” targets a nucleic acid and digests the nucleic acid by recognizing a certain site (i.e., certain short sequence) in the nucleic acid and then cleaving the nucleic acid. The recognizing site and the cleaving site can be the same or different in the nucleic acid. The protein having an activity of cleaving nucleic acid includes but is not limited to a restriction endonuclease, a Cas enzyme, an Ago enzyme, a ZFN enzyme, a TALEN enzyme, and a functional complex thereof.
A “functional complex” of a protein, such as an endonuclease, may include the endonuclease per se and molecule(s) capable of assisting the endonuclease to function. For example, a sgRNA or crRNA may be necessary for a Cas enzyme to function as a endonuclease. This is well known in the art. Therefore, for example, a functional complex as used herein include but is not limited to Cas enzyme/sgRNA complex, Ago/gDNA complex, or Cas12a/crRNA complex. Preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex. Preferably, the Ago/gDNA complex is a pfAgo/gDNA complex. Preferably, the Cas 12a/crRNA complex is a LbCas12a/crRNA complex. When referring to nucleic acids herein, “loci”, “site” or “position” can be used interchangeable.
The term “polymerase” refers to an enzyme that performs template-directed synthesis of polynucleotides by addition of nucleotide units to a nucleotide chain using DNA or RNA as a template. The term encompasses both a full length polypeptide and a domain that has polymerase activity. DNA polymerases are well-known in the art, and include but are not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, Bacteriophage T4 or modified versions thereof.
“Thermally stable polymerase” as used herein, refers to any enzyme that catalyzes polynucleotide synthesis through thermal cycling. “Isothermal polymerase” as used herein, refers to any enzyme that catalyzes polynucleotide synthesis at a constant temperature (e.g., 37-42° C.) without thermal cycling, such as DNA recombinase polymerase derived from Bacteriophage T4.
The term “nucleic acid amplification” or “amplification reaction” refers to any in vitro means for multiplying the copies of a target sequence of nucleic acid. Such means include but are not limited to polymerase chain reaction (PCR), DNA ligase chain reaction (LCR), isothermal DNA amplification, QBeta RNA replicase, and RNA transcription-based amplification reactions as well as others known in the art. Particularly, such means include but are not limited to: loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), or isothermal multiple displacement amplification (IMDA).
“Amplifying” refers to a step of submitting a solution to conditions sufficient to allow for amplification of a polynucleotide. Components of an amplification reaction include, e.g., primers, a polynucleotide template, polymerase, nucleotides, and the like. The term amplifying typically refers to an “exponential” increase in target nucleic acid. However, amplifying as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid, such as is obtained with cycle sequencing. In isothermal DNA amplification, the reaction also contains single stranded DNA binding (SSB). In one or more embodiments of the disclosure, the reaction also contains a protein having an activity of cleaving nucleic acid (such as restriction endonucleases, a Cas enzyme, an Ago enzyme, a ZFN enzyme, a TALEN enzyme, and a functional complex thereof).
“Isothermal DNA amplification” can be performed at a constant temperature without thermal cycling, including but not limited to: nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), the loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), as well as the recombinase polymerase amplification (RPA) or enzymatic recombinase amplification (ERA).
“Recombinase polymerase amplification” or “RPA” is a highly sensitive and selective isothermal amplification technique, operating at 37-42°° C. It has been used to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples. “Enzymatic recombinase amplification” or “ERA” is another version of RPA with different thermally stable polymerase.
The RPA process starts when a recombinase protein (e.g., uvsX) from T4-like bacteriophages bind to primers in the presence of ATP and a crowding agent (a high molecular polyethyleneglycol), forming a recombinase-primer complex. The complex then interrogates double stranded DNA seeking a homologous sequence and promotes strand invasion by the primer at the cognate site. In order to prevent the ejection of the inserted primer by branch migration, the displaced DNA strand is stabilized by single-stranded binding proteins. Finally, the recombinase disassembles and a strand displacing DNA polymerase (e.g. large fragment of Bacillus subtilis Pol 1, Bsu) binds to the 3′ end of the primer to elongate it in the presence of dNTPs. Cyclic repetition of this process results in the achievement of exponential amplification (
An “oligonucleotide primer” or “primer” refers to an oligonucleotide sequence that has a homologous sequence on a target nucleic acid and serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety of lengths and are often less than 50 nucleotides in length, for example 12-30 nucleotides in length. The length and sequences of primers for use in nucleic acid amplification (e.g., PCR or RPA) can be designed based on principles known in the art.
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).
The terms “about” and “approximately equal” are used herein to modify a numerical value and indicate a defined range around that value. If “X” is the value, “about X” or “approximately equal to X” generally indicates a value from 0.90X to 1.10X. Any reference to “about X” indicates at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.10X.
Thus, “about X” is intended to disclose, e.g., “0.98X.” Thus, “from about 6 to 8.5” is equivalent to “from about 6 to about 8.5.” When “about” is applied to the first value of a set of values, it applies to all values in that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%, about 9%, or about 11%.”
Disclosed include methods, compositions, and kits for sensitively, accurately and efficiently amplifying, enriching and/or detecting a target nucleic acid, especially in a small amount (for example, less than 20%, less than 10%, less than 5%, less than 3%, less than 1%, less than 0.5% or less than 0.1% based on the total nucleic acids in a sample), in a sample by digesting or degrading non-target nucleic acids in the sample before or during amplification and may detect the amplified target nucleic acids. In one embodiment, the non-target nucleic acids in the sample, which generally are background nucleic acid molecules or wild type nucleic acid molecules without the target mutation(s) at specified site(s), are recognized by one or more proteins having an activity of cleaving nucleic acid at the specified site before or during amplification and cleaved. As a result, only the target nucleic acids with the alternation/mutation of interest are amplified and no or basically no non-target nucleic acids are amplified. Interference of the non-target nucleic acids during detection will be minimized and the sensitivity, accuracy and efficiency of the detection will be greatly increased. These methods, materials, and kits are especially suitable for convenient, sensitive and specific detection of rare targets (such as genetic variants and mutations, cancer-related mutations, etc.) for early cancer diagnosis and precision medicine.
Provided are methods for amplifying or enriching target nucleic acids with alternation(s) of interest at a specified site in a sample, including digesting or degrading non-target nucleic acids without the altemation(s) of interest at the specified site in the sample by exposing the non-target nucleic acids to one or more proteins having an activity of cleaving nucleic acid that recognizes the base(s) at the specified site before or during amplification.
Also provided are methods for detecting a target nucleic acid with altemation(s) of interest at a specified site in a sample, including digesting or degrading non-target nucleic acids without the alternation(s) of interest at the specified site in the sample by exposing the non-target nucleic acids to one or more proteins having an activity of cleaving nucleic acid that recognize the specified site before or during amplification, and detecting the amplified target nucleic acid.
In one or more embodiments, exposing the nucleic acids to the one or more proteins having an activity of cleaving nucleic acid is performed by adding the protein(s) in an amplification mixture for amplifying the target nucleic acid. In some embodiments, the amplification mixture is an isothermal nucleic acid amplification mixture.
As used herein, “alternation” and “mutation” refer to different base(s) at a specified site of the target nucleic acids as compared to the non-target nucleic acid sequence, including deletion, substitution and insertion of one or more base(s) at the specified site(s). Accordingly, the “non-target nucleic acid” herein refers to any nucleic acid without an alternation at a specified site that is needed to be digested to increase the amplification efficiency of the target nucleic acids. Generally, non-target nucleic acids are background nucleic acid molecules or wild type nucleic acid molecules without the target mutation(s) at specified site(s). Alternation may include alternation of two or more continuous bases as compared to the non-target nucleic acid. In some embodiments, alternation is a mutation of the target nucleic acid as compared to WT sequence. The alternation may include alternations known in the art, which cause diseases such as drug-induced deafness and congenital deafness, lead to severity of diseases or drug resistances, etc., including, HBV drug resistance mutation, tumor mutation, tumor or drug resistance mutation, tuberculosis drug resistance mutation, SARS-COV-2 mutation, FLT3-D835 mutation or the like. Examples of alternations or mutations include those summarized in Table 1.
In the present disclosure, the protein having an activity of cleaving nucleic acid that cleave the nucleic acid by recognizing the specified site digests the nucleic acid sequence without the alternation of interest at the specified site and leaves behind variants (i.e., with an alteration of interest), to enrich the target nucleotide sequences with alternation of interest for further detection.
Examples of proteins having an activity of cleaving nucleic acid include restriction endonuclease, Cas enzyme, Ago enzyme, ZFN enzyme, TALEN enzyme, and functional complexes thereof.
Restriction endonuclease used in the present disclosure may be any restriction endonuclease known in the art, include but is not limited to BclI, BsaBI, AclWI, Bst4CI, Xmil, BlsI, PspFI, CviKI-1, CviJI, MscI, EcoP15I, BspACI, BseLI, BstKTI, PspN4I, BspLI, NlaIV, Bmil, MaiI, RsnAI, BspEI, HpaII, Mrol, Kpn2I, BcoDI, BstDEI, BpulOI, BtsIMutI, BasBI, BtsCI, NspI, Fail, EcoRV and Btsal.
As used herein, the Ago enzyme includes but is not limited to pfAgo, cbAgo, LrAgo, pfAgo-mut, Apol, pfAgo. TtAgo and MjAgo.
As used herein the Cas enzyme includes but is not limited to Cas 9, Cas 12, Cas 13 and Cas 14, especially includes but is not limited to SpCas9, SaCas9, HypaCas9, StlCas9, spCas9-NG, LbCas 12a, spCas9-mut, and ScCas9.
A functional complex formed by Cas or Ago with their respective partner, such as Cas enzyme/sgRNA complex, Ago/gDNA complex and Cas 12a/crRNA complex, can also be used as a protein having an activity of cleaving nucleic acid. Preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex. Preferably, the Ago/gDNA complex is a pfAgo/gDNA complex. Preferably, the Cas12a/crRNA complex is a LbCas 12a/crRNA complex.
In the subject application, the terms sgRNA, gDNA and crRNA have a meaning commonly acknowledged in the art. In some embodiments, the protein having an activity of cleaving nucleic acid is a Cas enzyme, and the digesting or degrading step requires a gRNA (guide RNA) which forms a Cas enzyme/gRNA complex. That is, the digesting or degrading step is CRISPR-based digestion. In these embodiments, the guide RNA has a targeting region which binds to the non-target sequence to direct Cas enzyme cleavage of the bound sequence at a specified site. Guide RNAs can be designed based on principles known in the art. In preferable embodiments, guide RNA is designed to recognize WT FLT3 D835 sequence (-GATATC-) and the Cas enzyme/gRNA complex digest such sequence.
The protein having an activity of cleaving nucleic acid can cleave the nucleic acid at the specified site or other sites, depending on the particular protein used. Therefore, the recognizing site (i.e., the specified site) and the cleaving site can be the same or different in the non-target nucleic acid.
As a result of digestion by the proteins having an activity of cleaving nucleic acid, the non-target sequences are cleaved or degraded and amplification thereof are stopped before the cleavage site.
Amplification may be performed with a conventional amplification method, including helicase-dependent amplification (HAD), polymerase chain reaction (PCR), DNA ligase chain reaction (LCR), isothermal DNA amplification, QBeta RNA replicase, RNA transcription-based amplification reactions, loop-mediated isothermal amplification (LAMP), RT-LAMP, recombinase polymerase amplification (RPA), reverse transcription-recombinase polymerase amplification (RT-RPA), helicase-dependent amplification (HDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA).
Polymerase used in amplification may be any known polymerases and may be selected according to the used amplification method. Suitable DNA polymerases include but are not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, Bacteriophage T4 or modified versions thereof.
In one or more preferred embodiments, a recombinase polymerase amplification (RPA) is used in the method of the subject application. In one embodiment, RPA are carried out in the presence of the protein(s) having an activity of cleaving nucleic acid. As such, cleavage of the non-target sequence and amplification of the target sequence are carried out sequentially or simultaneously, preferably in the same reaction system. As such, in a preferred embodiment, provided is a modified RPA method, which includes amplification of target nucleic acids with alternation of interest in the presence of the protein(s) having an activity of cleaving nucleic acid as described herein with which the non-target nucleic acids are digested or degraded before or during amplification.
Primers used in the amplification may be designed according to the sequence of the target nucleotide molecule or segment. This is well known in the art. Generally, the two primers of a primer pair are located on each side (i.e., downstream and upstream, respectively) of the site to be cleaved in the non-target nucleic acid. The cleaved sequence (e.g. wild-type sequence) cannot be amplified by the primer pair.
To detect the target nucleic acid, any suitable detection technique may be used, such as, DNA staining, nucleic acid amplification, spectrophotometry, sequencing, fluorescent probe hybridization, fluorescence resonance energy transfer, optical microscopy, electron microscopy, CRISPR-based detection, visual based detection, sensor-based detection, color detection, gold nanoparticle based detection, electrochemical detection, semiconductor-based sensing, or combinations thereof. Sequencing may be FGS or NGS. Nucleic acid amplification may be qPCR.
In one or more embodiments, the target nucleic acid is detected with one or more protein(s) capable of recognizing a specific nucleic acid sequence, or a functional complex thereof. The specific nucleic acid sequence generally includes the mutation site.
Protein(s) capable of recognizing a specific nucleic acid sequence, i.e., protein(s) for detection, include but is not limited to Cas enzyme, Ago enzyme, ZFN enzyme, TALEN enzyme, and functional complexes thereof. In some embodiments, the detection is a CRISPR-based detection based on any known Cas proteins
As used herein the Cas enzyme includes but is not limited to Cas 9, Cas 12, Cas 13 and Cas 14, especially includes but is not limited to SpCas9, SaCas9, HypaCas9, StlCas9, spCas9-NG, LbCas12a, spCas9-mut, and ScCas9.
As used herein, the Ago enzyme includes but is not limited to pfAgo, cbAgo, LrAgo, pfAgo-mut, Apol, pfAgo, TtAgo and MjAgo.
A functional complex formed by Cas or Ago with their respective partner, such as Cas enzyme/sgRNA complex, Ago/gDNA complex and Cas 12a/crRNA complex, can also be used as a protein for detection. Preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex. Preferably, the Ago/gDNA complex is a pfAgo/gDNA complex. Preferably, the Cas 12a/crRNA complex is a LbCas12a/crRNA complex.
In some embodiments, the protein capable of recognizing a specific nucleic acid sequence include but is not limited to LbCas 12a, FnCas12a, Lb5Cas12a, HkCas12a, TsCas 12a, BbCas12a, BoCas 12a, Lb4Cas 12a, LbuCas13a, LwaCas13a, LbaCas13a, PprCas13a, HheCas13a, EreCas13a, AsCas12a, TsCas12a, BbCas12a, BoCas 12a, Lb4Cas 12a, spCas9, pfAgo, cbAgo, LrAgo, Cas12b, Cas12a-mut, Cas 12b-mut, AapCas12b, BrCas 12b, CcaCas13b, PsmCas13b and AacCas12b, or functional complexes thereof.
Cleavage of the non-target sequence, amplification of the target sequence and the detection of the target sequence may be carried out sequentially or simultaneously, preferably in the same reaction system.
The methods of the present disclosure may be used for the detection of clinically actionable information about a subject or a tumor in a patient, to detect and describe mutations and/or alterations in DNA of hematologic cancer cell in a blood or plasma sample that also contains an abundance of “normal”, somatic DNA, to monitor cancer remission, to inform treatment, such as dosage regime or immunotherapy treatment, to be used with fetal DNA to detect, for example, mutations characteristic of inherited genetic disorders, to detect and describe mutations and/or alterations in circulating tumor DNA in a blood or plasma sample that also contains an abundance of “normal”, somatic DNA. The DNA may include circulating tumor DNA in a patient's blood or plasma, or fetal DNA in maternal blood or plasma.
The term “hematologic cancer” is a group of malignant diseases that arise from cells in the bone marrow or lymphatic tissues, including but not limited to leukemia, lymphoma and myeloma, such as acute lymphocytic leukemia (ALL).
The methods of the present disclosure may include detection or isolation of hematologic cancer cells from a blood sample. The methods of the present disclosure may include detection or isolation of lymphocyte (e.g., PBMC, WBC) from a blood sample of a subject suffering hematologic cancer. For example, to isolating WBCs, red cells in peripheral blood or bone marrow blood samples are lysed, and unlysed WBCs is separated from lysed RBCs simply by centrifuge. Genomic DNA can be extracted by nucleic acid releaser (e.g., Suzhou GenDx Biotech, China).
The methods of the present disclosure may include detection or isolation of circulating tumor cells (CTCs) from a blood sample. CTC The methods of the present disclosure may employ an enrichment step to optimize the probability of rare cell detection, achievable through immune-magnetic separation, centrifugation or filtration.
The methods of the present disclosure can be used to detecting a target RNA, which may include reverse transcription from RNA to DNA. Such method may further include isolation of RNA from a sample (such as virus). Means for isolation of RNA and/or reverse transcription of RNA are well known in the art.
When a genomic alteration is thus detected, a report may be provided to, for example, describe the alteration in a patient.
Knowledge of a mutational landscape of a tumor may be used to inform treatment decisions, monitor therapy, detect remissions, or combinations thereof. For example, where the report includes a description of multiple mutations, the report may also include an estimate of a tumor mutation burden (TMB) for a tumor. It may be found that TMB is predictive of success of immunotherapy in treating a tumor, and thus the methods described herein may be used for treating a tumor.
Examples of target nucleic acids and their respective mutations are listed in No. 1-85 as shown in Table 1. Proteins for cleavage and amplification method for purpose of amplification or enrichment and proteins for detection, if necessary, are also listed for each of target nucleic acids. It should be understood that the protein for cleavage, the amplification method and the protein for detection listed for each of target nucleic acids is not the sole protein for cleavage, amplification method and protein for detection for amplifying, enriching and detecting that target nucleic acid. It can be determined that a suitable protein for cleavage, a suitable amplification method and/or a suitable protein for detection for each of the target nucleic acids with the present disclosure and the prior art.
Also provided is a kit for amplifying or enriching and may detect target nucleic acids with alternation(s) of interest at a specified site in a sample, including reagent(s) for amplification of target nucleic acids with alternation(s) of interest at the specified site in the sample, and reagent(s) for digesting non-target nucleic acids without the alternation(s) of interest at the specified site in the sample.
The reagent(s) for amplification of target nucleic acids with alternation(s) of interest at the specified site in the sample may be any one or more reagents used in any known amplification methods, including but is not limited to helicase-dependent amplification (HAD), polymerase chain reaction (PCR), DNA ligase chain reaction (LCR), isothermal DNA amplification, QBeta RNA replicase, RNA transcription-based amplification reactions, loop-mediated isothermal amplification (LAMP), RT-LAMP, recombinase polymerase amplification (RPA), reverse transcription-recombinase polymerase amplification (RT-RPA), helicase-dependent amplification (HDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA).
In some preferred embodiments, the reagent(s) for amplification include one or more reagent(s) for PCR or isothermal amplification reaction.
Examples of the reagent(s) for amplification include one or more of reaction buffer, polymerase (thermally stable polymerase or isothermal polymerase), primers, dNTP, activator, ddH2O, or single stranded DNA binding (SSB). The buffer can contain one or more buffer components and salts. In some embodiments, the buffer component is Tris-HCl. In some embodiments, the salts are KCI and MgCl2. Isothermal amplification system includes the GenDx ERA Kit sold by Suzhou GenDx Biotech, China. As described herein, the two primers of a primer pair are located on each side (i.e., downstream and upstream, respectively) of the site to be cleaved in the non-target nucleic acid. As such, the cleaved sequence (e.g. wild-type sequence) cannot be amplified by the primer pair. Polymerase may be any known polymerase and may be selected according to the used amplification method. Suitable DNA polymerases include but are not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, Bacteriophage T4 or modified versions thereof.
In some preferred embodiments, the reagents for amplification include reagents used for RPA.
The reagent(s) for digesting non-target nucleic acids without the alternation(s) of interest at the specified site in the sample includes the protein having an activity of cleaving nucleic acid as described herein.
Examples of proteins having an activity of cleaving nucleic acid include restriction endonuclease, Cas enzyme, Ago enzyme, ZFN enzyme, TALEN enzyme, and functional complexes thereof.
Restriction endonuclease used in the present disclosure may be any restriction endonuclease known in the art, include but is not limited to BclI, BsaBI, AclWI, Bst4CI, Xmil, BlsI, PspFI, CviKI-1, CviJI, MscI, EcoP15I, BspACI, BseLI, BstKTI, PspN4I, BspLI, NlaIV, Bmil, MaiI, RsnAI, BspEI, HpaII, Mrol, Kpn21, BcoDI, BstDEI, BpulOI, BtsIMutI, BasBI, BtsCI, NspI, Fail, EcoRV and Btsal.
The Ago enzyme includes but is not limited to pfAgo, cbAgo, LrAgo, pfAgo-mut, Apol, pfAgo, TtAgo and MjAgo.
The Cas enzyme includes but is not limited to Cas 9, Cas 12, Cas 13 and Cas 14, especially includes but is not limited to SpCas9, SaCas9, HypaCas9, StlCas9, spCas9-NG, LbCas12a, spCas9-mut, and ScCas9.
A functional complex formed by Cas or Ago with their respective partner, such as Cas enzyme/sgRNA complex, Ago/gDNA complex and Cas 12a/crRNA complex, can also be used as a protein having an activity of cleaving nucleic acid. Preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex. Preferably, the Ago/gDNA complex is a pfAgo/gDNA complex. Preferably, the Cas12a/crRNA complex is a LbCas 12a/crRNA complex.
The kit can further include reagent(s) used for detecting the target nucleic acid. The reagent(s) used for detecting the target nucleic acids include one or more reagents used for DNA staining, nucleic acid amplification, spectrophotometry, sequencing, fluorescent probe hybridization, fluorescence resonance energy transfer, optical microscopy, electron microscopy, CRISPR-based detection, or combinations thereof.
In one or more embodiments, the reagent(s) used for detecting the target nucleic acids include one or more protein(s) capable of recognizing a specific nucleic acid sequence, or a functional complex thereof.
The protein(s) capable of recognizing a specific nucleic acid sequence, i.e., protein(s) for detection, include but is not limited to Cas enzyme, Ago enzyme, ZFN enzyme, TALEN enzyme, and functional complexes thereof. In some embodiments, the detection is a CRISPR-based detection based on any known Cas proteins.
The Cas enzyme includes but is not limited to Cas 9, Cas 12, Cas 13 and Cas 14, especially includes but is not limited to SpCas9, SaCas9, HypaCas9, StlCas9, spCas9-NG, LbCas12a, spCas9-mut, and ScCas9.
The Ago enzyme includes but is not limited to pfAgo, cbAgo, LrAgo, pfAgo-mut, Apol, pfAgo, TtAgo and MjAgo.
A functional complex formed by Cas or Ago with their respective partner, such as Cas enzyme/sgRNA complex, Ago/gDNA complex and Cas 12a/crRNA complex, can also be used as a protein for detection. Preferably, the Cas enzyme/sgRNA complex is a Cas9/sgRNA complex, more preferably, a spCas9/sgRNA complex. Preferably, the Ago/gDNA complex is a pfAgo/gDNA complex. Preferably, the Cas 12a/crRNA complex is a LbCas12a/crRNA complex.
In some embodiments, the protein capable of recognizing a specific nucleic acid sequence include but is not limited to LbCas 12a, FnCas12a, Lb5Cas12a, HkCas12a, TsCas12a, BbCas12a, BoCas 12a, Lb4Cas 12a, LbuCas13a, LwaCas 13a, LbaCas13a, PprCas13a, HheCas13a, EreCas13a, AsCas12a, TsCas12a, BbCas12a, BoCas 12a, Lb4Cas12a, spCas9, pfAgo, cbAgo, LrAgo, Cas12b, Cas12a-mut, Cas 12b-mut, AapCas12b, BrCas 12b, CcaCas13b, PsmCas13b and AacCas12b, or functional complexes thereof.
Preferably, the kit contains a mixture including reagent(s) for RPA and the reagent(s) for digesting the non-target sequence, especially protein(s) having an activity of cleaving nucleic acid as described herein. In some embodiments, the mixture further includes reagent(s) used for segment detection.
Most preferably, the kit includes a protein for cleavage listed in any one of the ID No. in Table A and reagent(s) for the amplification method listed in the same ID No., for amplifying or enriching the target of the same ID No. In some other embodiments, the kit further includes the protein for detection listed in the same ID No. for detection of the target of the same ID No.
For example, the kit including the protein for cleavage and reagent(s) for the amplification method listed in ID No. 1 may include spCas9 and reagent(s) for performing RT-RPA, and is used to amplify or enrich a fragment of 12S rRNA containing 1494C>T. The kit may further includes a protein for detection which is LbCas12a for detection of the fragment.
5 The kit may also include instructions or other materials such as pre-formatted report shells that receive information from the methods to provide a report. The reagents, instructions, and any other useful materials may be packaged in a suitable container. Kits of the disclosure may be made to order. For example, an investigator may use, e.g., an online tool to design guide RNA and reagents for the performance of methods herein. The guide RNAs may be synthesized using a suitable synthesis instrument. The synthesis instrument may be used to synthesize 10 oligonucleotides such as gRNAs or single-guide RNAs (sgRNAs). Any suitable instrument or chemistry may be used to synthesize a gRNA. The resultant reagents (e.g., guide RNAs, and endonuclease(s)) can be packaged in a container for shipping as a kit.
In present disclosure, restriction digestion was included in the amplification (e.g., Recombinase Polymerase Amplification (RPA)) step to destroy nucleic acids without an alteration or certain alteration (e.g., wild-type sequences), thus presumably enhancing detection sensitivity. Using FLT3 D835 mutations as a model, the inventor optimized the method by crRNAs and RPA primers screening, and compared it with conventional methods (FGS, NGS, and qPCR) on a series of plasmid templates and 112 clinical samples. A method herein reached a sensitivity of 0.001% for detecting FLT3 D835 mutations, which represented the highest level achieved by any mutation detection methods. The entire workflow (from sample preparation to data output) took only an hour and required only simple instruments and operations. Similar detection sensitivity and accuracy were also achieved for all the other cancer mutations, i.e., IDH2 R172K. EGFR L858R and e19del, and NRAS G12D, showing the method will be invaluable for point-of-care cancer diagnosis and precision medicine.
The present disclosure further provides use of one or more protein(s) having an activity of cleaving nucleic acid that recognizes base(s) at a specified site of non-target nucleic acids in the manufacture of a reagent or a kit for amplifying or enriching target nucleic acids with alternation(s) of interest at the specified site as compared to the target nucleic acids. Preferably, in addition to the protein(s), the reagent or the kit may further include reagents for performing amplification of the target nucleic acids, such as primers, buffer or the like. The amplification may be any one of the amplifications as disclosed herein.
Also provided is use of one or more protein(s) having an activity of cleaving nucleic acid that recognizes base(s) at a specified site of non-target nucleic acids in the manufacture of a reagent or a kit for detecting target nucleic acids with alternation(s) of interest at the specified site as compared to the target nucleic acids. Preferably, in addition to the protein(s), the reagent or the kit may further include reagents for performing amplification of the target nucleic acids, such as primers, buffer or the like, and/or reagents for detecting the amplified target nucleic acids. The amplification may be any one of the amplifications as disclosed herein, and the detection may be any one of the detection as disclosed herein.
The following example is provided in order to better enable one of ordinary skill in the art to make and use the disclosed compositions and methods, and is not intended to limit the scope of the disclosure in any way.
A total of 112 AML patient samples were collected from the Hematology Department in Zhongnan Hospital of Wuhan University under an approved Institutional Review Board protocol. For genomic DNA extraction, 200˜500 μl peripheral blood or bone marrow blood samples were mixed upside down four times with the red cell lysing reagent (Biosharp, Hefei, China). Then, 1 minute of minicentrifuge was used to separate lysed RBCs and unlysed WBCs. Finally, the precipitated WBCs were split by a 100 μl nucleic acid releaser (Suzhou GenDx Biotech, China) at 95° C. for 3 min to release genomic DNA. Two microliters of the treated sample were used for the subsequent assay.
A 675-bp DNA fragment covering the WT FLT3-D835 site was PCR amplified with primers P1 and P2 from a wild-type patient's genomic DNA, then fused and cloned into the pGem-T vector (Takara, China). After that, the recombinant plasmids were transformed into E. coli DH5α, extracted with an Axy Prep Plasmid Miniprep Kit (Axygen, CA, USA), and quantified by a Nanodrop2000 (Thermo Fisher Scientific, MA, USA). For the construction of FLT3-D835Y/H/V/F plasmids, primers P3 and P4 carrying the D835Y mutation, primers P5 and P6 carrying the D835H mutation, primers P7 and P8 carrying the D835V mutation, and primers P9 and P10 carrying the D835F mutation were used to amplify the wild-type Tvec-FLT3-D835 plasmid. Then, the amplified fragments were fused and cloned into the pGem-T vector (Takara, China). In crRNA screening, a 351-bp DNA fragment harboring the D835region was amplified with primers P11 and P12 from the above recombinant plasmids, then purified and quantified as mentioned above. The plasmid templates of IDH2-R172K, EGFR-L858R. NRAS-G12D, and their WT forms were constructed in the same way. And the plasmid templates of EGFR-e19del (E746-A750 deletion) and its corresponding WT form were directly synthesized by GenScript (Nanjing, China). The nucleotide sequences of all primers are listed in Table 2.
The Cas12a-based detection was performed according to a previous description with modifications (Wang X et al. Commun Biol 3, 62 (2020). [PubMed: 32047240]). Briefly, crRNAs were designed according to the target sequences and synthesized by GenScript (Nanjing, China). The nucleotide sequences of all crRNAs are listed in Table 3. Cas12a protein was expressed and purified as described previously (Creutzburg SCA et al. Nucleic Acids Res 48, 3228-3243 (2020). [PubMed: 31989168]). The 20 μl reaction system included Cas12a (200ng/μl, 1 μl), crRNA (100 nM, 1 μl), 10× NEBuffer 3.1 (2 μl, NEB, MA, USA), RNase inhibitor (1 μl, Novoprotein, China), ssDNA-FQ reporter (25 μM, 1 μl, Genewiz, NJ, USA), an appropriate amount of PCR product or 5 μl RPA product to test, and supplementary ddH2O. After sufficient mixing on the vortex shaker, the mixture was incubated at 37° C. for 20 min, and then the green fluorescence signal was visualized under a 485-nm blue lamp (Sangon, Shanghai, China). Fluorescence kinetics were monitored using a monochromator with excitation at 485 nm and emission at 520 nm.
Isothermal amplification of plasmids or patient genomic DNA was conducted by the GenDx ERA Kit (Suzhou GenDx Biotech, China). For CRISPR detection, the 50 μl RPA reaction system included 20 μl reaction buffer, 11 μl ERA basic buffer, 2.5 μl forward primer (10 nM), 2.5 μl reverse primer (10 nM), 2 μl DNA template, 2 μl activator, and supplementary ddH2O. For the Method of the disclosure, an additional 2 μl restriction enzyme was mixed into the above RPA system. Fastcut-EcoRV (Monad, Suzhou, China), FastDigest-BseLI, FastDigest-SaqAI, FastDigest-Mscl, and FastDigest-Bvel (Thermo Fisher Scientific, MA, USA) were used in Method detection of FLT3-D835Y, IDH2-R172K, EGFR-e19del, EGFR-L858R, and NRAS-G12D, respectively. Then, the mixture was incubated at 37° C. for 20 min. After RPA, 5 μl of the amplification product was transferred to the crRNA-guided Cas12a reaction. The primers for RPA are listed in Table 2.
For FGS, 25 μl PCR products or 25 μl RPA products were purified using an Axy Prep PCR Clean-up Kit (Axygen, CA, USA) and quantified by a Nanodrop2000 (Thermo Fisher Scientific, MA, USA). For each sample, approximately 300 ng amplified DNA fragments were sent to FGS by Tsingke (Beijing, China). For NGS, different barcoded primers were used to amplify the FLT3-D835 region of different samples. The PCR products were purified and mixed equally for NGS by the Illumina NextSeq 500 (2 x 150) platform at the CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China. The primers for NGS are listed in Table 4.
TaqMan qPCR probes and primers were designed and synthesized by Tsingke (Beijing, China). The probes were the complementary sequence to the FLT3-D835Y template with 5′ reporter dye FAM and 3′ MGB. The 20 μl qPCR system included 2× Taq Pro HS Universal Probe Master Mix (10 μl, Vazyme, Nanjing, China), qPCR-F (10 μM×0.4 μl), qPCR-R (10 μM×0.4 μl), TaqMan probe (10 μM×0.2 μl), Template DNA (1 μl), and ddH2O (8 μl). PCR cycling conditions were 95° C. for 30 s and 45 cycles of 95° C. for 10 s and 60° C. for 30 s. The sequences of qPCR primers and probes are listed in Table S4. Commercial kits performed the qPCR detection of EGFR-e19del, EGFR-L858R and NRAS-G12D mutations.
All experiments were repeated three times. Statistical analyses were carried out with GraphPad Prism 8.0. Unpaired two-tailed Student's t-test was used for comparison between two groups. Quantitative data are expressed as mean value±standard error. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001: ns., no significance.
As proof-of-concept, we used Method herein to detect the drug-resistant FLT3-D835 mutations in acute myeloid leukemia (AML), which includes four major mutations according to the cBioportal database: D835Y (c.2503G>T) with a frequency of 45%, D835H (c.2503G>C) with 22%, D835V (c.2504A>T) with 14%, and D835F (c.2503GA>TT) with less than 1% (
Next, to test the feasibility of EcoRV digestion in the RPA reaction, 5e10 copies of 100% WT and 100% D835Y/H/V/F PCR fragments were treated in the RPA mixture without primers, at 37° C. for 20 minutes, and then detected by Cas12a reaction. The fluorescence signal of WT D835 fragments was eliminated by EcoRV digestion (
The specificity of crRNAs in the Cas 12a reaction determines the accuracy of CRISPR detection. To screen for optimal crRNA for detecting FLT3-D835Y, we designed four crRNAs (Table 3), with FLT3-D835Y-crRNA1 perfectly matching the mutant sequence and FLT3-D835Y-crRNA2-4 bearing various mismatches (
To simplify the diagnosis of the four drug-resistant FLT3-D835 mutations, we pooled the D835Y/H/V/F crRNAs (MMT-crRNAs) and the 4 mutant templates into a single reaction, finding that it produced strong fluorescence signal (
We next sought to improve method sensitivity by optimizing the RPA amplification efficiency. To this end, three forward (RPA-F1-3) and reverse (RPA-R1-3) primers (Table 2) were designed (
With the optimized gRNA and primers, we set out to determine the detection limit of Method for FLT3-D835Y. We first quantified the effect of EcoRV on WT sequence, finding that EcoRV could almost completely inhibit the amplification of up to 1e6 copies of templates (
We next compared our method with the commonly used qPCR-based detection method. We designed two D835Y-specific TaqMan probes and a pair of qPCR primers (Table 5) for FLT3-D835Y detection (
After the tests on plasmid templates, frozen cell samples of AML patients were used for D835Y/V/H/F mutation detection. Briefly, genomic DNA of patient cells was released by a nucleic acid releaser and then treated by the present method. Finally, the results were visualized with naked eyes under a blue lamp (
Considering clinical detection of drug-resistant mutation by NGS is time-consuming, we aim to further simplify the whole Method diagnosis process of drug-resistant FLT3-D835 mutations. To this end, we developed a white blood cell (WBC) enrichment method to treat fresh peripheral blood drawn from AML patients (
We next benchmarked our method against the commonly used FGS for detecting FLT3-D835 mutations in 80 AML patients (P33-P112) with unknown FLT3-D835 mutation status, with the samples also analyzed by NGS as the gold standard. Method, but not FGS, was able to detect D835Y in P38, P59, P71, P80, P83, and P106 (
To verify the versatility of our method, we applied it to mutations at 4 other genes (IDH2, EGFR and NRAS). IDH2 R172K is a hotspot mutation in glioma and leukemia, and of prognostic and therapeutic value. EGFR e19del and L858R are the two main mutations sensitive to EGFR-TKIs, thus of great therapeutic value for patients with lung cancer. At the same time, the NRAS G12D is a driver mutation in leukemia and colorectal cancer. All four mutations are important testing items in the clinic. We first compared Method with the CRISPR detection method to detect 1e5 copies of plasmid templates of 1% or 0.1% mutation rate. The results showed that the WT fluorescence signals were strong while the signals of all the four mutations were weak in CRISPR detection. However, the WT signals were almost invisible while the mutation signals were significantly increased in the present method (
We also detected EGFR-e19del, EGFR-L858R, and NRAS-G12D mutations using commercial kits based on fluorescence qPCR. The tested samples were 1e5 copies of plasmid templates with a mutation rate of 10%, 1%, 0.1%, and 0% (WT), respectively. The results of all three sites showed that, the amplification curves of different samples were gradually shifted to right, consistent with the decreased mutation rate. However, we noticed the strong fluorescence signals in WT samples (
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.
The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/131956 | Nov 2021 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/133119 | 11/21/2022 | WO |
