ATTORNEY DOCKET NO. T19227WO001 (222112-2280) THERMOSTABLE AND/OR CHARGE-ENGINEERED CAS12A VARIANTS FOR NUCLEIC ACID DETECTION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to, and the benefit of, U.S. provisional application Serial No.63/611,813 filed December 19, 2023, and U.S. provisional application Serial No.63/596,070 filed November 3, 2023, each of which is hereby incorporated by reference in its entirety. CROSS REFERENCE TO SEQUENCE LISTING [0002] The genetic components described herein are referred to by sequence identifier numbers (SEQ ID NO). The sequence listing in written computer readable format (CRF) as a text file named “222112-2280_Sequence_Listing.xml” created on October 22, 2024, and having a size of 62,352 bytes, is incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0003] This invention was made with government support under grant number R21AI156321 awarded by the National Institutes of Health, grant number R21AI168795 awarded by the National Institutes of Health, and grant number 1R35GM147788-01 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND [0004] The discovery of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) systems has provided new platforms and approaches to the field of genome engineering (i.e., editing), diagnostics, and development of other advanced technologies in biology, agriculture, and biotechnology. Of note, CRISPR-based diagnostics have elevated nucleic acid detection in terms of sensitivity, specificity, and rapidness. [0005] Originally derived from various species of bacterial and archaeal adaptive immune systems, the 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. By binding with Cas, 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.” Some types of Cas proteins, once bound to a target sequence, also become active for collateral, non-specific cleavage, called
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) “trans-cleavage.” This trans-cleavage activity can be leveraged for detection technologies. However, currently-known detection technologies require extensive sample preparation, processing, and long analysis times. It would be desirable to develop an enzyme or system that requires fewer processing steps and that can be completed quickly. [0006] 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. [0007] Despite advances in nucleic acid detection research, there is still a scarcity of methods that can be completed quickly with minimal sample processing. An ideal method would leverage Cas enzymes with enhanced stability under extreme conditions to shorten processing times and/or to reduce sample processing steps. In some aspects, the Cas enzymes would have an increased net positive or negative charge relative to their wild-type counterparts and/or could be used for gene editing in extremophilic organisms. These needs and other needs are satisfied by the present disclosure. SUMMARY [0008] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, 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. In one aspect, 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. [0009] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another. BRIEF DESCRIPTION OF THE DRAWINGS [0010] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views. [0011] FIG.1 is a diagram showing PAM effects on CRISPR-Cas12a detection under various scenarios as known in the art. [0012] FIG.2 is a schematic showing detection of non-canonical PAM containing targets with PAMless Identification of Nucleic Acids with CRISPR/Cas12a (PICNIC). [0013] FIGs.3A-3D show a trans-cleavage assay at time = 30 min using a PAM library of dsDNA activators with wild-type (WT) versus the disclosed (PICNIC) method using LbCas12a. [0014] FIGs.4A-4B show PICNIC can be expanded to diverse type V CRISPR-Cas systems. A trans cleavage assay at time = 30 min using a WT versus PICNIC method for RFND BrCas12b (FIG.4A) and Cas12i1 (FIG.4B). [0015] FIG.5 shows PAM dependence of SPLENDID with RT-LAMP. A trans-cleavage assay is depicted at time = 30 min using a PAM library of dsDNA activators with and without RT-LAMP for RFND BrCas12b. [0016] FIG. 6 shows one-pot PAM-less detection with RFND BrCas12b at 62 °C. Error bars represent SEM. [0017] FIG.7 is a comparison of the originally-developed PICNIC workflow versus the disclosed, simplified workflow. [0018] FIG.8 shows extraction-free detection of positive SARS-CoV-2 saliva samples with and without a 5 minute heating step at 95 °C.
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) [0019] 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. [0020] FIG.10 shows aligning and identifying conserved positive residues in Cas12a enzymes. Sequences listed correspond to portions of SEQ ID NOs.7-29. [0021] 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). [0022] FIG.12 shows aligning and identifying conserved negative residues in Cas12a enzymes. Sequences listed correspond to portions of SEQ ID NOs.7-29. [0023] 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). [0024] 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. [0025] FIGs. 15A-15B show trans-cleavage functionality of WT ErCas12a, SuperPos, and SuperNeg variants at different temperatures. [0026] 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. [0027] 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). [0028] 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). [0029] FIGs. 19A-19B show efficiency of gene editing with WT, SuperPos, and SuperNeg ErCas12a variants in HEK293T cells. Percentage of indels recognized after RNP nucleofection are shown in FIG.19B. [0030] 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). [0031] 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. [0032] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. DETAILED DESCRIPTION [0033] In one aspect, disclosed herein is 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. In one aspect, 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. In a further aspect, 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. In one aspect, 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. [0034] In a further aspect, 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. [0035] In yet another aspect, 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. In an additional aspect, the disclosed platform eliminates the need for nucleic acid extraction, further simplifying the workflow and accelerating the time to result. In one aspect, 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. In another aspect, "SupErCas" is a novel variant of the CRISPR-Cas12a enzyme, specifically derived from the Eubacterium rectale (ErCas12a). In a further aspect, 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. [0037] In one aspect, disclosed herein is a library of ErCas12a enzymes that have been designed with increasing positive and negative charges. In a further aspect, 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. In one aspect, it was observed that increasing the overall charge of the enzyme led to an increase in its resilience to pH and temperature changes. In a further aspect, 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. In yet another aspect, it has also been demonstrated herein that one of the negatively charged variants characterized here possesses superior trans-cleavage activity that can be applied for highly sensitive detection of nucleic acid substrates. Cas12 Variant Enzymes [0038] In one aspect, disclosed herein is a Cas12 variant enzyme derived from and bearing an increased net charge relative to a wild-type Cas12 enzyme (SEQ ID NO.1). In an aspect, the increased net charge can be a positive charge or a negative charge. In a further aspect, when the charge is a positive charge, the Cas12 variant enzyme can include one or more of the following mutations: T29K, L88K, S123K, and A171K. In an alternative aspect, when the charge is a negative charge, the Cas12 variant enzyme can include one or more of the following mutations: Q32E, A107E, S537D, Y610E, and N826D. [0039] In any of these aspects, 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. In another aspect, 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. [0040] In one aspect, 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. In an aspect, the Cas12 variant enzyme exhibits higher trans-cleavage activity than the wild-type Cas12 enzyme. Method for Detecting a Target Nucleic Acid [0041] In an aspect, disclosed herein is a method for detecting a target nucleic acid in a biological sample, the method 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. [0042] In one aspect, 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). In a further aspect, the fluorescence-quencher reporter molecule can be 56-FAM-TTATT-3IABkFQ or 5HEX-TTTTTTTT-3IABkFQ. [0043] In one aspect, the target nucleic acid can be RNA and the reverse transcriptase can be included. Further in this aspect, the Cas12 enzyme can be Cas12b. In one aspect, RT-LAMP is typically used in the disclosed methods for RNA detection only. In another aspect, the target nucleic acid can be amplified prior to or during detection. In a further aspect, in RT-LAMP, 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. In a further aspect, the reaction can proceed while held at constant temperature using a hot water bath; temperature cycling is not needed. [0044] In an alternative aspect, the target nucleic acid can be RNA or DNA and the reverse transcriptase is not included. Further in this aspect, the Cas12 enzyme can be Cas12a. In another aspect, 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.
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) [0045] In one aspect, the Cas12 enzyme having enhanced stability can be a Type V Cas12 enzyme such as, for example, a Cas12b enzyme. In some aspects, 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). In one aspect, the at least one extreme condition can be low temperature, elevated temperature, acidic pH, alkaline pH, or any combination thereof. In another aspect, the Cas12 enzyme having enhanced stability can be the Cas12 variant enzyme having an increased net charge described herein. [0046] In one aspect, in the disclosed method, 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. [0047] In one aspect, in the disclosed method, a protospacer adjacent motif (PAM) is not required for detecting the target nucleic acid. In another aspect, the biological sample does not need to be subjected to an extraction or purification step prior to performing step (a). [0048] In an aspect, the target nucleic acid can be RNA or DNA. In one aspect, 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. In one aspect, the biological sample can be blood, urine, saliva, plasma, or a combination thereof. System for Site-Specific Knockdown of a Gene in a Double-Stranded DNA Target Sequence [0049] In one aspect, disclosed herein is a system for genetic modification of an organism, the system 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. Method for Site-Specific Knockdown of a Gene in a Double-Stranded Target DNA Sequence [0050] In one aspect, disclosed herein is a method for gene editing, the method 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. [0051] In one aspect, following the creation of a double-stranded DNA break, transcription and subsequent translation are disrupted, resulting in knockdown of the gene. [0052] In an aspect, the cell can be a eukaryotic cell or can be a prokaryotic cell such as, for example, an extremophile. In a further aspect, the cell can be a dividing cell or a non-dividing cell. In still another aspect, the system can be introduced to the cell using nucleofection. [0053] In one aspect, 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. [0054] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein. [0055] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. [0056] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. [0057] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation,
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. [0058] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation. [0059] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class. [0060] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. [0061] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. Definitions [0062] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, 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. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of. [0063] 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. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed. [0065] When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, 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’. Likewise, 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’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”. [0066] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, 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. [0067] As used herein, 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. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. [0068] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. [0069] As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” 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. In addition, 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. For instance, the term polynucleotide as used herein can include DNAs or RNAs as described herein that contain one or more modified bases. Thus, 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”, “nucleotide sequences” and “nucleic acids” 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. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or "polynucleotides" as that term is intended herein. As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined elsewhere herein. [0070] As used 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). [0071] As used herein, “cDNA” refers to a DNA sequence that is complementary to an RNA transcript in a cell. It is a man-made molecule. Typically, cDNA is made in vitro by an enzyme called reverse-transcriptase using RNA transcripts as templates. [0072] As used herein, “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) [0073] As used herein with reference to the relationship between DNA, cDNA, cRNA, RNA, protein/peptides, and the like “corresponding to” or “encoding” (used interchangeably herein) refers to the underlying biological relationship between these different molecules. As such, one of skill in the art would understand that 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. [0074] As used herein, the term “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). Typically, the introduced exogenous sequence is a recombinant sequence. [0075] As used herein, “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. [0076] As used herein, “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. [0077] As used herein, “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. As used herein, “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. [0078] 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). 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). Another portion of the guide sequence serves as a binding scaffold for the CRISPR-associated (Cas) nuclease. This portion of the guide sequence can be referred to as the tracrRNA. In one aspect, 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. As used herein 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. [0079] 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. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide polynucleotide to be tested, 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. Similarly, 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. [0080] As used herein, “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. In accordance with standard nomenclature, 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] As used herein, “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. “Identity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.1988, 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch (J. Mol. Biol., 1970, 48: 443-453) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides or polynucleotides of the present disclosure. [0082] As used herein, “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. As used herein, “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. [0083] As used herein, “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. [0084] As used herein, “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. T19227WO001 (222112-2280) [0085] As used herein, “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. [0086] As used herein, “similarity” is a quantitative term that defines the degree of sequence match between two compared polypeptide sequences. [0087] 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). [0088] As used herein, 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. 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. [0089] As used herein, “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. [0090] As used herein, “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] As used herein, 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. In some embodiments, 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). In some embodiments, 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. Examples of 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. [0092] Unless otherwise specified, atmospheres referred to herein are based on atmospheric pressure (i.e. one atmosphere) and temperatures are ambient. [0093] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure. ASPECTS [0094] The present disclosure can be described in accordance with the following numbered aspects, which should not be confused with the claims. [0095] Aspect 1. 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) [0096] Aspect 2. The Cas12 variant enzyme of aspect 1, wherein the increased net charge comprises a positive charge or a negative charge. [0097] Aspect 3. 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. The Cas12 variant enzyme of aspect 5, wherein the enzyme exhibits increased stability at alkaline pH relative to the wild-type Cas12 enzyme. [0101] Aspect 7 The Cas12 variant enzyme of any one of aspects 1-6, wherein the wild-type Cas12 enzyme comprises a Cas12a enzyme. [0102] Aspect 8. The Cas12 variant enzyme of aspect 7, wherein the wild-type Cas12 enzyme comprises a Eubacterium rectale Cas12a (SEQ ID NO.1). [0103] 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. [0104] Aspect 10. The Cas12 variant enzyme any one of aspects 1-9, wherein the Cas12 variant enzyme exhibits enhanced stability under at least one extreme condition. [0105] 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. [0106] 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. [0107] Aspect 13. The method of aspect 12, wherein the fluorophore comprises 5(6)- carboxyfluorescein (56-FAM), 5′-hexachlorofluorescein (5HEX), or any combination thereof. [0108] Aspect 14. The method of aspect 12 or 13, wherein the quencher comprises 3′-Iowa Black FQ (3IABkFQ). [0109] 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. [0110] 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. [0111] Aspect 17. The method of aspect 16, wherein the Cas12 enzyme comprises Cas12b. [0112] Aspect 18. 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. [0113] Aspect 19. The method of aspect 18, wherein the Cas12 enzyme comprises Cas12a. [0114] Aspect 20. The method of any one of aspects 12-19, further comprising amplifying the target nucleic acid during or prior to detection. [0115] Aspect 21. The method of any one of aspects 12-20, wherein the Cas12 enzyme having enhanced stability comprises a Type V Cas12 enzyme. [0116] Aspect 22. The method of aspect 21, wherein the Type V Cas12 enzyme comprises a Cas12b enzyme. [0117] Aspect 23. 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. [0118] Aspect 24. The method of aspect 22, wherein the Cas12b enzyme comprises a thermostable Cas12b enzyme.
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) [0119] Aspect 25. The method of aspect 24, wherein the thermostable Cas12b enzyme comprises a BrCas12b variant. [0120] Aspect 26. The method of aspect 25, wherein the BrCas12b variant comprises RFND BrCas12b (SEQ ID NO.4). [0121] Aspect 27. The method of any one of aspects 12-26, wherein the Cas12 enzyme having enhanced stability comprises the Cas12 variant enzyme of any one of aspects 1-11. [0122] Aspect 28. The method any one of aspects 12-27, wherein step (a) comprises heating the biological sample to 95 °C for 5 min. [0123] Aspect 29. The method of any one of aspects 12-28, wherein step (a) further comprises contacting the biological sample with a buffer. [0124] Aspect 30. The method of aspect 29, wherein the buffer comprises a buffer having a pH of at least 8.0. [0125] Aspect 31. The method any one of aspects 12-30, wherein step (a) denatures double stranded DNA to single stranded DNA. [0126] Aspect 32. The method of any one of aspects 12-31, wherein a protospacer adjacent motif (PAM) is not required for detecting the target nucleic acid. [0127] 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). [0128] Aspect 34. The method of any one of aspects 12-33, wherein the target nucleic acid comprises RNA or DNA. [0129] 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. [0130] Aspect 36. The method of any one of aspects 12-35, wherein the biological sample comprises blood, urine, saliva, plasma, or any combination thereof. [0131] 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, the method 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. [0133] Aspect 39. The method of aspect 38, wherein the cell comprises a prokaryotic cell or a eukaryotic cell. [0134] Aspect 40. The method of aspect 38 or 39, wherein the cell comprises an extremophile. [0135] Aspect 41. The method of any one of aspects 38-40, wherein the cell is a dividing cell or a non-dividing cell. [0136] Aspect 42. The method of any one of aspects 38-41, wherein the system is introduced to the cell using nucleofection. [0137] 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. [0138] Aspect 44. A cell comprising at least one gene knockdown introduced by the method of any one of aspects 38-43. EXAMPLES [0139] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) Example 1: Thermostable Cas Enzymes for Single-Pot PAM-Free and Extraction-Free Nucleic Acid Detection [0140] 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 (PICNIC) 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). [0141] 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). [0142] Herein 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). This is particularly important in urgent and point-of-care settings where fast and effective identification of nucleic acids in samples is needed for the guidance of medical decisions, such as, for example, detecting viruses such as SARS-CoV-2 (FIG.8). 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. Sanger sequencing was performed to identify the accuracy of desired substitutions and choose the most accurate plasmid. Proteins were produced and purified. Trans-cleavage, thermal stability, and gene editing capabilities of the protein were tested. [0144] 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). A separate sequence alignment shows conserved negative residues in Cas12a enzymes (FIG.12). 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. Melting temperatures were determined at pH values of 7, 8, and 9 for wild type and engineered net positive and net negative enzymes (FIGs. 14A-14C). Trans-cleavage functionality of WT ErCas12a, SuperPos, and SuperNeg variants were tested at different temperatures (FIGs.15A-15B) and different pH values (FIG.16). SuperNeg had high trans cleavage ability especially at pH values of 8 and 9. Two- dimensional time/pH dependence trans-cleavage ability was also tested (FIGs.17A-17C). Cis cleavage activity was also tested for WT, SuperPos, and SuperNeg ErCas12a at different temperatures and pH values (FIGs.18A-18C). [0145] 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.
[0146] 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). [0147] 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). [0148] SuperPos and SuperNeg also showed enhanced RNA detection using a PAM-containing 12 nucleotide dsDNA activator or linker DNA (FIGs.21A-21B). [0149] The variant with a net positive charge showed a shift in optimal pH, but variants with both net negative and net positive charges increased pH range as well a temperature range for enzyme operation. Variants retained functionality in cells. Both positive and negative variants showed increased catalytic activity, with the negative variant being especially promising. Both variants can be used for diagnostics in complex matrices such as blood and urine, and both show promise for gene editing in alkalphilic (negative variant) and low-temperature (positive variant) organisms. Enzymes and variants useful herein are shown in Table 2. 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. T19227WO001 (222112-2280) IYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYDNNAIILMRDN LYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSKTG 1
[0150] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) principles of the disclosure. Many variations and modifications may be made to the above- described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) IYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYDNNAIILMRDN LYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSKTG 1
[0150] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the
ATTORNEY DOCKET NO. T19227WO001 (222112-2280) principles of the disclosure. Many variations and modifications may be made to the above- described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.