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WO2025096367A1 - Variants de cas12a thermostables et/ou modifiés par charge pour détection d'acides nucléiques - Google Patents

Variants de cas12a thermostables et/ou modifiés par charge pour détection d'acides nucléiques Download PDF

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Publication number
WO2025096367A1
WO2025096367A1 PCT/US2024/053313 US2024053313W WO2025096367A1 WO 2025096367 A1 WO2025096367 A1 WO 2025096367A1 US 2024053313 W US2024053313 W US 2024053313W WO 2025096367 A1 WO2025096367 A1 WO 2025096367A1
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Prior art keywords
enzyme
cas12
nucleic acid
variant
sequence
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Piyush K. Jain
Santosh RANANAWARE
Katelyn MEISTER
Grace SHOEMAKER
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated
  • CRISPR-based diagnostics have elevated nucleic acid detection in terms of sensitivity, specificity, and rapidness.
  • CRISPR/Cas technology works by introducing a CRISPR associated (Cas) nuclease and a short guide RNA sequence with a region complimentary to a target sequence/site.
  • the guide RNA/Cas complex may be directed to a target.
  • This complex then acts as molecular pair of scissors to cut the target sequence at a specific site creating double- stranded cuts in the target DNA or a single-stranded cut in the target RNA.
  • This specific target recognition and cleavage is also referred to as “cis-cleavage.”
  • CRISPR technology can be used in the field of molecular diagnostics for swift, sensitive, and precise nucleic acid detection across a diversity of sample types.
  • the evolution of nucleic acid detection has been significantly accelerated by the advent of CRISPR-based diagnostic platforms, which exploit the collateral cleavage activity of enzymes such as Cas12 and Cas13. These platforms are prized for their speed, sensitivity, and accuracy, making them invaluable, particularly in resource-scarce settings. Nevertheless, there remains a pressing need to adapt these technologies to enhance their simplicity, efficiency, and suitability for field use and point-of- care applications.
  • the disclosure in one aspect, relates to a one-pot nucleic acid detection platform that is designed to optimize nucleic acid diagnostics while ensuring quick and dependable results.
  • the platform uniquely incorporates the thermostable Cas12b enzyme, enabling direct nucleic acid detection from untreated samples.
  • the disclosure also relates to positively- and negatively-charged designed variants of Cas12a and their use for detection of nucleic acids.
  • the pH and thermal conditions disclosed herein that allow the one pot reaction without extraction would work with any enzyme that is functional under these conditions.
  • FIG.1 is a diagram showing PAM effects on CRISPR-Cas12a detection under various scenarios as known in the art.
  • FIG.2 is a schematic showing detection of non-canonical PAM containing targets with PAMless Identification of Nucleic Acids with CRISPR/Cas12a (PICNIC).
  • FIGs.4A-4B show PICNIC can be expanded to diverse type V CRISPR-Cas systems.
  • FIG.5 shows PAM dependence of SPLENDID with RT-LAMP.
  • FIG. 6 shows one-pot PAM-less detection with RFND BrCas12b at 62 °C. Error bars represent SEM.
  • FIG.7 is a comparison of the originally-developed PICNIC workflow versus the disclosed, simplified workflow.
  • FIG.8 shows extraction-free detection of positive SARS-CoV-2 saliva samples with and without a 5 minute heating step at 95 °C.
  • FIG.9 shows a method for generating charged ErCas12a variants. WT ErCas12a is used as the template, and both net positive and net negative variants were purified.
  • FIG.10 shows aligning and identifying conserved positive residues in Cas12a enzymes. Sequences listed correspond to portions of SEQ ID NOs.7-29.
  • FIGs.11A-11B show wild type ErCas12a (FIG.11A) and SuperPos ErCas12a, where positive mutations are highlighted in a yellow circle.
  • SuperPos ErCas12a includes the mutations T29K, L88K, S123K, and A171K (SEQ ID NOs.2, 6).
  • FIG.12 shows aligning and identifying conserved negative residues in Cas12a enzymes. Sequences listed correspond to portions of SEQ ID NOs.7-29.
  • FIGs.13A-13D show models of WT ErCas12a and SuperNeg ErCas12a in main views (FIGs.13A-13B of WT and FIGs.13C-13D for SuperNeg).
  • SuperNeg ErCas12a includes the mutations Q32E, A107E, S537D, Y610E, and N826D. Negative charges are found more on the surface, where environmental interactions are expected to be observed (SEQ ID NOs.3, 5).
  • FIGs.14A-14C show melting temperatures of WT ErCas12a, SuperPos, and SuperNeg variants at pH 7 (FIG.14A), pH 8 (FIG.14B), and ph 9 (FIG.14C). Melting points are shown as inflection points in derivatives of fluorescence spectra.
  • FIGs. 15A-15B show trans-cleavage functionality of WT ErCas12a, SuperPos, and SuperNeg variants at different temperatures.
  • FIG.16 shows the trans cleavage functionality of WT ErCas12a, SuperPos, and SuperNeg variants at different pH values over a time period of 60 min.
  • FIGs.17A-17C show fold change in fluorescence activity indicating trans cleavage for WT, SuperPos, and Super Neg ErCas12a variants at different temperatures at pH 7 (FIG.17A), ph 8 (FIG.17B), and ph 9 (FIG.17C).
  • FIGs.18A-18C show fold change in fluorescence activity indicating cis cleavage for WT, SuperPos, and SuperNeg ErCas12a variants at different temperatures at pH 7 (FIG.18A), ph 8 (FIG.18B), and ph 9 (FIG.18C).
  • FIGs.18A-18C show fold change in fluorescence activity indicating cis cleavage for WT, SuperPos, and SuperNeg ErCas12a variants at different temperatures at pH 7 (FIG.18A), ph 8 (FIG.18B), and ph 9 (FIG.18C).
  • FIGs.20A-20B show a comparison between use of SupErNeg variants to crENHANCE ATTORNEY DOCKET NO. T19227WO001 (222112-2280) v1 and v2 at 37 °C for detection of a malaria gene (FIG.20A) and a SARS-CoV-2 nucleocapsid gene (FIG.20B).
  • FIG.21A shows a schematic of a split activator system for RNA detection using a PAM- containing 12 nucleotide dsDNA activator or a linker DNA, pre-attached to the crRNA.
  • FIG.21B shows detection of a synthetic RNA target using linker DNA.
  • CRISPR-ULTIMATE a simple diagnostic platform based on thermostable Cas12b enzymes that can quickly detect nucleic acid targets from unextracted samples in a single step.
  • the disclosed platform combines nucleic acid extraction, RT-LAMP based amplification, and fluorescence readout in a single reaction tube, thereby streamlining the assay preparation and reducing the risk of contamination.
  • CRISPR ULTIMATE is also capable of detecting any target sequence without the constraint of a PAM or TAM sequence, thus making it a powerful tool for nucleic acid detection.
  • CRISPR-ULTIMATE can be applied to the direct detection of SARS-CoV-2 RNA from crude saliva samples providing a sample-to-result in less than one hour.
  • the CRISPR-ULTIMATE protocol consists of two primary stages: an initial 5-minute sample lysis step, followed by a single-tube combined CRISPR and RT-LAMP reaction. Further in this aspect, the lysis step ensures the liberation of nucleic acids in the sample, facilitating their subsequent rapid amplification and detection.
  • a distinct advantage of CRISPR-ULTIMATE is its ability to detect target sequences without necessitating a Protospacer Adjacent Motif (PAM), distinguishing it from other CRISPR-based systems.
  • the disclosed platform eliminates the need for nucleic acid extraction, further simplifying the workflow and accelerating the time to result.
  • CRISPR-ULTIMATE has successfully been used to detect SARS-CoV-2 RNA in crude saliva samples, delivering precise results in under an hour.
  • ATTORNEY DOCKET NO. T19227WO001 (222112-2280) [0036]
  • the CRISPR-Cas12a system is a powerful tool for genetic research, enabling precise gene editing and nucleic acid detection.
  • SpentErCas is a novel variant of the CRISPR-Cas12a enzyme, specifically derived from the Eubacterium rectale (ErCas12a).
  • this enzyme has been biochemically engineered to exhibit an increased overall positive or negative charge compared to the wild-type enzyme (SEQ ID NO. 1), resulting in enhanced performance across a variety of applications.
  • SEQ ID NO. 1 wild-type enzyme
  • disclosed herein is a library of ErCas12a enzymes that have been designed with increasing positive and negative charges.
  • structure-guided mutagenesis was used to systematically substitute neutral amino acids with those containing either positively charged (Lys, Arg, His) or negatively charged (Glu, Asp) side chains, thereby enriching the local charge at different domains of the enzyme.
  • positively charged (Lys, Arg, His) or negatively charged (Glu, Asp) side chains thereby enriching the local charge at different domains of the enzyme.
  • Glu, Asp negatively charged
  • the disclosed charged Cas12a variants work at a broader temperature and pH range compared to the wild type Cas12a (SEQ ID NO.1), thus making them ideal candidates for diverse genomic applications.
  • Cas12 Variant Enzymes [0038]
  • a Cas12 variant enzyme derived from and bearing an increased net charge relative to a wild-type Cas12 enzyme (SEQ ID NO.1).
  • the increased net charge can be a positive charge or a negative charge.
  • the Cas12 variant enzyme can include one or more of the following mutations: T29K, L88K, S123K, and A171K.
  • the Cas12 variant enzyme when the charge is a negative charge, can include one or more of the following mutations: Q32E, A107E, S537D, Y610E, and N826D.
  • the enzyme can exhibit increased stability at extreme temperatures including, but not limited to, temperatures between about 25 °C and about 37 °C and/or above 65 °C, relative to a wild-type Cas12 enzyme.
  • the enzyme can exhibit increased stability across a broad pH range relative to a wild-type Cas12 enzyme such as, for example, increased stability at acid and/or alkaline pH levels.
  • the wild-type Cas12 enzyme can be a Cas12a enzyme such as a Eubacterium rectale Cas12a (SEQ ID NO.1), although Cas12a enzymes from other organisms ATTORNEY DOCKET NO. T19227WO001 (222112-2280) are also contemplated and should be considered disclosed.
  • the Cas12 variant enzyme exhibits higher trans-cleavage activity than the wild-type Cas12 enzyme.
  • a method for detecting a target nucleic acid in a biological sample including at least the steps of: (a) performing a high temperature lysis step on the biological sample to form a lysed sample; and (b) admixing the lysed sample with a Cas12 enzyme having enhanced stability under at least one extreme condition, a guide nucleic acid sequence, optionally a reverse transcriptase, and a fluorescence-quencher reporter molecule; wherein, in the presence of the target nucleic acid, the fluorescence-quencher reporter molecule is cleaved by the Cas12 enzyme, separating a fluorophore from a quencher and producing a fluorescence signal.
  • the fluorophore can be 5(6)-carboxyfluorescein (56-FAM), 5′- hexachlorofluorescein (5HEX), or any combination thereof, while the quencher can be 3′-Iowa Black FQ (3IABkFQ).
  • the fluorescence-quencher reporter molecule can be 56-FAM-TTATT-3IABkFQ or 5HEX-TTTTTT-3IABkFQ.
  • the target nucleic acid can be RNA and the reverse transcriptase can be included.
  • the Cas12 enzyme can be Cas12b.
  • RT-LAMP is typically used in the disclosed methods for RNA detection only.
  • the target nucleic acid can be amplified prior to or during detection.
  • the reverse transcriptase makes DNA (i.e. a cDNA molecule) from sample RNA using primers designed to work with the DNA to be detected. Amplification can then be conducted as known in the art using a DNA polymerase such as, for example, Bst DNA polymerase.
  • the reaction can proceed while held at constant temperature using a hot water bath; temperature cycling is not needed.
  • the target nucleic acid can be RNA or DNA and the reverse transcriptase is not included.
  • the Cas12 enzyme can be Cas12a.
  • the disclosed systems and methods can function to directly detect either DNA or RNA using SAHARA (Split Activator for Highly Accessible RNA Analysis) without the need for amplification reverse transcription, or strand displacement.
  • SAHARA Split Activator for Highly Accessible RNA Analysis
  • the Cas12 enzyme having enhanced stability can be a Type V Cas12 enzyme such as, for example, a Cas12b enzyme.
  • the Cas12b enzyme can be a thermostable enzyme, such as, for example, a BrCas12b variant including, but not limited to, RFND BrCas12b (SEQ ID NO.4).
  • step (a) includes heating the biological sample to 95 °C for 5 min. In some aspects, step (a) also includes contacting the biological sample with a buffer having an alkaline pH such as, for example, pH 8.0. In any of these aspects, step (a) denatures double stranded DNA to single stranded DNA.
  • a protospacer adjacent motif PAM is not required for detecting the target nucleic acid.
  • the biological sample does not need to be subjected to an extraction or purification step prior to performing step (a).
  • the target nucleic acid can be RNA or DNA.
  • the target nucleic acid can be a SARS-CoV-2 RNA, an HIV RNA, a malaria mRNA, or any RNA or mRNA associated with an RNA virus or clinically relevant disease or condition.
  • the biological sample can be blood, urine, saliva, plasma, or a combination thereof.
  • a system for genetic modification of an organism including at least the following components: (a) a Cas12 variant enzyme as disclosed herein; and (b) a guide nucleic acid sequence including at least one region of complementarity to a first strand of the double-stranded target DNA sequence and a core sequence that interacts with the Cas12 variant enzyme.
  • a method for gene editing including at least the step of contacting a double-stranded target DNA sequence with the system for site-specific knockdown of double-stranded DNA as disclosed herein; wherein the core sequence binds the Cas12 variant enzyme; ATTORNEY DOCKET NO. T19227WO001 (222112-2280) wherein the Cas12 variant enzyme creates a double-stranded break in the double- stranded target DNA sequence at a position complementary to the guide nucleic acid sequence.
  • the cell can be a eukaryotic cell or can be a prokaryotic cell such as, for example, an extremophile.
  • the cell can be a dividing cell or a non-dividing cell.
  • the system can be introduced to the cell using nucleofection.
  • performing the method results in knockdown of the gene in at least 25% of a population of cells contacted with the system. Also disclosed herein are cells including at least one gene knockdown introduced by the disclosed methods.
  • each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting ATTORNEY DOCKET NO. T19227WO001 (222112-2280) sense and may be used interchangeably.
  • the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.”
  • the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.
  • a target nucleic acid As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a target nucleic acid,” “a Cas12 enzyme,” or “an extreme environmental condition,” include, but are not limited to, mixtures or combinations of two or more such target nucleic acids, Cas12 enzymes, or extreme environmental conditions, and the like. [0064] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’.
  • the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein.
  • nucleic acid can be used interchangeably herein and can generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.
  • polynucleotide as used herein can refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions can be from the same molecule or from different molecules.
  • the regions may include all of one or more of the ATTORNEY DOCKET NO. T19227WO001 (222112-2280) molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • Polynucleotide and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
  • polynucleotide as used herein can include DNAs or RNAs as described herein that contain one or more modified bases.
  • DNAs or RNAs including unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein.
  • Polynucleotide also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.
  • Natural nucleic acids have a phosphate backbone, artificial nucleic acids can contain other types of backbones, but contain the same bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or "polynucleotides” as that term is intended herein.
  • nucleic acid sequence and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined elsewhere herein.
  • deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” can generally refer to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • RNA can be in the form of non-coding RNA such as tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), microRNA (miRNA), or ribozymes, aptamers, guide RNA (gRNA), CRISPR RNA (crRNA), Trans-activating crRNA (tracrRNA), or coding mRNA ( messenger RNA).
  • cDNA refers to a DNA sequence that is complementary to an RNA transcript in a cell. It is a man-made molecule.
  • cDNA is made in vitro by an enzyme called reverse-transcriptase using RNA transcripts as templates.
  • gene can refer to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism.
  • the term gene can refer to translated and/or untranslated regions of a genome.
  • Gene can refer to the specific sequence of DNA that is transcribed into an RNA transcript that can be translated into a polypeptide or be a catalytic RNA molecule, including but not limited to, tRNA, siRNA, piRNA, miRNA, long-non-coding RNA and shRNA.
  • ATTORNEY DOCKET NO. T19227WO001 (222112-2280)
  • corresponding to or “encoding” refers to the underlying biological relationship between these different molecules.
  • operatively “corresponding to” can direct them to determine the possible underlying and/or resulting sequences of other molecules given the sequence of any other molecule which has a similar biological relationship with these molecules. For example, from a DNA sequence an RNA sequence can be determined and from an RNA sequence a cDNA sequence can be determined.
  • exogenous DNA or “exogenous nucleic acid sequence” or “exogenous polynucleotide” refers to a nucleic acid sequence that was introduced into a cell, organism, or organelle via transfection.
  • Exogenous nucleic acids originate from an external source, for instance, the exogenous nucleic acid may be from another cell or organism and/or it may be synthetic and/or recombinant. While an exogenous nucleic acid sometimes originates from a different organism or species, it may also originate from the same species (e.g., an extra copy or recombinant form of a nucleic acid that is introduced into a cell or organism in addition to or as a replacement for the naturally occurring nucleic acid).
  • the introduced exogenous sequence is a recombinant sequence.
  • isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, do not require “isolation” to distinguish it from its naturally occurring counterpart.
  • variant can refer to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential and/or characteristic properties (structural and/or functional) of the reference polynucleotide or polypeptide.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. The differences can be limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in nucleic or amino acid sequence by one or more modifications at the sequence level or post- transcriptional or post-translational modifications (e.g., substitutions, additions, deletions, methylation, glycosylations, etc.).
  • a substituted nucleic acid may or may not be an unmodified nucleic acid of adenine, thiamine, guanine, cytosine, uracil, including any chemically, ATTORNEY DOCKET NO. T19227WO001 (222112-2280) enzymatically or metabolically modified forms of these or other nucleotides.
  • a substituted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. “Variant” includes functional and structural variants.
  • gene refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism.
  • synthetic gene can refer to a recombinant gene comprising one or more coding sequences for a protein of interest, or a synthetically purified protein that is not naturally occurring in its purified state.
  • guide polynucleotide As used herein, the terms “guide polynucleotide,” “guide sequence,” or “guide RNA” (gRNA or sgRNA) as can refer to any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence- specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide polynucleotide and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available
  • a guide polynucleotide (also referred to herein as a guide sequence and includes single guide sequences (sgRNA)) can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 90, 100, 110, 112, 115, 120, 130, 140, or more nucleotides in length.
  • the guide polynucleotide (gRNA or sgRNA) can include a nucleotide sequence that is complementary to a target DNA sequence. This portion of the guide sequence can be referred to as the complementary region of the guide RNA or the CRISPR RNA (crRNA).
  • crRNA/tracrRNA can also work with the disclosed approach. Further in this aspect, since crRNA is shorter, it may be easier to incorporate the desired DNA modifications to the crRNAs by ligation or synthesis compared to incorporation into sgRNAs. In a further aspect, and without wishing to be bound by theory, tracrRNAs are generally universal and work with any sequence of crRNAs and so the ATTORNEY DOCKET NO. T19227WO001 (222112-2280) crRNA/tracrRNA system may be more economical for use.
  • the guide sequence can also include one or more miRNA target sequences coupled to the 3’ end of the guide sequence.
  • the guide sequence can include one or more MS2 RNA aptamers incorporated within the portion of the guide strand that is not the complementary portion.
  • the term guide sequence can include any specially modified guide sequences, including but not limited to those configured for use in synergistic activation mediator (SAM) implemented CRISPR or suppression.
  • SAM synergistic activation mediator
  • a guide polynucleotide can be less than about 150, 125, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide polynucleotide to direct sequence- specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide polynucleotide to be tested and a control guide polynucleotide different from the test guide polynucleotide, and comparing binding or rate of cleavage at the target sequence between the test and control guide polynucleotide reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • polypeptides or “proteins” refers to amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus.
  • amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
  • Protein and “Polypeptide” can refer to a molecule composed of one or more chains of amino acids in a specific order.
  • the term protein is used interchangeable with “polypeptide.” The order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins can be involved in the structure, function, and regulation of various functions.
  • ATTORNEY DOCKET NO. T19227WO001 (222112-2280) [0081]
  • identity is a relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences.
  • heterologous refers to compounds, molecules, nucleotide sequences (including genes), and polypeptide sequences (including peptides and proteins) that are different in both activity (function) and sequence or chemical structure.
  • heterologous can also refer to a gene or gene product that is from a different organism. For example, a human GTP cyclohydrolase or a synthase can be said to be heterologous when expressed in yeast.
  • homolog refers to a polypeptide sequence that shares a threshold level of similarity and/or identity as determined by alignment of matching amino acids. Two or more polypeptides determined to be homologs are said to be homologs. Homology is a qualitative term that describes the relationship between polypeptide sequences that is based upon the quantitative similarity.
  • paralog refers to a homolog produced via gene duplication of a gene. In other words, paralogs are homologs that result from divergent evolution from a common ancestral gene. ATTORNEY DOCKET NO.
  • orthologs refers to homologs produced by speciation followed by divergence of sequence but not activity in separate species. When speciation follows duplication and one homolog sorts with one species and the other copy sorts with the other species, subsequent divergence of the duplicated sequence is associated with one or the other species. Such species specific homologs are referred to herein as orthologs.
  • similarity is a quantitative term that defines the degree of sequence match between two compared polypeptide sequences.
  • organism As used herein, "organism”, “host”, and “subject” refers to any living entity comprised of at least one cell.
  • a living organism can be as simple as, for example, a single isolated eukaryotic cell or cultured cell or cell line, or as complex as a mammal, including a human being, and animals (e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans).
  • animals e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans).
  • the term “recombinant” or “engineered” can generally refer to a non- naturally occurring nucleic acid, nucleic acid construct, or polypeptide.
  • Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc.
  • a nucleic acid sequences encoding a fusion protein e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments
  • a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promote
  • Recombinant or engineered can also refer to the polypeptide encoded by the recombinant nucleic acid.
  • Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.
  • “cell,” “cell line,” and “cell culture” include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.
  • “culturing” refers to maintaining cells under conditions in which they can proliferate and avoid senescence as a group of cells.
  • “Culturing” can also include conditions in which the cells also or alternatively differentiate.
  • ATTORNEY DOCKET NO. T19227WO001 (222112-2280) [0091]
  • the term “specific binding” or “preferential binding” can refer to non- covalent physical association of a first and a second moiety wherein the association between the first and second moieties is at least 2 times as strong, at least 5 times as strong as, at least 10 times as strong as, at least 50 times as strong as, at least 100 times as strong as, or stronger than the association of either moiety with most or all other moieties present in the environment in which binding occurs.
  • Binding of two or more entities may be considered specific if the equilibrium dissociation constant, K d , is 10 ⁇ 3 M or less, 10 ⁇ 4 M or less, 10 ⁇ 5 M or less, 10 ⁇ 6 M or less, 10 ⁇ 7 M or less, 10 ⁇ 8 M or less, 10 ⁇ 9 M or less, 10 ⁇ 10 M or less, 10 ⁇ 11 M or less, or 10 ⁇ 12 M or less under the conditions employed, e.g., under physiological conditions such as those inside a cell or consistent with cell survival.
  • specific binding can be accomplished by a plurality of weaker interactions (e.g., a plurality of individual interactions, wherein each individual interaction is characterized by a Kd of greater than 10 ⁇ 3 M).
  • specific binding which can be referred to as “molecular recognition,” is a saturable binding interaction between two entities that is dependent on complementary orientation of functional groups on each entity.
  • specific binding interactions include primer-polynucleotide interaction, aptamer-aptamer target interactions, antibody-antigen interactions, avidin-biotin interactions, ligand-receptor interactions, metal-chelate interactions, hybridization between complementary nucleic acids, etc.
  • atmospheres referred to herein are based on atmospheric pressure (i.e. one atmosphere) and temperatures are ambient.
  • a Cas12 variant enzyme derived from and bearing an increased net charge relative to a wild-type Cas12 enzyme ATTORNEY DOCKET NO. T19227WO001 (222112-2280)
  • Aspect 2 The Cas12 variant enzyme of aspect 1, wherein the increased net charge comprises a positive charge or a negative charge.
  • the Cas12 variant enzyme of aspect 2 wherein the increased net charge is a positive charge and the Cas12 variant enzyme includes the mutations T29K, L88K, S123K, and A171K (SEQ ID NO.2, 6). [0098] Aspect 4. The Cas12 variant enzyme of aspect 3, wherein the enzyme exhibits increased stability relative to the wild-type Cas12 enzyme at temperatures from about 25 °C to about 37 °C. [0099] Aspect 5. The Cas12 variant enzyme of aspect 2, wherein the increased net charge is a negative charge and the Cas12 variant enzyme includes the mutations Q32E, A107E, S537D, Y610E, and N826D (SEQ ID NO.3, 5). [0100] Aspect 6.
  • Aspect 7 The Cas12 variant enzyme of any one of aspects 1-6, wherein the wild-type Cas12 enzyme comprises a Cas12a enzyme.
  • Aspect 8 The Cas12 variant enzyme of aspect 7, wherein the wild-type Cas12 enzyme comprises a Eubacterium rectale Cas12a (SEQ ID NO.1).
  • Aspect 9 The Cas12 variant enzyme of any one of aspects 1-8, wherein the Cas12 variant enzyme exhibits higher trans-cleavage activity than the wild-type Cas12 enzyme.
  • Aspect 10 The Cas12 variant enzyme of aspect 5, wherein the enzyme exhibits increased stability at alkaline pH relative to the wild-type Cas12 enzyme.
  • the Cas12 variant enzyme any one of aspects 1-9, wherein the Cas12 variant enzyme exhibits enhanced stability under at least one extreme condition.
  • Aspect 11 The Cas12 variant enzyme of aspect 10, wherein the at least one extreme condition comprises low temperature, elevated temperature, acidic pH, alkaline pH, or any combination thereof.
  • Aspect 12 A method for detecting a target nucleic acid in a biological sample, the method comprising: (a) performing a high temperature lysis step on the biological sample to form a lysed sample; and ATTORNEY DOCKET NO.
  • T19227WO001 (222112-2280) (b) admixing the lysed sample with a Cas12 enzyme having enhanced stability under at least one extreme condition, a guide nucleic acid sequence, optionally a reverse transcriptase, and a fluorescence-quencher reporter molecule; wherein, in the presence of the target nucleic acid, the fluorescence-quencher reporter molecule is cleaved by the Cas12 enzyme, separating a fluorophore from a quencher and producing a fluorescence signal.
  • the fluorophore comprises 5(6)- carboxyfluorescein (56-FAM), 5′-hexachlorofluorescein (5HEX), or any combination thereof.
  • Aspect 14 The method of aspect 12 or 13, wherein the quencher comprises 3′-Iowa Black FQ (3IABkFQ).
  • Aspect 15 The method of any one of aspects 12-14, wherein the fluorescence-quencher reporter molecule comprises 56-FAM-TTATT-3IABkFQ or 5HEX-TTTTTTTT-3IABkFQ.
  • Aspect 16 The method of any one of aspects 12-15, wherein the target nucleic acid comprises RNA and where the reverse transcriptase is included.
  • Aspect 17 The method of aspect 16, wherein the Cas12 enzyme comprises Cas12b. [0112] Aspect 18.
  • Aspect 19 The method of any one of aspects 12-15, wherein the target nucleic acid comprises RNA or DNA and wherein the reverse transcriptase is not included.
  • Aspect 19 The method of aspect 18, wherein the Cas12 enzyme comprises Cas12a.
  • Aspect 20 The method of any one of aspects 12-19, further comprising amplifying the target nucleic acid during or prior to detection.
  • Aspect 21 The method of any one of aspects 12-20, wherein the Cas12 enzyme having enhanced stability comprises a Type V Cas12 enzyme.
  • Aspect 22 The method of aspect 21, wherein the Type V Cas12 enzyme comprises a Cas12b enzyme.
  • Aspect 23 The method of any one of aspects 12-15, wherein the target nucleic acid comprises RNA or DNA and wherein the reverse transcriptase is not included.
  • Aspect 24 The method of any one of aspects 12-22, wherein the at least one extreme condition comprises low temperature, elevated temperature, acidic pH, alkaline pH, or any combination thereof.
  • the Cas12b enzyme comprises a thermostable Cas12b enzyme. ATTORNEY DOCKET NO. T19227WO001 (222112-2280)
  • Aspect 25 The method of aspect 24, wherein the thermostable Cas12b enzyme comprises a BrCas12b variant.
  • Aspect 26 The method of aspect 25, wherein the BrCas12b variant comprises RFND BrCas12b (SEQ ID NO.4).
  • Aspect 27 The method of any one of aspects 12-22, wherein the at least one extreme condition comprises low temperature, elevated temperature, acidic pH, alkaline pH, or any combination thereof.
  • Aspect 28 The method any one of aspects 12-27, wherein step (a) comprises heating the biological sample to 95 °C for 5 min.
  • step (a) further comprises contacting the biological sample with a buffer.
  • Aspect 30 The method of aspect 29, wherein the buffer comprises a buffer having a pH of at least 8.0.
  • Aspect 31 The method any one of aspects 12-30, wherein step (a) denatures double stranded DNA to single stranded DNA. [0126] Aspect 32.
  • Aspect 33 the method of any one of aspects 12-32, wherein the biological sample is not subjected to an extraction or purification step prior to performing step (a).
  • Aspect 34 The method of any one of aspects 12-33, wherein the target nucleic acid comprises RNA or DNA.
  • Aspect 35 The method of aspect 34, wherein the target nucleic acid comprises a SARS- CoV-2 RNA, an HIV RNA, or a malaria mRNA.
  • Aspect 37 A system for site-specific knockdown of a gene in a double-stranded target DNA sequence, the system comprising: (a) the Cas12 variant enzyme of any one of aspects 1-11; and ATTORNEY DOCKET NO. T19227WO001 (222112-2280) (b) a guide nucleic acid sequence comprising at least one region of complementarity to a first strand of the double-stranded target DNA sequence and a core sequence that interacts with the Cas12 variant enzyme. [0132] Aspect 38.
  • a method for site-specific knockdown of a gene in a double-stranded target DNA sequence in a cell comprising contacting the double-stranded target DNA sequence with the system of aspect 37; wherein the core sequence binds the Cas12 variant enzyme; and wherein the Cas12 variant enzyme creates a double-stranded break in the double- stranded target DNA sequence at a position complementary to the guide nucleic acid sequence.
  • Aspect 39 The method of aspect 38, wherein the cell comprises a prokaryotic cell or a eukaryotic cell.
  • Aspect 40 The method of aspect 38 or 39, wherein the cell comprises an extremophile.
  • Aspect 42 The method of any one of aspects 38-41, wherein the system is introduced to the cell using nucleofection.
  • Aspect 43 The method of any one of aspects 38-42, wherein performing the method results in knockdown of the gene in at least 25% of a population of cells contacted with the system.
  • Aspect 44 A cell comprising at least one gene knockdown introduced by the method of any one of aspects 38-43.
  • Example 1 Thermostable Cas Enzymes for Single-Pot PAM-Free and Extraction-Free Nucleic Acid Detection
  • CRISPR-Cas12a systems have been used for nucleic acid detection, where trans- cleavage can be achieved with PAM (for dsDNA) and without PAM (ssDNA) (FIG.1).
  • PAMless identification of nucleic acids with CRISPR/Cas12a has also been demonstrated (FIG. 2).
  • LbCas12a is a particularly useful enzyme for the PICNIC system (FIGs.3A-3D), but other Type V CRISPR-Cas systems (e.g.
  • BrCas12b can also be used (FIGs.4A-4B).
  • the RFND variant of BrCas12b can be used in various nucleic acid detection methods including single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases (SPLENDID), which can have PAM dependence (FIG.5).
  • RFND BrCas12b (SEQ ID NO.4) can also be used for one-pot PAM-less detection (FIG.6).
  • a revised PICNIC method has been developed that eliminates extraction steps and uses thermostable Cas enzymes, allowing a shorter processing time and fewer steps in nucleic acids detection (FIG.7).
  • Example 2 Charge Engineering Cas12a Enzymes [0143] A wild-type ErCas12a (SEQ ID NO. 1) was used as a template to generate variants bearing different overall charges, including both positive and negative net charges (FIG.9). A backbone and 4 dsDNA fragments, each encoding a positively charged amino acid substitution, were amplified. Each fragment overlaps by 20-30 bp. These fragments were inserted into a vector and transfected into bacteria; colonies were selected using antibiotic resistance.
  • FIG. 10 A sequence alignment shows conserved positive residues in Cas12a enzymes (FIG.10).
  • FIGs. 11A-11B show wild type ErCas12a (FIG. 11A) and engineered net positive enzyme SuperPos ErCas12a (SEQ ID NO. 2, 6), where positive mutations are highlighted in a yellow circle.
  • SuperPos ErCas12a includes the mutations T29K, L88K, S123K, and A171K (SEQ ID NO. 2, 6).
  • FIGs.13A-13D show models of WT ErCas12a (SEQ ID NO.1) and engineered net ATTORNEY DOCKET NO. T19227WO001 (222112-2280) negative enzyme SuperNeg ErCas12a (SEQ ID NO.3, 5) in main views (FIGs.13A-13B of WT and FIGs.13C-13D for SuperNeg).
  • SuperNeg ErCas12a includes the mutations Q32E, A107E, S537D, Y610E, and N826D (SEQ ID NO.3, 5). Negative charges are found more on the surface, where environmental interactions are expected to be observed.
  • Table 1 shows SEQ ID NOs. for full sequences of which portions are shown in FIGs.10 and 12.
  • Table 1 Cas12a Enzymes Enzyme SEQ ID NO.
  • Gene editing efficiency of WT, SuperPos, and SuperNeg ErCas12a variants in HEK293T ATTORNEY DOCKET NO. T19227WO001 (222112-2280) cells was also tested. When a guide was present, both SuperPos and SuperNeg effectively generated indel mutations (FIGs.19A-19B).
  • SuperNeg was also used for detection of malaria and SARS-CoV-2 genes and found to be more effective than conventional crENHANCE methods (FIGs.20A-20B).
  • Table 2 Enzymes and Variants Useful in the Disclosed Systems and Methods s e ATTORNEY DOCKET NO. T19227WO001 (222112-2280) LINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPAAYTSKI DPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLFCFTFDYNNFITQNTVM es o , es o , , ATTORNEY DOCKET NO.

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Abstract

Selon un aspect, la divulgation concerne une plateforme de détection d'acide nucléique monotope qui est conçue pour optimiser des diagnostics d'acide nucléique tout en assurant des résultats rapides et dépendants. La plateforme incorpore de manière unique l'enzyme Cas12b thermostable, permettant une détection directe d'acide nucléique à partir d'échantillons non traités. La divulgation concerne également des variants conçus de Cas12a chargés positivement et négativement et leur utilisation pour la détection d'acides nucléiques. Selon un aspect, le pH et les conditions thermiques divulgués, qui permettent la réaction monotope sans extraction, fonctionneraient avec n'importe quelle enzyme qui est fonctionnelle dans ces conditions.
PCT/US2024/053313 2023-11-03 2024-10-29 Variants de cas12a thermostables et/ou modifiés par charge pour détection d'acides nucléiques Pending WO2025096367A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022174108A1 (fr) * 2021-02-12 2022-08-18 The Board Of Trustees Of The Leland Stanford Junior University Cas12a synthétique pour le contrôle et l'édition de gènes multiplex améliorés
WO2023081902A1 (fr) * 2021-11-05 2023-05-11 University Of Florida Research Foundation, Inc. Systèmes et procédés pour la détection de polynucléotides cibles avec crispr/cas12a à l'aide d'activateurs
WO2023104185A1 (fr) * 2021-12-09 2023-06-15 Beijing Institute For Stem Cell And Regenerative Medicine Protéines effectrices de cas12b modifiées et leurs procédés d'utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022174108A1 (fr) * 2021-02-12 2022-08-18 The Board Of Trustees Of The Leland Stanford Junior University Cas12a synthétique pour le contrôle et l'édition de gènes multiplex améliorés
WO2023081902A1 (fr) * 2021-11-05 2023-05-11 University Of Florida Research Foundation, Inc. Systèmes et procédés pour la détection de polynucléotides cibles avec crispr/cas12a à l'aide d'activateurs
WO2023104185A1 (fr) * 2021-12-09 2023-06-15 Beijing Institute For Stem Cell And Regenerative Medicine Protéines effectrices de cas12b modifiées et leurs procédés d'utilisation

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