Improved CRISPR-CAS technology

Abstract

The present disclosure provides improved CRISPR-Cas proteins (e.g., improved thermostability).

Description

[Background technology]

[0001] Various clustered regularly interspaced short palindromic repeats-CRISPR associated ("Cas") proteins have been discovered to have collateral (trans) cleavage activity and improved thermostability, useful, for example, in detection (e.g., diagnostic) systems for detecting specific nucleic acids of interest as therapeutics (e.g., gene editing). See, e.g., review by Sashital Genome Med 2018:10,32. [Prior art documents] [Non-patent literature]

[0002] [Non-Patent Document 1] Sashital Genome Med 2018:10,32 Summary of the Invention

[0003] The present disclosure provides improved CRISPR-Cas proteins characterized by thermostable activity and / or Cas protein collateral activity. The present disclosure also provides improved guide RNA technology. In particular, the present disclosure specifically identifies sources of problems with the use of certain Cas enzymes, including in certain collateral activity assays. For example, the present disclosure notes that certain uses, including, for example, certain such collateral activity assays, include steps that involve incubation at elevated temperatures for periods of time, and that various Cas enzymes may not be stable enough to maintain sufficient levels of activity (e.g., collateral activity) under such conditions. In many embodiments, such steps may be or may include nucleic acid extension and / or amplification steps.

[0004] Alternatively, or in addition, the present disclosure provides the consideration that particularly preferred embodiments of various reactions utilizing Cas enzymes, including, for example, certain collateral activity assays, can be performed in a single reaction vessel (i.e., so-called "one-pot") assay. The present disclosure acknowledges that Cas enzymes whose activity (e.g., collateral cleavage activity) is not stable enough to maintain sufficient activity through any and all high temperature step(s) (which may be or may include, for example, one or more nucleic acid extension and / or amplification step(s)) may not be useful in such one-pot assays. The present disclosure further notes that certain Cas protein(s) (e.g., Cas13 and Cas12) are not stable enough at the appropriate temperature(s), for example, at temperatures at which nucleic acid extension and / or amplification reactions are typically performed (e.g., above about 60-65°C).

[0005] The present disclosure encompasses the recognition that thermostable variants of various Cas proteins (e.g., Cas9) have been described and / or are otherwise publicly available (see, e.g., Mougiakos et al. Nat Commun. 8:1647, 2017). One of skill in the art can compare such thermostable variants to related non-thermostable homologs (e.g., orthologs) to investigate sequence changes and / or elements that may be necessary and / or sufficient to achieve thermostability, and can further identify such sequence changes and / or elements in and / or introduce such sequence changes and / or elements into other homologs (e.g., orthologs). Furthermore, one of skill in the art is well aware of potential sources of naturally occurring thermostable Cas proteins (e.g., in microorganisms that survive high temperature conditions, such as in water vents, or other thermophilic microorganisms). Thus, one of skill in the art, upon reading the present disclosure, will be able to rapidly identify and / or develop thermostable Cas proteins suitable for the uses described herein.

[0006] In some embodiments, a useful thermostable Cas protein is a Cas12 or Cas13 homolog (e.g., ortholog). In some embodiments, a useful thermostable Cas protein is a Cas enzyme that comprises an amino acid sequence having 80%, 85%, 90%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-10.

[0007] Alternatively, or in addition, in some embodiments, useful thermostable Cas proteins can be expressed at temperatures above about 50°C; in some embodiments, at about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C, about 100°C, about 101°C, about 102°C, about 103°C, about 104°C, about 105°C, about 106°C, about 107°C, about 108°C, about 109°C, about 110°C, about 111°C, about 112°C, about 113°C, about 114°C, about 115°C, about 116°C, about 117°C, about 118°C, about 119°C, about ℃, about 78 ° C, about 79 ° C, about 80 ° C, about 81 ° C, about 82 ° C, about 83 ° C, about 84 ° C, about 85 ° C, about 86 ° C, about 87 ° C, about 88 ° C, about 89 ° C, about 90 ° C, about 91 ° C, about 92 ° C, about 93 ° C, about 94 ° C, about 95 ° C, about 96 ° C, about 97 ° C, about 98 ° C, about 99 ° C, about 100 ° C, or a combination thereof. In many embodiments, useful thermostable Cas proteins perform (e.g., perform their collateral cleavage activity function fully) at temperatures greater than about 60 ° C.

[0008] In some embodiments, a useful thermostable Cas protein will (e.g., fully perform its collateral cleavage activity) within the temperature range at which the nucleic acid extension and / or amplification reaction(s) are performed; one of skill in the art is familiar with a variety of such reactions and the temperature ranges at which they are performed. In some embodiments, such temperature ranges may be greater than a temperature selected from the group consisting of about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C, about 100°C, or a combination thereof. In some embodiments, the temperature range may be from about 60°C to about 90°C. In some embodiments, the temperature range may be from about 60°C to about 80°C. In some embodiments, the temperature range may be from about 60°C to about 75°C. In some embodiments, the temperature range may be from about 65°C to about 90°C. In some embodiments, the temperature range may be from about 60°C to about 80°C. In some embodiments, the temperature range may be from about 60°C to about 75°C.

[0009] Thus, as described herein, in some embodiments, a useful thermostable Cas protein is a Cas enzyme that comprises an amino acid sequence having 80%, 85%, 90%, 99%, or 100% sequence identity to a Cas12 or Cas13 homolog (e.g., ortholog), e.g., greater than about 50°C, and in some embodiments greater than about 60°C, e.g., within about 60-65°C and / or greater than about 60-65°C. One of skill in the art reading this disclosure will specifically understand that in some embodiments, a useful thermostable Cas protein is a Cas12 (e.g., SEQ ID NOs: 1-10, or a variant thereof having, e.g., at least 90%, 95%, 99% or more amino acid sequence identity thereto) or a Cas13 (or a variant thereof having, e.g., at least 90%, 95%, 99% or more amino acid sequence identity thereto) whose activity (e.g., target binding and collateral cleavage activity) is sufficiently thermostable to perform the assays described herein (e.g., in some embodiments, one-pot assays) at temperatures in the range of, e.g., 60-65° C. For example, in some embodiments, a sufficient thermostable activity is an activity that is reasonably comparable (e.g., within about 25%) to an appropriate reference thermostable Cas protein described herein (e.g., Aac or RS9).

[0010] In some embodiments, the present disclosure describes a detection method comprising contacting a CRISPR-Cas complex comprising a Cas protein having collateral cleavage activity that is thermostable at least above 60-65° C. and a guide RNA selected or engineered to be complementary to the target nucleic acid sequence with a sample potentially containing nucleic acid of the target nucleic acid sequence.

[0011] In some embodiments, the contacting step comprises contacting the CRISPR-Cas complex and the sample with a reporter susceptible to cleavage by Cas protein collateral activity. In some embodiments, the contacting step comprises incubating above the temperature for a period of time. In some embodiments, the detection method further comprises amplifying nucleic acid present in the sample. In some embodiments, the amplifying step utilizes a thermostable nucleic acid polymerase. In some embodiments, the amplifying and contacting steps are performed in a single vessel.

[0012] In some embodiments, the Cas protein is a Cas12 protein. In some embodiments, the Cas protein has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1. In some embodiments, the Cas protein has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the Cas protein has an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs:1-10. In some embodiments, the Cas protein has an amino acid sequence that has 80% sequence identity to any one of SEQ ID NOs:1-10.

[0013] In some embodiments, in a method of performing a detection assay utilizing a Cas protein having collateral cleavage activity, the improvement comprises utilizing a Cas protein having thermostable collateral cleavage activity. In some embodiments, the Cas protein is a Cas12 protein. In some embodiments, the Cas protein has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1. In some embodiments, the Cas protein has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the Cas protein has an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs:1-10. In some embodiments, the method of performing a detection assay is performed in a single reaction vessel. In some embodiments, the thermostable collateral cleavage activity is thermostable at temperatures above about 60° C. In some embodiments, the thermostable collateral cleavage activity is thermostable at temperatures above about 65° C.

[0014] Those skilled in the art understand the classification systems for Cas proteins, e.g., used to define Cas12-type and Cas13-type Cas proteins, and in particular, are familiar with sequence elements characteristic of Cas12 and Cas13. For example, Koonin et al., Curr Opin Microbiol.,2017 June;37:67-78, Makarova et al., Nat Rev Microbiol.,2015 November;13(11):722-736, Shmakov et al.,Mol Cell.,2015 November 5;60(3):385-397, Yan and Hunnewell et al. al.,Science,2018 Dec 6, Yan et al.,Mol Cell.,2018 April 19;70, 327-339, Makarova et al.,Nat Rev Microbiol.,2011 June;9(6):467-477, Makarova et al.,CRISPR Journal,2018,Volume 1,Number 5, Shmakov et al.,Nat Rev Microbiol.,2017 March;15(3):169-182, Yan and Hunnewell et al.,Science,2019 Jan 4;363,88-91, Abudayyeh et al.,Science,2016 August 5;353,6299, Gootenberg and Abudayyeh et al.,Science,2017 April 28,356,438-442, Gootenberg and See Abudayyeh et al., Science, 2018 April 27;360,439-444.

[0015] One of skill in the art will further appreciate that in many embodiments, a Cas12 provided herein (e.g., a thermostable Cas12) is characterized by overall sequence similarity to the exemplary Cas proteins (e.g., any of SEQ ID NOs: 1-10) and / or the presence of one or more sequence elements characteristic of Cas12, Cas13, subspecies thereof, and / or thermostable Cas proteins. In some embodiments, the presence of characteristic sequence elements and specific overall sequence identity, which may be moderately low (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the characteristic sequence elements, is indicative of a provided Cas protein described herein. Alternatively, in some embodiments, the provided Cas proteins described herein exhibit high sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to an exemplary Cas (e.g., any of SEQ ID NOs: 1-10) regardless of the presence of such distinctive sequence elements. In some embodiments, both one or more distinctive sequence elements and high (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity are present.

[0016] In some aspects, the disclosure provides a method of detection comprising contacting a CRISPR-Cas complex comprising a Cas protein having collateral cleavage activity that is thermostable at least above 60-65° C. and a guide RNA selected or engineered to be complementary to the target nucleic acid sequence with a sample potentially containing the target nucleic acid sequence. In some embodiments, the contacting step comprises contacting the CRISPR-Cas complex and the sample with a reporter that is susceptible to cleavage by the Cas protein collateral activity. In some embodiments, the contacting step comprises incubating above the temperature for a period of time.

[0017] In some embodiments, provided detection methods include (e.g., further include) amplifying nucleic acid present in the sample. In some embodiments, the amplifying step can utilize a thermostable nucleic acid polymerase. In some embodiments, the amplifying and contacting steps are performed in a single container and / or without intervening component removal step(s) and / or wash step(s).

[0018] In some embodiments, the technology described herein utilizes a Cas protein that is a Cas12 protein. In some embodiments, such a Cas protein has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the utilized Cas protein has an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs:1-10. In some embodiments, such a Cas protein has an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10.

[0019] In some aspects, the disclosure provides improved methods of performing detection assays utilizing a Cas protein with collateral cleavage activity, the improvement comprising utilizing a Cas protein with thermostable collateral cleavage activity. In some embodiments, the Cas protein utilized in such embodiments is a Cas12 protein. In some embodiments, the Cas protein utilized has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the Cas protein utilized has an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10.

[0020] In some embodiments, provided methods (e.g., improved methods) for performing detection assays are performed in a single reaction vessel. In some embodiments of the provided technology utilizing a Cas protein with thermostable collateral cleavage activity, such activity is thermostable at temperatures above about 60° C. In some embodiments, such activity is thermostable at temperatures above about 65° C. In some embodiments, the Cas protein has an amino acid sequence having at least 80% sequence identity to one or more of SEQ ID NOs: 1-10.

[0021] In some aspects, the disclosure provides compositions (and in particular recombinant or otherwise non-natural compositions) that include: (a) a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C; and (b) at least one guide capable of forming a complex with such thermostable Cas protein and directing binding of the complex to a target nucleic acid sequence. In some embodiments, the Cas protein utilized has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the Cas protein utilized has an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10. In some embodiments, the at least one guide utilized comprises two guide sequences that can hybridize to two different target nucleic acid sequences or to different regions of a single target nucleic acid sequence. In some embodiments, the at least one guide utilized comprises multiple guide sequences that can hybridize to multiple different target nucleic acid sequences or to multiple different regions of a single target nucleic acid sequence. In some embodiments, the guide sequence utilized is capable of hybridizing to one or more target nucleic acid sequences in a prokaryotic cell, hi some embodiments, the guide sequence utilized is capable of hybridizing to one or more target nucleic acid sequences in a eukaryotic cell.

[0022] In some aspects, the disclosure provides compositions (and in particular recombinant or otherwise non-natural compositions) that include: (a) a polynucleotide encoding a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C; and at least one guide capable of forming a complex with the Cas protein and directing binding of the complex to a target nucleic acid sequence. In some embodiments, the Cas protein utilized has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the Cas protein utilized has an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10. In some embodiments, the at least one guide utilized comprises two guide sequences that can hybridize to two different target nucleic acid sequences or to different regions of a single target nucleic acid sequence. In some embodiments, the at least one guide utilized comprises multiple guide sequences that can hybridize to multiple different target nucleic acid sequences or to multiple different regions of a single target nucleic acid sequence. In some embodiments, the guide sequence utilized is capable of hybridizing to one or more target nucleic acid sequences in a prokaryotic cell, hi some embodiments, the guide sequence utilized is capable of hybridizing to one or more target nucleic acid sequences in a eukaryotic cell.

[0023] In some aspects, the disclosure provides compositions (and particularly recombinant or otherwise non-natural compositions) for modifying nucleotides in a target nucleic acid comprising a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C. In some embodiments, the composition comprises (e.g., further comprises) at least one guide sequence capable of forming a complex with the Cas protein and directing binding of the complex to a target nucleic acid sequence. In some embodiments, the Cas protein utilized has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the Cas protein utilized has an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10. In some embodiments, the Cas protein utilized is modified to reduce off-target effects. In some embodiments, modification of nucleotides in a target nucleic acid treats a disease caused by a point mutation. In some embodiments, modification of nucleotides in a target nucleic acid inactivates a gene encoded by the target nucleic acid sequence. In some embodiments, modification of nucleotides in the target nucleic acid modifies the gene product encoded by the target nucleic acid sequence. In some embodiments, modification of nucleotides in the target nucleic acid alters the expression level of the gene product encoded by the target nucleic acid sequence. In some embodiments, the at least one guide utilized comprises two guide sequences that can hybridize to two different target nucleic acid sequences or different regions of one target nucleic acid sequence. In some embodiments, the at least one guide utilized comprises multiple guide sequences that can hybridize to multiple different target nucleic acid sequences or multiple different regions of one target nucleic acid sequence. In some embodiments, the guide sequence utilized can hybridize to one or more target nucleic acid sequences in a prokaryotic cell. In some embodiments, the guide sequence utilized can hybridize to one or more target nucleic acid sequences in a eukaryotic cell.

[0024] In some aspects, the disclosure provides a vector system comprising one or more vectors comprising (a) a first control element operably linked to a nucleotide sequence encoding a Cas protein having collateral cleavage activity that is thermostable at least at a temperature above 60-65° C.; and (b) a second control element operably linked to a nucleotide sequence encoding a guide. In some embodiments, the nucleotide sequence encodes a Cas protein having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the nucleotide sequence encodes a Cas protein having an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10. In some embodiments, the nucleotide sequence encoding the Cas protein is codon optimized. In some embodiments, (a) and (b) are comprised in a single vector. In some embodiments, (a) and (b) are comprised in separate vectors. In some embodiments, the vector system comprises a viral vector.

[0025] In some aspects, the disclosure provides a method of cleaving at least one target nucleic acid in a cell, the method comprising contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide and capable of causing a disruption in the at least one target nucleic acid.

[0026] In some aspects, the disclosure provides a method of altering expression of at least one target nucleic acid in a cell, comprising contacting the cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide and capable of causing a disruption in the at least one target nucleic acid.

[0027] In some aspects, the disclosure provides a method of altering expression of at least one target nucleic acid in a cell, comprising contacting the cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide and is capable of editing the at least one target nucleic acid sequence.

[0028] In some aspects, the disclosure provides a method of modifying at least one target nucleic acid in a cell, comprising contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide and is capable of editing the at least one target nucleic acid sequence.

[0029] In some embodiments, editing the target nucleic acid comprises inserting a payload nucleic acid at the target nucleic acid sequence. In some embodiments, the nucleotide sequence encodes a Cas protein having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the nucleotide sequence encodes a Cas protein having an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10. In some embodiments, the at least one guide utilized comprises two guide sequences that can hybridize to two different target nucleic acid sequences or different regions of a target nucleic acid. In some embodiments, the at least one guide utilized comprises multiple guide sequences that can hybridize to multiple different target nucleic acid sequences or multiple different regions of a target nucleic acid. In some embodiments, the guide sequence utilized can hybridize to one or more target nucleic acid sequences in a prokaryotic cell. In some embodiments, the guide sequence utilized can hybridize to one or more target nucleic acid sequences in a eukaryotic cell.

[0030] In some aspects, the disclosure provides nucleic acids encoding a Cas protein with collateral cleavage activity that is thermostable at least at temperatures above 60-65° C. In some embodiments, the nucleotide sequence encodes a Cas protein having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the nucleotide sequence encodes a Cas protein having an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10.

[0031] In some aspects, the disclosure provides a method of treating a disorder or disease in a subject in need thereof, comprising administering to the subject a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C, and at least one guide capable of hybridizing to a target nucleic acid. In some embodiments, the nucleotide sequence encodes a Cas protein having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the nucleotide sequence encodes a Cas protein having an amino acid sequence that has at least 80% sequence identity to one or more of SEQ ID NOs:1-10. In some embodiments, the at least one guide utilized comprises two guide sequences that can hybridize to two different target nucleic acid sequences or different regions of a target nucleic acid. In some embodiments, the at least one guide utilized comprises multiple guide sequences that can hybridize to multiple different target nucleic acid sequences or multiple different regions of a target nucleic acid. In some embodiments, the at least one guide utilized can hybridize to one or more target nucleic acid sequences in a prokaryotic cell. In some embodiments, at least one guide sequence is utilized that can hybridize to one or more target nucleic acid sequences in a eukaryotic cell. In some embodiments, a Cas protein is utilized that can form a complex with a guide and cause destruction in a target nucleic acid. In some embodiments, a Cas protein is utilized that can form a complex with a guide and edit a target nucleic acid sequence.

[0032] In some aspects, the present disclosure provides a composition in which a Cas protein is associated with a modifying element. In some embodiments, the modifying element is an adenosine deaminase. In some embodiments, the modifying element is a cytidine deaminase.

[0033] In some aspects, the present disclosure provides pharmaceutical compositions comprising a Cas protein of the present disclosure.

[0034] In some aspects, the disclosure provides methods for characterizing Cas proteins, including investigating one or more of: (a) cis-cleavage activity; (b) trans-cleavage activity; (c) sensitivity; (d) selectivity for RNA or DNA target nucleic acids; (e) selectivity for RNA or DNA non-target nucleic acids; and (f) enzymatic stability. [Brief description of the drawings]

[0035] [Figure 1] Exemplary characterization of candidate thermostable Cas proteins Pal1, Pal2 low MW, Pal2 high MW, and Pal3 is shown.

[0036] [Diagram 2] Exemplary Pal1 and Pal2 activities at 56° C. are shown.

[0037] [Diagram 3] Exemplary activities of Pal1 at 37° C., 56° C., and 70° C. with different exemplary guides are shown.

[0038] [Figure 4] Exemplary activities of Pal1 compared to controls are shown at 56° C. and 70° C. These data suggest that the activity of Pal1 is specific for the target DNA.

[0039] [Diagram 5] 1 shows an exemplary temperature profile of Pal1.

[0040] [Figure 6] 1 shows exemplary activities of Pal2 high MW at 37° C., 56° C., and 70° C. with different exemplary guides.

[0041] [Figure 7] 1 shows an exemplary activity of Pal2 high MW compared to a control at 56° C. These data suggest that the activity of Pal2 high MW is specific for the target DNA.

[0042] [Figure 8] 1 shows an exemplary temperature profile for Pal2 high MW.

[0043] [Figure 9] An exemplary protein thermal shift assay was performed according to the vendor's (ThermoFisher) recommended protocol. Briefly, Cas enzymes (PAL1, PAL2, PAL3, PAL4, PAL5, PAL6, PAL8, PAL9, or PAL10) (500ng / uL) were mixed with protein thermal shift dye (8x) protein thermal shift buffer and placed in a QuantStudio 5 qPCR instrument to track the fluorescence change as the temperature was slowly increased. Data analysis was performed to extract the melting temperature (Tm) by taking the first derivative of the raw fluorescence intensity in the X4-M4 channel.

[0044] [Figure 10] Collateral activity signals of thermostable PAL5 Cas12b complexed with engineered single guide RNAs (sgRNAs) targeting either the N gene or the Orf1ab gene of SARS CoV2 are shown. Non-targeting controls (NTCs) are shown in grey. Targets are provided as purified in vitro transcribed (IVT) RNA.

[0045] [Figure 11] Collateral activity signals of thermostable PAL5 Cas12b complexed with engineered single guide RNAs (sgRNAs) targeting either the N or Orf1ab genes of SARS CoV2 are shown. Non-targeting controls (NTCs) are shown in grey. Targets provided at low concentrations were first amplified using LAMP and the products were simultaneously detected using PAL5 Cas12b.

[0046] [Figure 12]Collateral activity signal of thermostable PAL8 Cas12b complexed with recombinant single guide RNA (sgRNA) targeting the N gene of Sars CoV2. There are two variants of sgRNA tested (N-1) and (N-5) that show clear signal above background. Non-targeting control (NTC) is shown in grey. Targets are provided as purified in vitro transcribed (IVT) RNA.

[0047] [Figure 13] Collateral activity signals of thermostable PAL8 Cas12b complexed with recombinant single guide RNA (sgRNA) targeting the N gene of SARS CoV2 (SCoV2-N1). Non-targeting control (NTC) is shown in grey. Target provided at low concentration is first amplified using LAMP and the product is simultaneously detected using PAL8 Cas12b.

[0048] [Figure 14] Collateral activity signal of thermostable PAL9 Cas12b complexed with recombinant single guide RNA (sgRNA) targeting the N gene of SARS CoV2. There are three variants of sgRNA tested, (N-1), (N-2), and (N-3), that show clear signal above background. Non-targeting control (NTC) is shown in grey. Targets are provided as purified in vitro transcribed (IVT) RNA.

[0049] [Figure 15] Collateral activity signal of thermostable PAL9 Cas12b complexed with recombinant single guide RNA (sgRNA) targeting the N gene of Sars CoV2. Non-targeting control (NTC) is shown in grey. There are three variants of sgRNA tested, (N-1), (N-2) and (N-3), that show clear signal above background. Targets provided at low concentration are first amplified using LAMP and the products are simultaneously detected using PAL9 Cas12b.

[0050] [Figure 16] Collateral activity signal of thermostable PAL10 Cas12b complexed with recombinant single guide RNA (sgRNA) targeting the N gene of SARS CoV2. There are two variants of sgRNA tested (N-1) and (N-3) that show clear signal above background. Non-targeting control (NTC) is shown in grey. Target is provided as purified in vitro transcribed (IVT) RNA.

[0051] [Figure 17] Collateral activity signal of thermostable PAL10 Cas12b complexed with recombinant single guide RNA (sgRNA) targeting the N gene of Sars CoV2. Non-targeting control (NTC) is shown in grey. There are two variants of sgRNA tested, (N-1) and (N-3), that show clear signal above background. Target provided at low concentration is first amplified using LAMP and the product is simultaneously detected using PAL10 Cas12b. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] definition Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system. Those skilled in the art will recognize the various routes available for administration to a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, transdermal (e.g., may be or include one or more of topical skin, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may include intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses spaced apart over a period of time) dosing. In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected period of time.

[0053] Agent: As used herein, the term "agent" can refer to any chemical class of compound, molecule, or element, including, for example, small molecules, polypeptides, nucleic acids, sugars, lipids, metals, or combinations or complexes thereof. In some embodiments, the term "agent" can refer to a compound, molecule, or element that includes a polymer. In some embodiments, the term can refer to a compound or element that includes one or more polymeric moieties. In some embodiments, the term "agent" can refer to a compound, molecule, or element that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term can refer to a compound, molecule, or element that lacks or is substantially free of any polymer or polymeric moiety.

[0054] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to compounds and / or substances that may be, are, or are incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-naturally occurring amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. In some embodiments, an amino acid may include structural modifications compared to the general structures above, including the carboxy- and / or amino-terminal amino acids in a polypeptide. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) relative to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide comprising the modified amino acid compared to one comprising the otherwise identical amino acid without the modification. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide comprising the modified amino acid compared to one comprising the otherwise identical amino acid without the modification. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid. In some embodiments, the term may be used to refer to an amino acid residue of a polypeptide.

[0055] Analog: As used herein, the term "analog" refers to a substance that shares one or more specific structural features, components, constituents, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity with the reference substance, e.g., sharing a core or consensus structure, but also differs in one or more specific, distinct ways. In some embodiments, an analog is a substance that can be generated from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated by performing a synthetic process that is substantially similar (e.g., shares multiple steps) to that which generates the reference substance. In some embodiments, an analog can be generated by performing a synthetic process that is different from that used to generate the reference substance.

[0056] Animal: As used herein, refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, the animals may be transgenic animals, genetically modified animals, and / or clones.

[0057] Approximately: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest means a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction of the stated reference value (greater or less than), unless otherwise stated or otherwise clear from the context (except when such number exceeds 100% of possible values).

[0058] Binding: As used herein, the term "binding" is typically understood to mean a non-covalent association of two or more elements. "Direct" binding involves physical contact between the elements or moieties, and indirect binding involves physical interaction through physical contact by one or more intermediate elements. Binding between two or more elements can typically be evaluated in any of a variety of contexts, including when the interacting elements or moieties are studied alone or in the context of a more complex system (e.g., covalently or otherwise associated with a carrier element, and / or in a biological system or cell). Binding between two elements can be considered "specific" if, under the conditions investigated, the associated elements are more likely to associate with each other than with other available binding partners.

[0059] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source of interest (e.g., tissue or organism or cell culture) as described herein. In some embodiments, the source of interest includes an organism such as an animal or human. In some embodiments, the biological sample is or includes a biological tissue or fluid. In some embodiments, the biological sample can be or include bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, suspended nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological body fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as ductal lavage or bronchoalveolar lavage, aspirates, scrapings, bone marrow specimens, tissue biopsy specimens, surgical specimens, feces, other body fluids, secretions, and / or excretions, and / or cells therefrom, and the like. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells derived from the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspirate or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.), and the like. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing (e.g., by removing one or more components and / or adding one or more agents) of a primary sample. For example, filtration using a semi-permeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as, for example, amplification or reverse transcription of mRNA, isolation and / or purification of specific components.

[0060] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, tumors may be or include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, the associated cancer may be characterized as a solid tumor. In some embodiments, the associated cancer may be characterized as a hematological tumor. In general, examples of various types of cancers known in the art include, for example, hematopoietic cancers including leukemia, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), myeloma and myeloproliferative disorders; sarcomas, melanomas, adenomas, and carcinomas of solid tissues; squamous cell carcinoma of the mouth, throat, larynx, and lung; liver cancer; genitourinary cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer, and renal cell carcinoma; bone cancer; pancreatic cancer; skin cancer; cutaneous or intraocular melanoma; cancers of the endocrine system; cancer of the thyroid gland; cancer of the parathyroid gland; head and neck cancer; breast cancer; gastrointestinal cancer; cancer of the nervous system; benign lesions such as papilloma, and the like.

[0061] Carrier: As used herein, refers to a diluent, adjuvant, excipient, or vehicle used in administering a composition. In some exemplary embodiments, the carrier may comprise sterile liquids, such as, for example, water, and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid components.

[0062] Composition: As used herein, one of ordinary skill in the art will understand that the term "composition" may be used to mean a physically separate entity that contains one or more specific components. In general, unless otherwise specified, a composition may be in any form, such as, for example, a gas, a gel, a liquid, a solid, etc.

[0063] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but that other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it should also be understood that any composition or method described as "comprising" (or "comprises") one or more named elements or steps also represents a corresponding, more limited composition or method that "consistes essentially of" (or "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps, and may include additional elements or steps that do not materially affect the basic and novel property(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also represents a corresponding, more limited, close-ended composition or method that "consists of" (or "consists of") the named elements or steps, excluding any other elements or steps not named. In any composition or method disclosed herein, known or disclosed equivalents of any of the required named elements or steps may be substituted for that element or step.

[0064] Designed: As used herein, the term "designed" means that (i) the structure is selected or chosen by the hand of man, (ii) is produced by a process involving the hand of man, and / or (iii) is distinct from natural materials and other known agents.

[0065] Determining: Many methodologies described herein include a "determining" step. Those skilled in the art will understand upon reading this specification that such "determining" may utilize or be accomplished by use of any of a variety of techniques available to those skilled in the art, including, for example, certain techniques explicitly mentioned herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and / or manipulation of data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining involves receiving the relevant information and / or material from a source. In some embodiments, determining involves comparing one or more characteristics of the sample or element to a comparable reference.

[0066] Recombined: In general, the term "recombined" refers to an aspect that has been manipulated by the hand of man. For example, a polynucleotide can be considered "recombined" when two or more sequences that are not naturally linked to each other in that order are manipulated by the hand of man to be directly linked to each other in a recombinant polynucleotide, and / or when certain residues in the polynucleotide are combined by the action of the hand of man with elements or moieties that are non-natural and / or not naturally linked. For example, in some embodiments of the invention, a recombinant polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, and that is joined by the hand of man into operative association with the second coding sequence. Similarly, a cell or organism is considered "recombined" when, upon being subjected to manipulation, its genetic, epigenetic, and / or phenotypic identity is altered compared to an appropriate reference cell, such as an otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or includes a genetic operation in which the genetic information is altered (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutations, or mating protocols). In some embodiments, a recombinant cell is one that has been engineered to contain and / or express a particular agent of interest (e.g., a protein, nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and understood by those of skill in the art, the progeny of a recombinant polynucleotide or cell is usually still referred to as "recombined," even though actual manipulation has been performed on the previous elements.

[0067] Excipient: as used herein means a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0068] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0069] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized. A biological molecule can have two functions (i.e., it can be bifunctional) or it can have many functions (i.e., it can be multifunctional).

[0070] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes coding sequences (i.e., sequences that code for a particular product), and in some embodiments, a gene includes non-coding sequences. In some particular embodiments, a gene can include both coding (e.g., exon) and non-coding (e.g., intron) sequences. In some embodiments, a gene can include one or more regulatory elements that can, for example, control or affect one or more aspects of gene expression (e.g., cell type specific expression, inducible expression, etc.).

[0071] Gene product or expression product: As used herein, the terms "gene product" or "expression product" generally refer to the RNA transcribed from a gene (before and / or after processing) or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0072] Genome: As used herein, the term "genome" means the total genetic information contained in an individual organism or cell and represented by the complete DNA sequence of its chromosomes.

[0073] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar.

[0074] Host cell: as used herein, refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those skilled in the art will understand upon reading this disclosure that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotic and eukaryotic (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions, e.g., hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3 A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above-mentioned cells. In some embodiments, the cell comprises one or more viral genes.

[0075] Human: In some embodiments, the human is an embryo, fetus, infant, child, teenager, adult, or elderly.

[0076] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second sequences, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated in the ALIGN program (version 2.0) can be used to determine the percent identity between two nucleotide sequences. In some exemplary embodiments, comparison of nucleic acid sequences using the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix.

[0077] "Improve", "Increase", "Inhibit" or "Decrease": As used herein, the terms "improve", "increase", "inhibit", "decrease", or their grammatical equivalents, refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of a suitable comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in a comparable system known or expected to respond in a particular manner in the presence of a relevant agent or treatment.

[0078] Intraperitoneal: As used herein, the phrases "intraperitoneal administration" and "administered intraperitoneally" have their art-recognized meaning of administering a compound or composition to the peritoneum of a subject.

[0079] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0080] In vivo: As used herein, refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term is also used to refer to events that occur within living cells (e.g., not in an in vitro system).

[0081] Isolated: As used herein, refers to a substance and / or element that is (1) separated from at least some of the components with which it was associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) that has been artificially designed, produced, prepared, and / or manufactured. An isolated substance and / or element can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of other components with which it was originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may be considered to be "isolated" or even "pure" even after it has been combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the isolation rate or purity of a substance is calculated without including such carriers or excipients. By way of example, in some embodiments, a biological polymer, such as a polypeptide or polynucleotide, occurring in nature is considered to be "isolated" if: a) by its origin or source of derivation, it is not associated with some or all of the components that accompany it in its natural state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; or c) it is expressed or otherwise associated with components from cells or other expression systems other than the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide.Alternatively, or in addition, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components with which it a) is associated in nature and / or b) is associated when originally produced.

[0082] Linker: As used herein, it is used to refer to a portion of a multi-element agent that connects different elements to each other. For example, one of skill in the art will understand that a polypeptide whose structure includes two or more functional or organizational domains will often include a stretch of amino acids between them that connects such domains. In some embodiments, a polypeptide that includes a linker element has an overall structure of the general form S1-L-S2, where S1 and S2, which may be the same or different, represent two domains that are associated with each other by the linker. In some embodiments, the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids in length. In some embodiments, linker is characterized by not tending to adopt a rigid three-dimensional structure, but rather providing flexibility to the polypeptide.A variety of different linker elements that can be appropriately used in recombining polypeptides (e.g., chimeric systems) are known in the art (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1 121-1123).

[0083] Moiety: One of skill in the art will understand that a "moiety" is a defined chemical group or element having a particular structure and / or activity as described herein.

[0084] Nucleic acid: as used herein in its broadest sense means any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" means individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" means an oligonucleotide chain that includes individual nucleic acid residues. In some embodiments, "nucleic acid" is or includes RNA. In some embodiments, "nucleic acid" is or includes DNA. In some embodiments, a nucleic acid is, includes, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, includes, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, and are considered within the scope of the present invention. Alternatively, or in addition, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to that of a natural nucleic acid. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length. In some embodiments, the nucleic acid is partially or completely single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide, hi some embodiments, the nucleic acid has enzymatic activity.

[0085] Operably linked: As used herein, refers to a juxtaposition in a relationship permitting the components described to function in their intended manner. A control element "operably linked" to a functional element is associated such that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest, and in some embodiments the control element acts in trans or remote from the functional element of interest.

[0086] Oral: As used herein, the phrases "oral administration" and "orally administered" have their art-understood meaning and refer to administration of a compound or composition by mouth.

[0087] Patient: As used herein, the term "patient" refers to any organism to which a provided composition is or can be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes the presence of cancer or one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular treatment to diagnose and / or treat a disease, disorder, or condition.

[0088] Payload: In general, as used herein, the term "payload" refers to an agent that can be delivered or transported by association with another moiety. In some embodiments, such association can be or include a covalent bond, and in some embodiments, such association can be or include a non-covalent interaction(s). In some embodiments, the association can be direct, and in some embodiments, the association can be indirect. The term "payload" is not limited to a particular chemical identity or type. For example, in some embodiments, the payload can be or include, for example, any chemical class of member, including, for example, lipids, metals, nucleic acids, polypeptides, sugars (e.g., polysaccharides), small molecules, or combinations or complexes thereof. In some embodiments, the payload can be or include, a biological modifier, a detectable agent (e.g., dyes, fluorophores, radiolabels, etc.), a detection agent, a nutrient, a therapeutic agent, etc., or a combination thereof. In some embodiments, the payload can be or include, a cell or organism, or a fraction, extract, or component thereof. In some embodiments, the payload may be or include a natural product found in nature and / or obtained from nature. Alternatively, or in addition, in some embodiments, the term can be used to mean one or more elements that are engineered, engineered, and / or created by the action of the hand of man and / or are man-made in that they are not found in nature. In some embodiments, the payload may be or include an agent in isolated or pure form. In some embodiments, such an agent may be in crude form.

[0089] Pharmaceutically acceptable: As used herein, the phrase "pharmacologically acceptable" means compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0090] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in the transport or transfer of the subject compounds from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which may function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0091] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutical acceptable salts" refers to salts of compounds that are suitable for use in pharmaceutical situations, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al., describe pharmaceutical acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutical acceptable salts include, but are not limited to, non-toxic acid addition salts, which are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. In some embodiments, pharma- ceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydrogen phosphate, glycerol ... Examples of the salts include oxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Exemplary alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, etc. In some embodiments, pharma- ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1-6 carbon atoms, sulfonates, and arylsulfonates.

[0092] Polypeptide: as used herein refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered, in that it is artificially designed and / or created. In some embodiments, a polypeptide may comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications, e.g., modifications of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, PEGylation, and the like, including combinations thereof. In some embodiments, a polypeptide may be cyclic and / or include a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be added to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to polypeptides that share a related activity or structure and thus can be considered members of the same class or family of polypeptides. For each such class, the present specification provides, and / or one of skill in the art will be aware of, exemplary polypeptides within the class whose amino acid sequence and / or function are known.In some embodiments, such an exemplary polypeptide is a reference polypeptide of a class or family of polypeptides. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (in some embodiments, with all polypeptides in the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range) with the reference polypeptide of the class (in some embodiments, with all polypeptides in the class). For example, in some embodiments, member polypeptides exhibit at least one region (e.g., a conserved region, which in some embodiments may be or may include a distinctive sequence element) that exhibits an overall degree of sequence homology or identity of at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and / or often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%, with a reference polypeptide. Such conserved regions typically encompass at least 3-4, and often up to 20 or more amino acids, and in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids. In some embodiments, a related polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a polypeptide useful as a polypeptide may comprise or consist of multiple fragments, each of which are found in the same parent polypeptide in a different spatial arrangement compared to each other than that found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or the fragments may be present in a different order in the polypeptide of interest compared to the parent), such that the polypeptide of interest is a derivative of its parent polypeptide.In some embodiments, a "polypeptide" may be referred to as a "protein" (e.g., the term "Cas protein" may be used to refer to a "Cas polypeptide" as defined herein. In some embodiments, a "Cas12 protein" may be distinguished from a "Cas13 protein" by, for example, taking into account percent homology or identity with the appropriate reference polypeptides described herein and / or shared characteristic sequence elements).

[0093] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a series of at least two amino acids linked together by peptide bonds). A protein can include moieties other than amino acids (e.g., it can be a glycoprotein, a proteoglycan, etc.) and / or can be otherwise processed or modified. Those of skill in the art will understand that a "protein" can be an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may include, for example, two or more polypeptide chains linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a unique portion thereof.

[0094] Pure: As used herein, an agent or element is "pure" if it is substantially free of other components. For example, a preparation containing more than about 90% of a particular agent or element is typically considered a pure preparation. In some embodiments, the agent or element is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0095] Recombinant: as used herein, a polypeptide that is designed, modified, prepared, expressed, made, produced, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; transgenic or otherwise engineered for one or more genes or genetic components that encode and / or direct the expression of a polypeptide, or one or more component(s), portion(s), element(s), or domain(s) thereof, It is intended to mean a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that expresses it; and / or a polypeptide prepared, expressed, produced, or isolated by splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or any other method that involves otherwise generating a nucleic acid that encodes and / or directs the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, such one or more selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from a natural or synthetic source, such as, for example, the germline of a source organism of interest (e.g., human, mouse, etc.).

[0096] Reference: as used herein refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or measured substantially simultaneously with the test or measurement of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those of skill in the art, the reference or control is determined or analyzed under conditions or circumstances comparable to those to be evaluated. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control. In some embodiments, suitable reference Cas protein sequences can be found in literature reports, or sequence databases (e.g., GENBANK).To name a few examples known to those of skill in the art, in some embodiments, a suitable reference Cas12 protein, or a suitable reference Cas13 protein, can be, for example, those described in Koonin et al., Curr Opin Microbiol., 2017 June; 37: 67-78; Makarova et al., Nat Rev Microbiol., 2015 November; 13(11): 722-736; Shmakov et al., Mol Cell., 2015 November 5; 60(3): 385-397; Yan and Hunnewell et al., Science, 2018 Dec 6; Yan et al., Mol Cell., 2018 April 19; 70, 327-339; Makarova et al., Nat Rev Microbiol., 2011 June; 9(6): 467-477; Makarova et al., CRISPR Journal, 2018, Volume 1, Number 5, Shmakov et al., Nat Rev Microbiol., 2017 March;15(3):169-182, Yan and Hunnewell et al., Science, 2019 Jan 4;363,88-91, Abudayyeh et al., Science, 2016 August 5;353,6299, Gootenberg and Abudayyeh et al., Science, 2017 April 28,356,438-442, Gootenberg and Abudayyeh et al., Science, 2018 April 27;360,439-444. Alternatively, in some embodiments, an exemplary Cas protein described herein (e.g., one of SEQ ID NOs: 1-10) may serve as a reference to which other proteins are compared.

[0097] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained or derived from a source of interest, as described herein. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest can be or include a cell or an organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid can be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, the biological fluid can be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or cellular permeate fluid. In some embodiments, the biological fluid can be or include plant exudates. In some embodiments, the biological tissue or sample can be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or irrigation (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or irrigation). In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components and / or adding one or more agents). For example, filtration using a semipermeable membrane.Such a "processed sample" can include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques, such as, for example, amplification or reverse transcription of nucleic acids, isolation and / or purification of specific components.

[0098] Single Nucleotide Polymorphism (SNP): As used herein, the term "single nucleotide polymorphism" or "SNP" refers to a specific base position in a genome where alternative bases are known to distinguish certain alleles from one another. In some embodiments, one or a few SNPs and / or CNPs are sufficient to distinguish complex genetic variants from one another, so that for analysis purposes, one or a set of SNPs and / or CNPs can be considered characteristic of a particular variant, trait, cell type, individual, species, etc., or set thereof. In some embodiments, one or a set of SNPs and / or CNPs can be considered to define a particular variant, trait, cell type, individual, species, etc., or set thereof.

[0099] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments prenatal human forms). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is one who has one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or treatment is to be administered and / or has been administered.

[0100] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting a complete or near-total degree or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0101] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that partially or completely alleviates, improves, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of subjects who do not exhibit symptoms of the relevant disease, disorder, and / or condition, and / or subjects who exhibit only early signs of the disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be of subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who have been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of subjects known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the relevant disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic. In some embodiments, the treatment may be therapeutic.

[0102] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may include precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is an indication of cancer. In some embodiments, a tumor may be a dispersed tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0103] Vector: As used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors, etc.). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture, as well as transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes. (Mode for carrying out the invention)

[0104] CRISPR-Cas technology Typically, Cas proteins and / or guides are the main components of CRISPR-Cas technology. CRISPR-Cas technology or CRISPR-Cas system refers collectively to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA, or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guides (also referred to as "spacers" in the context of endogenous CRISPR systems), or "RNA(s)" as used herein (e.g., RNA(s) that guide Cas, such as Cas proteins disclosed herein, e.g., CRISPR RNA and transactivating (tracr) RNA, or single guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from CRISPR loci. Generally, CRISPR-Cas technology features an element (also called a protospacer in the context of endogenous CRISPR systems) that promotes CRISPR complex formation at the location of the target nucleic acid. In some embodiments, tracrRNA is not required.

[0105] In some embodiments of the recombinant or non-naturally occurring techniques of the present disclosure, the direct repeats can include naturally occurring or non-naturally occurring sequences. In some embodiments, the direct repeats (DR) can be of naturally occurring length and / or sequence. In some embodiments, the direct repeats can be 36 nucleotides (nt) long, but can be altered to be longer or shorter. For example, in some embodiments, the direct repeats can be 30 nt or more, e.g., 30-100 nt or more. For example, the direct repeats can be 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 70 nt, 80 nt, 90 nt, 100 nt or more in length. In some embodiments, the direct repeats of the present disclosure can include a synthetic nucleotide sequence inserted between the 5' and 3' ends of the naturally occurring direct repeat. In certain embodiments, the inserted sequence can be self-complementary, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% self-complementary. Furthermore, the direct repeats of the present disclosure may contain nucleotide insertions, such as aptamers or sequences that bind to adaptor proteins (to associate with functional domains). In certain embodiments, one end of the direct repeat containing such an insertion is approximately the first half of the short DR, and the other end is approximately the second half of the short DR. In some embodiments, the direct repeats can be identified in silico.

[0106] In some embodiments, the Cas protein is a thermostable Cas protein.

[0107] In the context of forming a CRISPR complex, "target nucleic acid sequence" refers to a sequence to which a guide, including a guide sequence, has (e.g., is designed to have) complementarity, and hybridization of the target nucleic acid sequence with the guide sequence promotes the formation of a CRISPR complex. In some embodiments, the target nucleic acid comprises any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target nucleic acid is located in the nucleus or cytoplasm of a cell. In some embodiments, the target nucleic acid is present ex vivo. In some embodiments, the target nucleic acid is present in an in vitro system. In some embodiments, the target nucleic acid is present in a sample, such as a biological sample or an environmental sample.

[0108] In some embodiments, the guide (e.g., constant domain and / or spacer) is 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, 100, 125, 150, 160, 170, 180, 190 or more nucleotides in length. In some embodiments, the guide is about 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. In some embodiments, the guide is 10-30 nucleotides long, e.g., 20-30 or 20-40 nucleotides long or more, e.g., 30 nucleotides long or about 30 nucleotides long, relative to the CRISPR-Cas effector. In certain embodiments, the guide is 10-30 nucleotides long, e.g., 20-30 nucleotides long, e.g., 30 nucleotides long. In certain embodiments, the guide is 90-200 nucleotides long, e.g., 100-190 nucleotides long, e.g., 110-180 nucleotides long, e.g., 120-170 nucleotides long. The ability of the guide to direct sequence-specific binding of the CRISPR complex to the target nucleic acid can be investigated by any suitable assay. For example, in some embodiments, sufficient components of the CRISPR system to form a CRISPR complex, including the guide sequence to be tested, are provided to a host cell having the corresponding target nucleic acid sequence, e.g., by transfecting a vector or vector system encoding components of the CRISPR-Cas technology, as discussed elsewhere herein, followed by investigating preferential cleavage within the target nucleic acid sequence. Similarly, in some embodiments, cleavage of a target nucleic acid is investigated in a test tube, for example, by providing a target nucleic acid, components of a CRISPR complex (a guide sequence to be tested, and a control guide sequence that differs from the test guide sequence), and comparing the binding or rate of cleavage at the target nucleic acid sequence in the test guide sequence reaction and the control guide sequence reaction. Other assays are possible and will occur to those skilled in the art.

[0109] In some embodiments, the degree of complementarity between a guide sequence and its corresponding target nucleic acid sequence can be about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%, or about greater than these percentages. In some embodiments, a guide or RNA or crRNA can be 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 or more nucleotides in length, or about greater than these values. Alternatively, in some embodiments, a guide or RNA or crRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides in length. And advantageously, the tracrRNA is 30 or 50 nucleotides long. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target nucleic acid sequence is 94.5%, or 95%, or 95.5%, or 96%, or 96.5%, or 97%, or 97.5%, or 98%, or 98.5%, or 99%, or 99.5%, or more than 99.9%, or 100%. In some embodiments, the off-target is 100%, or 99.9%, or 99.5%, or 99%, or 99%, or 98.5%, or 98%, or 97.5%, or 97%, or 96.5%, or 96%, or 95.5%, or 95%, or 94.5%, or 94%, or 93%, or 92%, or 91%, or 90%, or 89%, or 88%, or 87%, or Advantageously, the off-target has less than 86%, or 85%, or 84%, or 83%, or 82%, or 81%, or 80% complementarity, and the off-target has 100%, or 99.9%, or 99.5%, or 99%, or 99%, or 98.5%, or 98%, or 97.5%, or 97%, or 96.5%, or 96%, or 95.5%, or 95%, or 94.5% complementarity between the target nucleic acid sequence and the guide.

[0110] In some embodiments, modulation of cleavage activity (e.g., efficiency) can be utilized by introducing mismatches, e.g., one or more mismatches (such as one or two mismatches), between the guide sequence and the target nucleic acid sequence, including the position of the mismatch along the guide sequence / target nucleic acid sequence. Without wishing to be bound by any one theory, in some embodiments, the cleavage activity (e.g., efficiency) can be modulated by selecting the position of the mismatch along the guide sequence. For example, in some embodiments, if less than 100% cleavage of the target (e.g., in a cell population) is desired, one or more, e.g., preferably two, mismatches can be introduced into the guide sequence between the guide sequence and the target nucleic acid sequence. In some embodiments, the more central the guide sequence of the mismatch position, the lower the cleavage activity (e.g.,

[0111] In some embodiments, the formation of a CRISPR complex (including a guide sequence hybridized to a target nucleic acid sequence and complexed with one or more Cas proteins) results in cleavage at or near the target nucleic acid sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target nucleic acid sequence). Without wishing to be bound by any one theory, the location of the cleavage site at or near the target nucleic acid sequence may depend on the secondary structure, for example, especially in the case of RNA targets. In some cases, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence hybridized to a target nucleic acid sequence and complexed with one or more Cas proteins) results in cleavage of one or (if applicable) both strands at or near the target nucleic acid sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target nucleic acid sequence).

[0112] Thermostable Cas Proteins As described herein, the present disclosure identifies a source of problems with certain Cas proteins, e.g., Cas proteins with collateral activity, as described above, in that the activity of such certain proteins is not sufficiently stable at elevated temperatures. This can prove to be particularly challenging, for example, when utilizing Cas proteins in systems or assays that benefit from or require elevated temperatures (e.g., at temperatures where nucleic acid extension and / or amplification is performed and / or in cells or organisms (e.g., thermophiles that survive or grow only at elevated temperatures). Moreover, the present disclosure further surprisingly shows that for some proteins, loss of activity upon temperature increase can be irreversible. This reality heightens the importance of the considerations provided by the present disclosure that Cas proteins with thermostable activity (e.g., "thermostable Cas proteins"), including specifically Cas proteins with thermostable collateral activity, are particularly desirable.

[0113] Thus, the present disclosure provides improved Cas proteins, in particular improved Cas proteins having collateral activity, and further provides improved techniques that utilize thermostable Cas proteins as described herein (e.g., where the collateral activity is thermostable).

[0114] In some embodiments, the disclosure provides non-naturally occurring or recombinant compositions for binding to, detecting, and / or modifying nucleotides in a target nucleic acid comprising a thermostable Cas protein described herein (e.g., a Cas protein having collateral cleavage activity that is thermostable at least at a temperature above 60-65° C.). In some such embodiments, the compositions provided comprise non-naturally occurring or recombinant compositions comprising a thermostable Cas protein described herein (e.g., a Cas protein having collateral cleavage activity that is thermostable at least at a temperature above 60-65° C.) and at least one guide capable of forming a complex with the thermostable Cas protein and directing binding of the complex to the target nucleic acid. In some embodiments, the compositions provided comprise a Cas protein / guide complex. In some embodiments, the compositions provided comprise a Cas protein / guide complex bound to a target nucleic acid. In some embodiments, the compositions provided comprise a Cas protein in combination with a nucleic acid susceptible to collateral cleavage. In some such embodiments, such a sensitive nucleic acid does not contain a guide binding site and / or does not bind to and / or otherwise associate with a target nucleic acid that does not contain such a guide binding site. In some embodiments, the sensitive nucleic acid is labeled such that its cleavage (e.g., by collateral activity of a Cas protein provided herein) is detectable (e.g., by emission of fluorescence or visible light).

[0115] In some embodiments, the disclosure provides non-naturally occurring or recombinant compositions that include a polynucleotide encoding a thermostable Cas protein described herein (e.g., a Cas protein having collateral cleavage activity that is thermostable at least at temperatures above 60-65° C.). In some such embodiments, such compositions can be combined (e.g., used in combination with) at least one guide that is capable of forming a complex with the Cas protein and directing binding of the complex to a target nucleic acid sequence.

[0116] In some embodiments, the Cas enzymes provided herein (e.g., having thermostable collateral cleavage activity) are homologs (e.g., orthologs) of Cas enzymes that either do not have apparent collateral cleavage activity, or that have apparent collateral cleavage activity but lose such activity above the relevant temperatures described herein.

[0117] In some embodiments, the Cas enzyme with thermostable collateral cleavage activity described herein is a Cas12 (e.g., Cas12a or Cas12b) enzyme. In some embodiments, the Cas enzyme with thermostable collateral cleavage activity described herein is a Cas enzyme comprising an amino acid sequence having 80%, 85%, 90%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-10. In some embodiments, the improved collateral activity assay described herein is performed with a Cas enzyme comprising an amino acid sequence having 80%, 85%, 90%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-10.

[0118] Codon Optimization Without wishing to be bound by any one theory, some species exhibit codon bias (i.e., differences in codon usage by organisms) that can be correlated with the translation efficiency of mRNA by the abundance of tRNA species for a codon in a particular organism utilizing that codon in the corresponding mRNA. Various methods for codon optimization are well known in the art. In some embodiments, codons are optimized by algorithmic methods.

[0119] In some embodiments, codon optimization refers to the modification of a nucleic acid sequence to improve expression in a cell by replacing at least one codon (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more codons) with respect to a suitable reference sequence (e.g., a natural sequence). In some embodiments, the codons of the suitable reference sequence are replaced with codons that are more frequently or most frequently used in the genes of the cell while maintaining the natural amino acid sequence encoded by the nucleic acid sequence.

[0120] In some embodiments, the nucleotide sequence encoding the Cas protein of the present disclosure is codon-optimized. In some embodiments, the nucleotide sequence encoding the Cas protein of the present disclosure is codon-optimized for expression in eukaryotic cells. In some such embodiments, the eukaryotic cell is a human cell. In some embodiments, the nucleotide sequence encoding the Cas protein of the present disclosure is codon-optimized for expression in prokaryotic cells.

[0121] Qualifying element In some embodiments, the Cas proteins of the disclosure are associated with (e.g., fused, i.e., covalently or non-covalently linked to) one or more modifying elements that can modify (e.g., edit) a nucleic acid sequence (e.g., a target nucleic acid sequence) and / or a nucleic acid (e.g., a target nucleic acid) structure, e.g., by chemically modifying nucleotide bases, as a chimeric system.

[0122] In some embodiments, the modifying element has base editing activity. In some embodiments, the modifying element is a deaminase. In some such embodiments, the modifying element is a cytidine deaminase or a functional fragment thereof. In some embodiments, the cytidine deaminase or a functional fragment thereof comprises 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more sequence (i.e., nucleotide or amino acid) identity with any cytidine deaminase known in the art. In some embodiments, the cytidine deaminase or a functional fragment thereof exhibits cytidine deaminase activity (e.g., converting C to U). In some such embodiments, the modifying element is an adenosine deaminase or a functional fragment thereof. In some embodiments, the adenosine deaminase or functional fragment thereof comprises 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more sequence (i.e., nucleotide or amino acid) identity to any adenosine deaminase known in the art. In some embodiments, the adenosine deaminase or functional fragment thereof exhibits adenosine deaminase activity (e.g., converting A to I).

[0123] In some embodiments, the modifying element modifies the target nucleic acid sequence in a site-specific manner.In some embodiments, for example, the modifying element activity comprises methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, or nuclease activity, any of which can modify DNA or DNA-related polypeptide (e.g., histone or DNA-binding protein).

[0124] In some embodiments, a chimeric system of the present disclosure comprises one modifying element. In some embodiments, a chimeric system comprises multiple modifying elements (e.g., at least two modifying elements). In some embodiments, a chimeric system comprises a modifying element C-terminal to a Cas protein. In some embodiments, a chimeric system comprises a modifying element N-terminal to a Cas protein. In some embodiments, the modifying element and Cas protein of the chimeric system are directly linked. In some embodiments, the modifying element and Cas protein of the chimeric system are indirectly linked (e.g., by a linker).

[0125] Exemplary characterization of thermostable Cas proteins Among other things, the disclosure provides methods for characterizing Cas proteins (e.g., thermostable Cas proteins of the disclosure). In some embodiments, the Cas proteins are characterized for one or more of cis (e.g., target nucleic acid) cleavage activity, trans or "collateral" (e.g., non-target nucleic acid) cleavage activity, sensitivity, selectivity for RNA and / or DNA target nucleic acids, selectivity for RNA and / or DNA non-target nucleic acids, and / or enzymatic stability.

[0126] In some embodiments, the Cas protein is characterized by one guide. In some embodiments, the Cas protein is characterized by two or more guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 1,000, or more guide sequences). In some such embodiments, the two or more guides comprise different guide sequences.

[0127] In some embodiments, the Cas protein is characterized at a particular temperature. In some such embodiments, the particular temperature comprises 25° C., 30° C., 35° C., 37° C., 40° C., 42° C., 45° C., 47° C., 50° C., 52° C., 54° C., 55° C., 56° C., 57° C., 58° C., 60° C., 62° C., 65° C., 67° C., 70° C., 72° C., 80° C., 90° C., or 100° C. In some embodiments, the Cas protein is characterized over a range of temperatures. In some such embodiments, the range of temperatures includes 10-100° C., 20-90° C., 30-80° C., 40-70° C., 50-70° C., 10-90° C., 10-80° C., 10-70° C., 20-100° C., 20-80° C., 20-70° C., 30-100° C., 30-90° C., or 30-70° C. In some embodiments, the Cas protein is characterized at multiple temperatures. In some such embodiments, the multiple temperatures include any combination of any temperatures between 10-100° C.

[0128] Cis-cleavage activity In some embodiments, the cis (e.g., target nucleic acid) cleavage activity of the thermostable Cas proteins described herein is characterized. In some embodiments, the cis cleavage activity is characterized by an in vitro cleavage assay. In some such embodiments, the in vitro cleavage assay includes, for example, expressing the Cas protein in a host cell, preparing a cell lysate, preparing (e.g., amplifying) a target nucleic acid (e.g., DNA and / or RNA), incubating the Cas-containing cell lysate with the target nucleic acid and guide, and examining the cleavage activity (e.g., by gel electrophoresis, reporter) relative to a suitable reference standard. In some embodiments, a suitable reference standard is a host cell expressing a control protein (e.g., green fluorescent protein) that does not exhibit cis cleavage activity.

[0129] In some embodiments, cis cleavage activity is characterized by an ex vivo cleavage assay. In some such embodiments, the ex vivo cleavage assay includes, for example, expressing a Cas protein in a host cell and a guide, extracting a polynucleotide of interest from the host cell (e.g., DNA and / or RNA), and sequencing to measure cleavage activity (e.g., insertion, deletion, and / or mutation patterns). In some embodiments, the sequencing includes deep sequencing. In some embodiments, the sequencing includes next-generation sequencing.

[0130] In some embodiments, the cis cleavage activity is characterized by an end-point assay. In some embodiments, the cis cleavage activity is characterized by a kinetic assay.

[0131] Trans-cleavage activity In some embodiments, the trans, or "collateral" (e.g., non-target nucleic acid) cleavage activity of the Cas proteins described herein is characterized. Trans, or "collateral" activity is the non-specific cleavage activity of non-target nucleic acids after the Cas protein binds to the target nucleic acid (e.g., becomes an "activated" Cas protein). In some embodiments, upon recognition of the target nucleic acid, the trans cleavage activity of the Cas protein is activated, resulting in cleavage of the non-target nucleic acid (DNA or RNA, or both, depending on the enzyme). In some embodiments, the trans cleavage activity is investigated by a reporter. In some such embodiments, the reporter comprises an associated cleavable nucleic acid (e.g., DNA or RNA) and is appropriately configured (e.g., labeled) such that its cleavage is detectable as a result of activated collateral activity (e.g., separation of a fluorophore from a quencher, resulting in detectable fluorescence, etc.). In some embodiments, a negative control (e.g., a control without target nucleic acid) is used. In some such embodiments, the trans cleavage activity is investigated in vitro.

[0132] In some embodiments, the trans-cleavage activity is characterized by an end-point assay. In some embodiments, the trans-cleavage activity is characterized by a kinetic assay.

[0133] sensitivity In some embodiments, the sensitivity of the Cas proteins described herein is characterized. In some embodiments, the sensitivity is examined by determining the lowest concentration of target nucleic acid that can be detected, cleaved, and / or modified 80%, 85%, 90%, 95%, 99% or more of the time. In some such embodiments, the sensitivity is measured by contacting the Cas enzyme with dilutions (e.g., serial dilutions) of the target nucleic acid and examining the detection, cleavage, and / or modification of the target nucleic acid.

[0134] Selection of RNA or DNA target nucleic acid and / or non-target nucleic acid In some embodiments, the Cas proteins described herein are characterized for their selectivity for RNA or DNA target nucleic acids (e.g., cis cleavage activity) and / or non-target nucleic acids (e.g., trans or "collateral" cleavage activity).

[0135] In some embodiments, the selection of the Cas proteins described herein for RNA or DNA target nucleic acids (e.g., cis cleavage activity) is characterized. In some such embodiments, the selection is characterized by examining cis Cas protein activity by comparing the cleavage activity when the Cas protein contacts a DNA target nucleic acid to when the Cas protein contacts an RNA target nucleic acid. In some embodiments, the DNA target nucleic acid is double-stranded DNA. In some embodiments, the DNA target nucleic acid is single-stranded DNA.

[0136] In some embodiments, the selection of RNA or DNA non-target nucleic acids (e.g., trans, or "collateral" cleavage activity) of the Cas proteins described herein is characterized. In some such embodiments, the selection is characterized by examining the trans-Cas protein activity by comparing the trans-cleavage activity when the Cas protein contacts a DNA non-target nucleic acid to when the Cas protein contacts an RNA non-target nucleic acid. In some embodiments, the DNA non-target nucleic acid is double-stranded DNA. In some embodiments, the DNA non-target nucleic acid is single-stranded DNA. In some embodiments, the non-target nucleic acid includes a reporter (e.g., a reporter that separates a fluorophore from a quencher upon cleavage, such that fluorescence is detectable). In some such embodiments, the reporter is DnaseAlert. In some such embodiments, the reporter is RnaseAlert.

[0137] Enzyme stability In some embodiments, the enzyme stability of the thermostable Cas proteins described herein is characterized. In some such embodiments, the enzyme stability is characterized by studying the enzyme denaturation (e.g., using a proteolysis method). In some embodiments, studying the enzyme denaturation includes mixing the Cas protein with a buffer and a dye and generating a melting curve. Without wishing to be bound by any one theory, in some embodiments, as the temperature is increased, the Cas protein unfolds, exposing hydrophobic regions to which the dye can bind. Upon dye binding, the dye fluoresces. In some such embodiments, the change in fluorescence with temperature change is plotted against the temperature of the melting curve. In some embodiments, the melting temperature of the Cas protein can be measured and compared to the melting temperature of an appropriate reference standard (e.g., a Cas protein with known thermostability, e.g., Aac and / or RS9). In some such embodiments, the change in melting temperature correlates to a change in protein stability (e.g., thermostability) and activity.

[0138] target nucleic acid In some embodiments, the target nucleic acid useful according to the present disclosure is not limited to a particular length. In some embodiments, the target nucleic acid is a nucleotide (oligonucleotide or polynucleotide) of any length, including the sequence to which the guide sequence hybridizes. In some embodiments, the target nucleic acid comprises a three-dimensional structure. In some embodiments, the target nucleic acid sequence comprises coding and / or non-coding regions. In some embodiments, the target nucleic acid sequence comprises exons, introns, mRNA, tRNA, rRNA, siRNA, shRNA, miRNA, ribozymes, cDNA, plasmids, vectors, exogenous nucleotide sequences, and / or endogenous nucleotide sequences. In some embodiments, the target nucleic acid sequence comprises modified nucleotides, including, for example, methylated nucleotides or nucleotide analogs. In some embodiments, the target nucleic acid sequence may be interspersed with non-nucleic acid components. In some embodiments, the target nucleic acid is single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0139] In some embodiments, the target nucleic acid is recognized by CRISPR-Cas technology and binds to a Cas protein as described herein. In some embodiments, the target nucleic acid is modified or truncated, or the gene encoded by the target nucleic acid has altered expression as a result of Cas protein binding and activity. In some embodiments, the target nucleic acid sequence contains a specific, recognizable protospacer adjacent motif (PAM).

[0140] guide In some embodiments of the present disclosure, the CRISPR-Cas technology includes at least one guide comprising a guide sequence. In some embodiments, the guide sequence is a nucleotide sequence that has sufficient complementarity such that the guide can hybridize to a specific target nucleic acid. In some embodiments, the guide comprises a guide sequence that is complementary to a target nucleic acid sequence. In some embodiments, the guide sequence is 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more complementary to the target nucleic acid sequence. In some embodiments, the guide sequence is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length.

[0141] In some embodiments, the disclosed technology utilizes multiple guides. In some embodiments, the multiple guides include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1,000, or more guides. In some embodiments, two or more guides include the same guide sequence. In some embodiments, two or more guides further include different guide sequences. In some embodiments, two or more guide sequences hybridize to different target sites of the same target nucleic acid. In some embodiments, two or more guide sequences hybridize to different target nucleic acid sequences.

[0142] In some embodiments, the ability of a guide to direct sequence-specific binding of CRISPR-Cas technology to a target nucleic acid can be investigated by any suitable assay. For example, the components of the CRISPR-Cas technology disclosed herein are sufficient to form a CRISPR complex containing the guide sequence to be tested, and can be subjected to a cell having a corresponding (e.g., complementary) target nucleic acid sequence, for example, by transfecting a vector encoding the components of the CRISPR-Cas technology (e.g., described elsewhere herein) and then characterizing selective cleavage in the target nucleic acid sequence. Similarly, cleavage of a target nucleic acid can be investigated in a test tube, for example, by providing a target nucleic acid, components of the CRISPR-Cas technology (including the guide to be tested and a control guide that includes a guide sequence different from the test guide sequence), and comparing the binding or rate of cleavage at the target nucleic acid in the test guide sequence reaction and the control guide sequence reaction. Other assays are possible and will occur to those skilled in the art. A guide sequence can be selected to target any target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence within the genome of a cell. Exemplary target nucleic acids are those that are unique to the target genome.

[0143] In some embodiments, the composition comprises a Cas protein and a heterologous guide sequence, for example, the guide sequence and the Cas protein do not naturally occur in the same cell or in the same species.

[0144] In some embodiments, the CRISPR-Cas technology described herein uses a crRNA or similar polynucleotide that includes a guide sequence, where the polynucleotide is RNA, DNA, or a mixture of RNA and DNA, and / or the polynucleotide includes one or more nucleotide analogs. In some such embodiments, the sequence can include any structure of a naturally occurring crRNA, including, but not limited to, a bulge, hairpin, or stem-loop structure. In some embodiments, the polynucleotide that includes the guide sequence forms a duplex with a second polynucleotide sequence, which can be an RNA or DNA sequence.

[0145] In some embodiments, the guide of the present disclosure comprises a nucleic acid that does not occur naturally, and / or a non-naturally occurring nucleotide and / or nucleotide analog, and / or a chemical modification. Non-naturally occurring nucleic acids can include, for example, a mixture of naturally occurring and non-naturally occurring nucleotides. In some embodiments, the non-naturally occurring nucleotide and / or nucleotide analog is modified at the ribose, phosphate, and / or base moiety. In some embodiments, the guide comprises a ribonucleotide and a non-ribonucleotide. In some such embodiments, the guide comprises one or more ribonucleotides and one or more deoxyribonucleotides. In some embodiments, the guide comprises one or more non-naturally occurring nucleotides or nucleotide analogs, such as a nucleotide having a phosphorothioate bond, a boranophosphate bond; a locked nucleic acid (LNA) nucleotide that comprises a methylene bridge between the 2' and 4' carbons of the ribose ring; or a bridged nucleic acid (BNA). Other examples of modified nucleotides include, but are not limited to, 2'-0-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2'-fluoro analogs. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine (Y), N1-methylpseudouridine (me1Y), 5-methoxyuridine (5moU), inosine, 7-methylguanosine. Examples of guide RNA chemical modifications include, but are not limited to, the incorporation of 2'-0-methyl (M), 2'-0-methyl-3'-phosphorothioate (MS), S-constrained ethyl (cEt), or 2'-0-methyl-3'-thioPACE (MSP) at one or more terminal nucleotides.In some embodiments, such chemically modified guide RNAs can include increased stability and increased activity compared to unmodified guide RNAs, but with less predictable on-target and off-target specificity (Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038 / nbt.3290, published online 29 June 2015; Allerson et al., J. Med. Chem. 2005, 48:901-904; Bramsen et al., Front. Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875; Sharma et al., MedChemComm., 2014, 5:1454-1471; Li et al., Nature Biomedical Engineering, 2017, 1,0066 See DOI:10.1038 / s41551-017-0066).

[0146] In some embodiments, the 5' and / or 3' ends of the guide are modified with various functional moieties, including fluorescent dyes, polyethylene glycol, cholesterol, proteins, or detection tags (see Kelly et al., 2016, J. Biotech. 233:74-83). In some embodiments, the guide comprises ribonucleotides in the region that binds to the target nucleic acid and one or more deoxyribonucleotides, and / or nucleotide analogs in the region that binds to the Cas protein. In some embodiments, deoxyribonucleotides and / or nucleotide analogs are incorporated into the guide sequence, including but not limited to the 5' and / or 3' ends, stem-loop regions, and seed regions. In some embodiments, the modifications are absent in the 5' handle of the stem-loop region. In some embodiments, chemical modifications at the 5' handle of the stem-loop region of the guide can result in loss of its function (see Li, et al., Nature Biomedical Engineering, 2017, 1:0066). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, or more nucleotides of the guide sequence are chemically modified. In some embodiments, 3-5 nucleotides at either the 3' or 5' end of the guide are chemically modified. In some embodiments, only minor modifications, such as 2'-F modifications, are introduced in the seed region. In some embodiments, 2'-F modifications are introduced at the 3' end of the guide. In some embodiments, 3-5 nucleotides at the 5' and / or 3' end of the guide are chemically modified with TO-methyl (M), 2'-0-methyl-3'-phosphorothioate (MS), S-constrained ethyl (cEt), or 2'-0-methyl-3'-thioPACE (MSP). In some embodiments, such modifications can improve genome editing efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9):985-989).In some embodiments, all of the phosphodiester bonds of the guide are replaced with phosphorothioate (PS) to improve the degree of gene disruption. In some embodiments, six or more nucleotides at the 5' and / or 3' end of the guide are chemically modified with 2'-OMe, 2'-F, or S-constrained ethyl (cEt). In some embodiments, such chemically modified guides can mediate improved degree of gene disruption (see Ragdarm et al., 0215, PNAS, E7110-E7111). In some embodiments, the guide is modified to include a chemical moiety at its 3' and / or 5' end. In some embodiments, such moieties include, but are not limited to, amine, azide, alkyne, thio, dibenzocyclooctyne (DBCO), or rhodamine. In some embodiments, the chemical moiety is conjugated to the guide sequence by a linker, such as an alkyl chain. In some embodiments, the chemical moiety of the modified guide can be used to attach the guide to another molecule, such as DNA, RNA, a protein, or a nanoparticle. In some embodiments, such chemically modified guides can be used, for example, to identify or enrich for cells that have been gene edited by the CRISPR system (see Lee et al., eLife, 2017, 6:e25312, DOI:10.7554).

[0147] In some embodiments, the modification to the guide is a chemical modification, an insertion, a deletion, or a split. In some embodiments, the chemical modification includes, but is not limited to, the incorporation of 2'-0-methyl (M) analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, 2'-fluoro analogs, 2-aminopurine, 5-bromo-uridine, pseudouridine (Y), N1-methylpseudouridine (me 1 Y), 5-methoxyuridine (5moU), inosine, 7-methylguanosine, 2'-O-methyl-3'-phosphorothioate (MS), S-constrained ethyl (cEt), phosphorothioate (PS), or 2'-0-methyl-3'-thioPACE (MSP). In some embodiments, the guide comprises one or more phosphorothioate modifications. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemically modified. In some embodiments, one or more nucleotides in the seed region are chemically modified. In some embodiments, one or more nucleotides at the 3' end are chemically modified. In some embodiments, none of the nucleotides in the 5'-handle are chemically modified. In some embodiments, the chemical modification in the seed region is a minor modification, such as the incorporation of a 2'-fluoro analog. In some embodiments, such chemical modification at the 3' end of Cpfl CrRNA improves gene cleavage efficiency (see Li, et al., Nature Biomedical Engineering, 2017, 1:0066).

[0148] In some embodiments, the loop of the 5' handle of the guide is modified. In some embodiments, the loop of the 5' handle of the guide is modified to have a deletion, insertion, split, or chemical modification. In some embodiments, the loop comprises 3, 4, or 5 nucleotides.

[0149] In some embodiments, the guide sequence includes moieties that are chemically linked or conjugated by non-phosphodiester bonds. In some embodiments, the guide sequence includes direct repeat sequence moieties and targeting sequence moieties that are chemically linked or conjugated by non-nucleotide loops, in non-limiting examples. In some embodiments, the moieties are linked by non-phosphodiester covalent linkers. Examples of covalent linkers include, but are not limited to, chemical moieties selected from the group consisting of carbamates, ethers, esters, amides, imines, amidines, aminotridines, hydrozones, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, sulphonates, sulphoxides, ureas, thioureas, hydrazides, oximes, triazoles, photolabile bonds, C-C bond-forming groups (such as Diels-Alder cycloaddition pairs or ring-closing metathesis pairs, and Michael reaction pairs).

[0150] In some embodiments, the guide moiety is first synthesized using standard phosphoramidite synthesis procedures (Herdewijn, P., ed., Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methods and Applications, Humana Press, New Jersey (2012)). In some embodiments, the non-target guide moiety can be functionalized to contain appropriate functional groups for ligation using standard procedures well known in the art (Hermanson, GT, Bioconjugate Techniques, Academic Press (2013)). Examples of functional groups include, but are not limited to, hydroxyl, amine, carboxylic acid, carboxylic acid halide, carboxylic acid active ester, aldehyde, carbonyl, chlorocarbonyl, imidazolylcarbonyl, hydrozide, semicarbazide, thiosemicarbazide, thiol, maleimide, haloalkyl, sulfonyl, allyl, propargyl, diene, alkyne, and azide. In some embodiments, once the non-target portion of the guide is functionalized, a covalent chemical bond or linkage can be formed between the two oligonucleotides. Examples of chemical bonds include, but are not limited to, those based on carbamates, ethers, esters, amides, imines, amidines, aminotridines, hydrozones, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, sulfones, sulfoxides, ureas, thioureas, hydrazides, oximes, triazoles, photolabile bonds, CC bond-forming groups (such as Diels-Alder cycloaddition pairs or ring-closing metathesis pairs, and Michael reaction pairs).

[0151] In some embodiments, one or more portions of the guide can be chemically synthesized using an automated solid-phase oligonucleotide synthesizer using 2'-acetoxyethyl orthoester (2'-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317:3-18) or 2'-thionocarbamate (2'-TC) chemistries (Dellinger et al., J. Am. Chem. Soc. (2011) 133:11540-11546; Hendel et al., Nat. Biotechnol. (2015) 33:985-989).

[0152] In some embodiments, guide moieties can be covalently attached using various bioconjugation reactions, loops, crosslinks, and sugar modifications, internucleotide phosphodiester bonds, non-nucleotide linkages via purine and pyrimidine residues. Sletten et al., Angew. Chem. Int. Ed. (2009) 48:6974-6998; Manoharan, M. Curr. Opin. Chem. Biol. (2004) 8:570-9; Behlke et al., Oligonucleotides (2008) 18:305-19; Watts, et al., Drug. Discov. Today (2008) 13:842-55; Shukla, et al., ChemMedChem (2010) 5:328-49.

[0153] In some embodiments, the guide moiety can be covalently attached using click chemistry. In some embodiments, the guide moiety can be covalently attached using a triazole linker. In some embodiments, the guide moiety can be covalently attached using a Huisgen 1,3-dipolar cycloaddition reaction involving an alkyne and an azide to yield a highly stable triazole linker (He et al., ChemBioChem (2015) 17:1809-1812; WO 2016 / 186745). In some embodiments, the guide moiety is covalently attached by ligation of a 5'-hexyne moiety and a 3'-azide moiety. In some embodiments, either or both of the 5'-hexyne and 3'-azide guide moieties can be protected with 2'-acetoxyethyl orthoester (2'-ACE) groups, which can then be removed using the Dharmacon procedure (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317:3-18).

[0154] In some embodiments, the guide moiety can be covalently linked by a linker (e.g., a non-nucleotide loop) including moieties such as spacers, attachments, bioconjugates, chromophores, reporter groups, dye-labeled RNA, and non-naturally occurring nucleotide analogs. More specifically, spacers suitable for the purposes of the present invention include, but are not limited to, polyethers (e.g., polyethylene glycol, polyalcohols, polypropylene glycol, or mixtures of ethylene and propylene glycol), polyamine groups (e.g., spennine, spermidine, and polymeric analogs thereof), polyesters (e.g., poly(ethyl acrylate)), polyphosphodiesters, alkylenes, and combinations thereof. In some embodiments, suitable attachments include any moiety that is attached to the linker to add additional properties to the linker, such as, but not limited to, a fluorescent label. Suitable bioconjugates include, but are not limited to, peptides, glycosides, lipids, cholesterol, phospholipids, diacylglycerols and dialkylglycerols, fatty acids, carbohydrates, enzyme substrates, steroids, biotin, digoxigenin, carbohydrates, polysaccharides.Suitable chromophores, reporter groups, and dye-labeled RNAs include, but are not limited to, fluorescent dyes (such as fluorescein and rhodamine), chemiluminescent, electrochemiluminescent, and bioluminescent marker compounds.Exemplary linker designs for conjugating two RNA components are also described in WO2004 / 015075.

[0155] In some embodiments, useful linkers (e.g., non-nucleotide loops) according to the present disclosure are not limited to a particular length. In some embodiments, useful linkers are linkers of any length. In some embodiments, the linker has a length equal to about 0-16 nucleotides. In some embodiments, the linker has a length equal to about 0-8 nucleotides. In some embodiments, the linker has a length equal to about 0-4 nucleotides. In some embodiments, the linker has a length equal to about 2 nucleotides. Exemplary linker designs are also described in WO2011 / 008730.

[0156] multiplexing In some embodiments, the CRISPR-Cas technology described herein utilizes multiple guides. Without wishing to be bound by any one theory, the use of multiple guides allows multiple target nucleic acids to be targeted. In some embodiments, the multiple guides are arranged in tandem, and can be optionally separated by a nucleic acid sequence (e.g., DR sequence). In some embodiments, the position of the two or more guides in tandem does not affect activity.

[0157] In some embodiments, the CRISPR-Cas technology described herein utilizes multiple guides for multiplexing. In some embodiments, two or more Cas proteins are used. In some embodiments, a single Cas protein is utilized. In some such embodiments, a single Cas protein is delivered with multiple guides, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 750, at least 1,000, or more guides.

[0158] In some embodiments, a guide or multiple guides hybridize to multiple target nucleic acids. In some embodiments, the CRISPR-Cas technology cleaves and / or edits multiple target nucleic acids. In some such embodiments, the cleavage and / or editing mutates, inserts, and / or deletes nucleotides in the target nucleic acid or multiple target nucleic acids. In some embodiments, the mutation, insertion, and / or deletion of nucleotides results in a change in gene expression of a gene encoded by the target nucleic acid or controlled by a control element of the target nucleic acid.

[0159] In some embodiments, the multiple guide sequences can hybridize to different target nucleic acids or different regions (e.g., sequences) of the same target nucleic acid. In some embodiments, methods are provided for altering expression of multiple gene products using CRISPR-Cas technology with multiple guides as described herein. In some embodiments, the method includes contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least at a temperature above 60-65°C and at least one guide sequence capable of hybridizing to at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide and capable of causing a disruption in the at least one target nucleic acid. In some embodiments, the method includes contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least at a temperature above 60-65°C and at least one guide capable of hybridizing to at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide and capable of editing the at least one target nucleic acid sequence.

[0160] Method for modifying a target nucleic acid sequence In some embodiments, the CRISPR-Cas technology described herein can be used for modifying a target nucleic acid sequence (e.g., gene editing). In some embodiments, modifying a target nucleic acid sequence can result in gene silencing or a change in expression level (e.g., an increase or decrease) in the expression of a gene product controlled or encoded by the target nucleic acid sequence. In some embodiments, the CRISPR-Cas technology of the present disclosure can be used for site-specific modification of a target nucleic acid sequence. In some embodiments, site-specific modification of a target nucleic acid sequence results in gene silencing or a change in expression level (e.g., an increase or decrease) in the expression of a gene product controlled or encoded by the target nucleic acid sequence. Thus, in some embodiments, the CRISPR-Cas technology described herein is used in a method of modifying a target nucleic acid sequence. In some embodiments, the CRISPR-Cas technology described herein is used in a method of modifying a target nucleic acid in a cell(s) (e.g., a prokaryotic or eukaryotic cell).

[0161] In some embodiments, the disclosed methods include inducing one or more nucleotide modifications in a cell, including delivering to the cell a vector or vector system discussed elsewhere herein. In some embodiments, the mutation(s) include the introduction, deletion, or substitution of one or more nucleotides or payloads in each target nucleic acid sequence of the cell(s) by the guide(s), RNA(s), or sgRNA(s). In some embodiments, the mutation(s) include the introduction, deletion, or substitution of 1-75 nucleotides in each target nucleic acid sequence of the cell(s) by the guide(s). In some embodiments, the mutation(s) comprise the introduction, deletion, or substitution of 1, 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, or 75 nucleotides in each target nucleic acid sequence of the cell(s) by the guide(s). In some embodiments, the mutation(s) comprise the introduction, deletion, or substitution of 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, or 75 nucleotides in each target nucleic acid sequence of the cell(s) by the guide(s). In some embodiments, the mutation(s) comprise the introduction, deletion, or substitution of 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, or 75 nucleotides in each target nucleic acid sequence of the cell(s) by the guide(s). In some embodiments, the mutation(s) comprise the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides in each target nucleic acid sequence of the cell(s) by the guide(s). In some embodiments, the mutation(s) comprise the introduction, deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400, or 500 nucleotides in each target nucleic acid sequence of the cell(s) by the guide(s).

[0162] In some embodiments, the concentration of delivered Cas mRNA or protein and guide(s) is controlled to minimize toxicity and off-target effects. In some embodiments, the optimal concentration of Cas mRNA or protein and guide(s) is determined, for example, by testing different concentrations in a cell or eukaryotic animal model and analyzing the extent of modification at potential off-target genomic loci using deep sequencing.

[0163] In some embodiments, the disclosed technology provides a method of cleaving a target nucleic acid in a cell, comprising contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide sequence capable of hybridizing to at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide sequence and capable of causing a disruption in the at least one target nucleic acid.

[0164] In some embodiments, the disclosed technology provides a method of altering expression of a gene controlled or encoded by a target nucleic acid in a cell, comprising contacting the cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide sequence capable of hybridizing to at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide sequence and capable of causing a disruption in the at least one target nucleic acid.

[0165] In some embodiments, the disclosed technology provides a method of altering expression of a gene controlled or encoded by a target nucleic acid in a cell, comprising contacting the cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide sequence capable of hybridizing to at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide sequence and is capable of editing the at least one target nucleic acid sequence.

[0166] In some embodiments, the disclosed technology provides a method of modifying a target nucleic acid in a cell, comprising contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide sequence capable of hybridizing to at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide sequence and is capable of editing the at least one target nucleic acid sequence.

[0167] In some embodiments, the method includes binding CRISPR-Cas technology to a target nucleic acid and cleaving the target nucleic acid. In some embodiments, the CRISPR-Cas technology cleaves the target nucleic acid duplex (e.g., a DNA or RNA duplex) by introducing a double-stranded break. In some embodiments, the CRISPR-Cas technology cleaves the target nucleic acid duplex (e.g., a DNA or RNA duplex) by introducing a single-stranded break.

[0168] In some embodiments, the CRISPR-Cas technology described herein includes an exogenous donor template nucleic acid (e.g., a DNA molecule or an RNA molecule) that includes a nucleic acid sequence of interest (e.g., a donor template nucleic acid sequence). In some embodiments, the donor template nucleic acid sequence is not identical to the genomic sequence it replaces. In some embodiments, the donor template nucleic acid sequence can contain at least one or more single base changes, insertions, deletions, inversions, or rearrangements relative to the genomic sequence, so long as there is sufficient homology to support homology-directed repair. Without wishing to be bound by any one theory, during repair of the cleavage event induced by the CRISPR-Cas technology described herein, the molecular machinery of the cell utilizes the exogenous donor template nucleic acid to repair and / or repair the cleavage event. Alternatively, the molecular machinery of the cell can utilize an endogenous donor template to repair and / or repair the cleavage event.

[0169] In some embodiments, the donor template nucleic acid sequence contains sufficient homology, e.g., 70%, 80%, 85%, 90%, 95%, or 100%, to the genomic sequence at the cleavage site, with the nucleotide sequence flanking the cleavage site, e.g., within about 50 bases, 40 bases, 30 bases, 20 bases, 15 bases, 10 bases, 5 bases, or 1 base from the cleavage site. Without wishing to be bound by any one theory, the homology with the nucleotide sequence flanking the cleavage site supports homology-directed repair between the nucleotide sequence and the homologous genomic sequence. For example, in some embodiments, sequence homology of approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides (or any integer value between 10-200 nucleotides or more) between the donor and the genomic sequence supports homology-directed repair.

[0170] In some embodiments, the donor template nucleic acid useful according to the present disclosure is not limited to a specific length.In some embodiments, the donor template nucleic acid is any length of nucleotide (oligonucleotide or polynucleotide).For example, in some embodiments, the donor template nucleic acid comprises 10 nucleotides or more, 25 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1,000 nucleotides or more, 500 nucleotides or more, etc.

[0171] Insert In some embodiments, the CRISPR-Cas technology described herein is used to edit a target nucleic acid sequence by inserting one or more nucleotides into the target nucleic acid. In some embodiments, the insertion is a intact insertion (e.g., inserting a nucleic acid sequence of interest into the target nucleic acid such that upon recovery of the cleavage event, no unintended additional nucleic acid sequence is generated).

[0172] In some embodiments, the insertion results in a frameshift mutation within the coding region of the target nucleic acid sequence that encodes a gene product. In some embodiments, the CRISR-Cas technology provided herein results in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% insertion formation in the target nucleic acid.

[0173] In some embodiments, to calculate the frequency of insertion, the sequence read is scanned for exact matches to two 10bp sequences that flank the window where insertion can occur. For example, if no exact match is located, the read is excluded from analysis. If the length of this insertion window exactly matches the reference sequence, the read is classified as not containing an insertion. If the insertion window is more than 2 bases longer than the reference sequence, the sequence read is classified as an insertion. In some embodiments, the modifying element provided herein can restrict the formation of insertions in a region of a nucleic acid. In some embodiments, the region is located within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the modifying element or the nucleotide targeted by the modifying element.

[0174] In some embodiments, the number of insertions formed in the target nucleic acid may depend on the amount of time the nucleic acid (e.g., the target nucleic acid in the genome of the cell) is exposed to the modifying element. In some embodiments, the number or rate of insertions is measured at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days after the target nucleic acid (e.g., the nucleic acid in the genome of the cell) is exposed to the modifying element. It should be understood that the properties of the modifying elements described herein are, in some embodiments, applicable to any of the chimeric systems or methods using the chimeric systems provided herein.

[0175] Deletion In some embodiments, the CRISPR-Cas technology described herein is used to edit a target nucleic acid sequence by deleting one or more nucleotides of the target nucleic acid.

[0176] In some embodiments, the deletion results in a frameshift mutation within the coding region of the target nucleic acid sequence that encodes a gene product. In some embodiments, the CRISPR-Cas technology provided herein can be used to detect less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10 ... or less than 0.01%, resulting in deletion formation in less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01%.

[0177] In some embodiments, to calculate deletion frequency, sequence reads are scanned for exact matches to two 10bp sequences that flank the window in which insertion can occur. For example, if no exact match is located, the read is excluded from analysis. If the length of this deletion window exactly matches the reference sequence, the read is classified as not containing an insertion. If the insertion window is 2 or more bases shorter than the reference sequence, the sequence read is classified as a deletion. In some embodiments, the modifying element provided herein can limit deletion formation within a region of a nucleic acid. In some embodiments, the region is located within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the modifying element or the nucleotide targeted by the modifying element.

[0178] In some embodiments, the number of deletions formed in the target nucleic acid may depend on the amount of time the target nucleic acid (e.g., the target nucleic acid in the genome of the cell) is exposed to the modifying element. In some embodiments, the number or percentage of deletions is measured at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days after the target nucleotide sequence (e.g., the nucleic acid in the genome of the cell) is exposed to the modifying element. It should be understood that the properties of the modifying elements described herein are, in some embodiments, applicable to any of the chimeric systems or methods using the chimeric systems provided herein.

[0179] mutation In some embodiments, the CRISPR-Cas technology described herein can be used to edit a target nucleic acid sequence by mutating one or more nucleotides of the target nucleic acid. In some embodiments, the mutation is a point mutation. In some embodiments, the mutation is a silent mutation (e.g., the mutation does not result in a change in the amino acid sequence relative to the relevant reference amino acid sequence). In some embodiments, the mutation introduces a non-naturally occurring stop codon. In some embodiments, the mutation introduces a non-naturally occurring start codon. In some embodiments, the mutation removes a naturally occurring stop codon. In some embodiments, the mutation removes a naturally occurring start codon.

[0180] In some embodiments, it is desirable to generate and / or use chimeric systems (e.g., including a Cas protein and a modifying element described elsewhere herein) that efficiently modify (e.g., mutate or deaminate) specific nucleotides within a target nucleic acid sequence without generating large numbers of insertions or deletions in the target nucleic acid sequence. In some embodiments, any of the chimeric systems disclosed herein are capable of generating a modification of interest (e.g., point mutations or deaminations) at a higher rate than insertions / deletions.

[0181] In some embodiments, the chimeric system of the present disclosure modifies a single nucleotide in a target nucleic acid, hi some embodiments, the modification repairs and / or corrects a GA or CT point mutation, a TC or AG point mutation, or a pathogenic single nucleotide polymorphism.

[0182] In some embodiments, any of the chimeric systems disclosed herein can efficiently generate mutations of interest, such as, for example, point mutations, in a target nucleic acid sequence (e.g., a nucleic acid in a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, any of the chimeric systems provided herein can generate at least 0.01% of mutations of interest (i.e., at least 0.01% base editing efficiency). In some embodiments, any of the chimeric systems provided herein can generate at least 0.01%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of mutations of interest.

[0183] In some embodiments, the chimeric systems described herein can generate a ratio of point mutations of interest to insertions / deletions or unintended point mutations of greater than 1: 1. In some embodiments, the base editors provided herein can generate a ratio of insertions / deletions or unintended point mutations of at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 8.5:1, at least 9:1, at least 10:1, at least 11:1, at least 12:1, at least 13:1, at least Ratios of desired point mutations to insertions / deletions or unintended point mutations of 14:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1 or more can be produced.

[0184] In some embodiments, the number of mutations and insertions / deletions of interest can be determined by a method described in, e.g., International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632); Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” It can be measured using any suitable method, as described in Science Advances 3:eaao4774 (2017), the entire contents of which are incorporated herein by reference.

[0185] Changes in gene expression levels In some embodiments, the CRISPR-Cas technology of the present disclosure is used to alter (e.g., increase or decrease) gene expression of a gene product controlled or encoded by a target nucleic acid. In some embodiments, gene expression levels are altered, for example, by gene or promoter insertion, deletion, mutation, inactivation of gene expression, activation of gene expression, enzymatic recombination, directed evolution, knowledge-based design, random mutagenesis, gene shuffling, and / or codon optimization.

[0186] In some embodiments, the change in gene expression level comprises targeting DNA. In some embodiments, targeting DNA comprises targeting a control element. In some such embodiments, the control element comprises, for example, a promoter, an enhancer, an internal ribosome entry site (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and polyU sequences).

[0187] In some embodiments, altering gene expression levels comprises targeting RNA. In some embodiments, targeting RNA comprises targeting RNA processing. In some such embodiments, targeting RNA processing comprises targeting, for example, RNA splicing (including alternative splicing), RNA polymerase, viral replication, tRNA biogenesis, and RNA activation.

[0188] payload In some embodiments, the disclosure provides methods for targeting the insertion of a payload nucleic acid at a location in a target nucleic acid. In some such embodiments, the methods include contacting the target nucleic acid with a technique described herein and a payload nucleic acid (e.g., a donor template nucleic acid that includes a payload nucleic acid). In some embodiments, the disclosure provides methods for targeting the removal of a payload nucleic acid from a location in a target nucleic acid, the method including contacting the target nucleic acid with a technique described herein.

[0189] In some embodiments, the donor template nucleic acid is delivered either in a vector, such as an AAV viral vector, or as a linear single-stranded or double-stranded DNA fragment. In some embodiments, for insertion of the donor template nucleic acid by homology directed repair (HDR), the donor template nucleic acid comprises the payload nucleic acid to be inserted at the locus of interest, plus flanking sequences homologous to endogenous sequences adjacent to the desired insertion site. In some embodiments, for inserting short payloads, e.g., less than 1 kb in length, the flanking homologous sequences can be short, e.g., in the range of 15-200 nucleotides in length. In other cases, long homologous flanking sequences are required to facilitate efficient HDR, e.g., greater than 200 nucleotides in length, for inserting long payloads, e.g., 1 kb in length or longer. In some embodiments, cleavage of the target genomic locus for HDR between sequences homologous to the template DNA flanking regions can significantly increase the frequency of HDR. In some embodiments, cleavage events that facilitate HDR include, but are not limited to, dsDNA cleavage, double nicking, and single-strand nicking activity.

[0190] In some embodiments, the payload nucleic acid is not limited to a particular nucleic acid. In some embodiments, the payload nucleic acid includes any nucleic acid of interest. In some embodiments, for example, the payload nucleic acid is linear or circular. In some embodiments, for example, the payload nucleic acid is a plasmid, a viral genome, an RNA and / or a DNA polynucleotide. In some embodiments, the payload nucleic acid is a modified nucleic acid. In some embodiments, the donor template nucleic acid is double-stranded (e.g., DNA or RNA). In some embodiments, the donor template nucleic acid is single-stranded (e.g., DNA or RNA). Methods for designing exogenous donor template nucleic acids are described, for example, in WO2016094874, the entire contents of which are expressly incorporated herein by reference. In some embodiments, the payload nucleic acid is a nucleic acid useful in the treatment, prevention, and / or diagnosis of a disorder and / or disease.

[0191] Vector systems and vectors In some embodiments, the CRISPR-Cas technology of the present disclosure comprises a system for delivering and / or expressing the Cas protein(s), chimeric system(s), guide(s), and / or donor template nucleic acid(s). In some embodiments, the system comprises a vector and / or vector system. Methods for delivering guide and donor template nucleic acids, as well as methods for exogenously expressing proteins and polypeptides, are well known in the art, and one of skill in the art will recognize that a variety of techniques may be successfully employed.

[0192] Recombinant polynucleotides (e.g., DNA or RNA) encoding Cas proteins and / or chimeric systems or providing guide or donor template nucleic acids of the present disclosure can be prepared by a variety of available methods. For example, desired sequences can be removed from DNA using restriction enzymes, amplified from plasmid or genomic polynucleotide sequences using, for example, the polymerase chain reaction, or synthesized using chemical synthesis techniques. In some embodiments, a combination of well-known methods is used to prepare the recombinant polynucleotides.

[0193] In some embodiments, a recombinant polynucleotide encoding a Cas protein and / or chimeric system of the present disclosure is cloned into a vector capable of expressing the Cas protein and / or chimeric system. In some embodiments, a recombinant polynucleotide providing a guide or donor template nucleic acid of the present disclosure is cloned into a vector. Cloning can be performed by a variety of available methods (e.g., Gibson assembly, restriction enzyme digestion and ligation, etc.). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a plasmid.

[0194] In some embodiments, the vector capable of expression comprises a recombinant polynucleotide encoding a Cas protein and / or chimeric system of the present disclosure operably linked to sequence(s) that control expression (e.g., promoter, start signal, stop signal, polyadenylation signal, activator, repressor, etc.). In some embodiments, the sequence(s) that control expression are selected to achieve a desired level of expression. In some embodiments, two or more sequences that control expression (e.g., promoters) are utilized. In some embodiments, two or more sequences that control expression (e.g., promoters) are utilized to achieve a desired level of expression of multiple recombinant polynucleotides encoding multiple proteins and / or polypeptides. In some embodiments, multiple recombinant proteins and / or polypeptides are expressed from the same vector (e.g., bicistronic vector, tricistronic vector, multicistronic). In some embodiments, multiple recombinant polypeptides are expressed, each of which is expressed from a separate vector.

[0195] In some embodiments, a vector comprising a recombinant polynucleotide encoding the Cas protein and / or chimeric system of the present disclosure is used to express the Cas protein and / or chimeric system by in vitro protein synthesis.

[0196] In some embodiments, an expression vector comprising a recombinant polynucleotide encoding the disclosed Cas protein or chimeric system is used to express the Cas protein or chimeric system in a host cell. In some embodiments, a vector capable of delivering the disclosed guide and / or donor template nucleic acid is used to deliver the guide and / or donor template nucleic acid to a host cell. The host cell can be selected from a variety of available and well-known host cells suitable for expressing the CRISPR-Cas technology disclosed herein (e.g., human embryonic kidney (HEK) cells, suspension HEK293 cells, Chinese hamster ovary cells).

[0197] Various methods of introducing vectors into host cells are well known in the art. In some embodiments, vectors can be introduced into host cells using transfection. In some embodiments, transfection is completed using, for example, calcium phosphate transfection, lipofection, or polyethyleneimine-mediated transfection. In some embodiments, vectors can be introduced into host cells using transfection.

[0198] In some embodiments, the transformed host cells are cultured after the vector is introduced into the host cells to allow expression of the recombinant polynucleotide. In some embodiments, the transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or more. The transformed host cells are cultured under growth conditions (e.g., temperature, carbon dioxide level, growth medium) according to the requirements of the selected host cells. Those skilled in the art will recognize that the culture conditions for the selected host cells are well known in the art.

[0199] use The CRISPR-Cas technology described herein has a wide variety of uses, including modifying (e.g., deleting, inserting, mutating, translocating, inactivating, or activating) target nucleic acid sequences, e.g., in multiple cell types and tissues, and detecting target nucleic acids (e.g., DNA and / or RNA), e.g., in specific sensitive enzyme reporter unlock (SHERLOCK)-based assays. In some embodiments, the thermostable Cas proteins described herein are particularly useful in gene editing and / or detection of target nucleic acids in thermophilic organisms. Additional applications include, but are not limited to, tracking and labeling nucleic acids, enrichment assays (e.g., extracting desired sequences from a sample), detecting circulating tumor DNA, preparing next-generation libraries, screening drugs, providing diagnosis and prognosis of diseases and disorders, treating various genetic and non-genetic diseases or disorders, or enhancing health through genome manipulation.

[0200] Gene editing In some embodiments, the CRISPR-Cas technology described herein is used for gene editing. In some embodiments, gene editing results in a gene silencing event or expression change (e.g., increase or decrease) in the expression of a desired target gene. Thus, in some embodiments, the CRISPR-Cas technology described herein is used in a method for changing the expression level of a gene product controlled or encoded by a target nucleic acid. In some embodiments, the CRISPR-Cas technology described herein is used in a method for modifying a target nucleic acid in a desired cell. In some embodiments, the technology disclosed herein provides a method for site-specific modification of a target nucleic acid in a cell (e.g., a eukaryotic or prokaryotic cell) to achieve a desired correction of gene expression or function of the expressed gene product.

[0201] In some embodiments, the disclosure provides engineered non-naturally occurring CRISPR-Cas technology comprising a Cas protein having collateral cleavage activity that is thermostable at least above 60-65° C., and at least one guide sequence capable of forming a complex with the thermostable Cas protein and directing binding of the complex to at least one target nucleic acid.

[0202] In some embodiments, the disclosure provides a method of cleaving at least one target nucleic acid in a cell, comprising contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C, and at least one guide sequence capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide sequence and capable of causing a disruption in the at least one target nucleic acid.

[0203] In some embodiments, the disclosure provides a method of altering expression of at least one target nucleic acid in a cell, comprising contacting the cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide sequence capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the guide sequence and is capable of editing the at least one target nucleic acid sequence.

[0204] In some embodiments, the disclosure provides a method of modifying at least one target nucleic acid in a cell, comprising contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C, and at least one guide sequence capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the guide sequence and is capable of editing the at least one target nucleic acid sequence.

[0205] In some embodiments, the disclosure provides a method of altering expression of at least one target nucleic acid in a cell, comprising contacting a cell with a Cas protein having collateral cleavage activity that is thermostable at least above 60-65°C and at least one guide capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide and is capable of editing the at least one target nucleic acid sequence.

[0206] Thus, in some embodiments, the Cas protein has about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 1-10. In some embodiments, the Cas protein is identical to SEQ ID NO: 1. In some embodiments, the Cas protein is identical to SEQ ID NO: 2.

[0207] In some embodiments, the method of the present disclosure includes contacting at least one target nucleic acid with a CRISPR-Cas system of the present disclosure and performing cleavage of at least one target nucleic acid. In some embodiments, the CRISPR-Cas technology cleaves the target DNA or RNA duplex by introducing a double-stranded break. In some embodiments, the CRISPR-Cas technology cleaves the target DNA or RNA by introducing a single-stranded break or nick.

[0208] In some embodiments, the CRISPR-Cas technology comprises a chimeric system comprising a modifying element that modifies target DNA in a site-specific manner, the modifying activity comprising a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, a demyristoylating activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, or a nuclease activity, any of which can modify DNA or a DNA-associated polypeptide (e.g., a histone or a DNA-binding protein).

[0209] In some embodiments, the CRISPR-Cas technology comprises a chimeric system that comprises a modification element (s) capable of editing DNA sequences by chemically modifying nucleotide bases, including deaminase enzymes, capable of modifying adenosine or cytosine bases and functioning as site-specific modification elements. A variety of modification elements are well known in the art and can be used in the methods and systems described herein. Exemplary modification elements throughout this disclosure are described, for example, in Rees and Liu Nature Review Genetics, 2018, 19(12):770-788, the entire contents of which are incorporated herein by reference.

[0210] In some embodiments, the modifier activity results in the introduction of a stop codon(s), e.g., silencing a gene(s). In some embodiments, the modifier activity results in the removal of a stop codon(s). In some embodiments, the modifier activity results in the introduction of a start codon(s). In some embodiments, the modifier activity results in the removal of a start codon(s), e.g., silencing a gene(s). In some embodiments, the modifier element results in a change in protein function by altering the amino acid sequence.

[0211] In some embodiments, the Cas proteins of the present disclosure epigenetically modify the target nucleic acid by fusion with histones. In some embodiments, the Cas proteins epigenetically modify the target nucleic acid by fusion with epigenetically modifying enzymes such as reader, writer, or eraser proteins. In some embodiments, the Cas proteins are fused with histone modifying enzymes to alter the histone modification pattern at selected regions of the target nucleic acid. Histone modifications can occur in many different ways, including, for example, methylation, acetylation, ubiquitination, phosphorylation, and many different combinations, resulting in structural changes to the DNA. In some embodiments, the histone modifications result in repression or activation of transcription.

[0212] In some embodiments, the Cas proteins of the present disclosure control transcription of a target nucleic acid by increasing or decreasing transcription through fusion with a transcription activator protein, a transcription repressor protein, a small molecule / drug responsive transcription regulator, or an inducible transcription regulator. In some embodiments, CRISPR-Cas technology is used to control expression of the mRNA encoding the target (i.e., a protein-coding gene) where binding results in increased or decreased gene expression.

[0213] In some embodiments, CRISPR-Cas technology is used to control gene regulation by editing genetic control elements such as promoters or enhancers.

[0214] In some embodiments, CRISPR-Cas technology is used to control the expression of target non-coding RNAs, including tRNAs, rRNAs, snoRNAs, siRNAs, miRNAs, and long ncRNAs.

[0215] In some embodiments, CRISPR-Cas technology is used for the targeted recombination of chromatin loop structure.Without wishing to be bound by any one theory, the targeted recombination of chromatin loop between regulatory genomic regions provides a means to manipulate endogenous chromatin structure, and allows new enhancer-promoter connections to be formed, and overcome genetic defects or inhibit abnormal enhancer-promoter connections.

[0216] In some embodiments, CRISPR-Cas technology is used for imaging of live cells. For example, in some embodiments, fluorescently labeled Cas proteins are targeted to repetitive genomic regions such as centromeres and telomeres to track natural chromatin loci through the cell cycle and measure the differences in the location of transcriptionally active and inactive regions within the 3D nuclear space.

[0217] Treatment As will be readily appreciated by those of skill in the art, in some embodiments, the CRISPR-Cas technology described herein is useful in one or more of a variety of therapeutic applications. Thus, in some embodiments, a method of treating a disorder or disease in a subject in need of treatment is provided. In some such embodiments, the method comprises administering to the subject a Cas protein having collateral cleavage activity that is thermostable at least at temperatures above 60-65° C., and at least one guide sequence capable of hybridizing to a target nucleic acid.

[0218] In some embodiments, the CRISPR-Cas technology disclosed herein can be used to edit a target nucleic acid sequence to modify the target nucleic acid (e.g., by inserting, deleting, or mutating one or more nucleotides). For example, in some embodiments, the CRISPR-Cas technology described herein includes an exogenous donor template nucleic acid (e.g., a DNA molecule or an RNA molecule) that includes a desired nucleic acid sequence (e.g., a payload nucleic acid). Without wishing to be bound by any one theory, upon recovery of a cleavage event induced by the CRISPR-Cas technology described herein, the molecular machinery of the cell can utilize the exogenous donor template nucleic acid to repair and / or repair the cleavage event. Alternatively, or in addition, in some embodiments, the molecular machinery of the cell can utilize an endogenous template to repair and / or repair the cleavage event. In some embodiments, the CRISPR-Cas technology described herein can be used to modify a target nucleic acid to result in an insertion, deletion, and / or point mutation. In some such embodiments, the insertion is an intact insertion (i.e., the insertion of a nucleic acid sequence of interest into a target nucleic acid such that upon recovery of the cleavage event, no unintended additional nucleic acid sequence is generated).

[0219] In some embodiments, the CRISPR-Cas technology disclosed herein is used to treat various diseases and disorders, such as genetic diseases, single gene diseases, diseases treatable by nuclease activity, various cancers, etc. In some embodiments, the methods described herein are used to treat a subject, e.g., a mammal, such as a human patient. In some embodiments, the mammalian subject can be a domesticated mammal, such as, but not limited to, a dog, cat, horse, monkey, rabbit, rat, mouse, cow, goat, or sheep.

[0220] In some embodiments, the CRISPR-Cas technology disclosed herein is used to correct pathogenic mutations by inserting beneficial clinical variants or suppressor mutations.

[0221] In some embodiments, the CRISPR-Cas technology disclosed herein is used to treat diseases caused by overexpression of RNA, toxic RNA, and / or mutant RNA (e.g., splicing defects or truncations).

[0222] In some embodiments, the CRISPR-Cas technology disclosed herein targets trans-acting mutations that affect RNA-dependent functions that cause various diseases.

[0223] In some embodiments, the CRISPR-Cas technology disclosed herein is used to target mutations that disrupt the cis-acting splicing code, which can lead to splicing defects and disease.

[0224] In some embodiments, the CRISPR-Cas technology disclosed herein is used for antiviral activity, especially against RNA viruses.In some embodiments, the RNA virus is, for example, Arenaviridae, Arteriviridae, Astroviridae, Birnaviridae, Bornaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Flaviviridae, Filoviridae, Hepeviridae, Nodaviridae, Nymaviridae, Orthmyxoviridae, Paramyxoviridae, Picobirnaviridae, Picornaviridae, Pneumoviridae, Reoviridae, Rhabdoviridae or Togaviridae family virus.In some embodiments, Cas protein targets viral RNA with suitable RNA guide selected to target viral RNA sequence.

[0225] In some embodiments, the CRISPR-Cas technology disclosed herein is used to treat cancer in a subject (e.g., a mammalian subject, e.g., a human subject). For example, the Cas proteins described herein, in some embodiments, have been found to be programmed with guides that target aberrant (e.g., containing point mutations or alternatively spliced) RNA molecules in cancer cells to induce cell death (e.g., by apoptosis) in the cancer cells.

[0226] Furthermore, in some embodiments, the CRISPR-Cas technology described herein is used to treat infectious diseases in a subject. For example, the Cas protein described herein is programmed with a guide sequence that targets an RNA molecule expressed by an infectious agent (e.g., a bacterium, a virus, a parasite, or a protozoan) in some embodiments to target and induce cell death in infectious agent cells. In some embodiments, the CRISPR-Cas technology disclosed herein treats diseases in which an intracellular infectious agent infects cells of a host subject. In some embodiments, cells infected with an infectious agent are targeted and cell death is induced by programming the Cas protein to target an RNA molecule encoded by an infectious agent gene.

[0227] In some embodiments, the CRISPR-Cas technology disclosed herein is useful for generating cells for therapeutic delivery. In some embodiments, the CRISPR-Cas technology is useful for generating, for example, chimeric antigen receptor (CAR) T cells, somatic cells (e.g., hematopoietic stem cells (HSC), mesenchymal stem cells (MSC)), and immortalized cell lines (e.g., the neural stem cell line CTX). In some such embodiments, the cells generated by the CRISPR-Cas technology are administered to a subject (e.g., for the treatment of a disorder and / or disease).

[0228] In some embodiments, provided herein are compositions, pharmaceutical compositions, vectors, host cells, and kits that comprise any of the recombinant proteins and / or polynucleotides described herein.

[0229] Collateral Activity Assay Those of skill in the art will readily appreciate that the techniques provided herein are broadly applicable to achieve detection of a wide range of nucleic acids, including, for example, nucleic acids from infectious agents (e.g., viruses, microorganisms, parasites, etc.), nucleic acids indicative of a particular physiological state or condition (e.g., the presence or state of a disease, disorder, or condition, such as, for example, cancer or an inflammatory or metabolic disease, disorder or condition, etc.), prenatal nucleic acids, and the like.

[0230] In some embodiments, the target nucleic acid is detected by an assay comprising a Cas enzyme described herein and a guide. In some embodiments, the structure of the guide can affect the activity of the Cas protein / guide complex. In some embodiments, the structure of the Cas protein / guide complex contributes to the thermostability of the Cas collateral activity.

[0231] Those skilled in the art are well aware of the burgeoning number of capable detection (e.g., diagnostic) assays that have been developed and are in the process of being developed using Cas protein collateral activity. See, e.g., Sashital Genome Med 2018:10,32. Furthermore, those skilled in the art are well aware that a "detailed classification of CRISPR / Cas biosensing systems" based on the collateral activity of Cas proteins has recently become publicly available. See review by Li et al Trends Biotechnol.37:730,July 2019.

[0232] Formats of particular interest include Cas13-based (e.g., Cas13a or Cas13b-based) systems, including those referred to as the "SHERLOCK" and / or "HUDSON" systems (see, e.g., Gootenberg et al., Science 356:438, 2017; Gootenberg et al., Science 360:339, 2018; Myhrvold et al., Science 360:444, 2018; see also US10266887), as well as Cas12-based (e.g., Cas12a or Cas12b-based) systems, including those referred to as the "HOLMES" or "DETECTR" systems (see, e.g., Cheng et al. CN Patent Application CN107488710A; PCT / CN18 / 82769 and US16 / 631,157; ​​Li et al. Cell Disc. 4:20, 2018; Chen et al. Science 360:436, 2018; Li, L. et al. bioRxiv Published online July 26, 2018. http: / / dx.doi.org / 10.1101 / 362889; see US10253365). Both Cas13a and Cas13b enzymes have been used in the SHERLOCK and / or HUDSON systems, as have both Cas12a and Cas12b.

[0233] As known in the art and described in the references cited herein, a typical detection assay utilizing the collateral cleavage activity of a Cas protein involves contacting a sample that may contain a target nucleic acid with an appropriate CRISPR-Cas complex, including a Cas protein with collateral activity and a guide that is complementary to a target nucleic acid sequence of interest. Upon recognition of the target nucleic acid sequence, the collateral activity of the Cas protein is activated, causing the Cas protein to cleave unrelated nucleic acids (DNA or RNA, or both, depending on the enzyme). A reporter of the relevant cleavable nucleic acid is provided that is appropriately configured (e.g., labeled) such that its cleavage is detectable as a result of the activated collateral activity (e.g., the separation of a fluorophore from a quencher, resulting in detectable fluorescence, etc.).

[0234] In many assays, a target nucleic acid sequence is generated and / or amplified (e.g., copied from RNA to DNA and / or amplified by primer extension, DNA replication (e.g., by polymerase chain reaction), and / or transcription). See, e.g., Figures 3 and 4 in the review by Li, supra (Li et al. Trends Biotechnol. 37:730, July 2019).

[0235] Thus, in many embodiments, collateral activity assays include (1) a target nucleic acid copying and / or amplification step, (2) a target nucleic acid binding step, and (3) a signal emission and / or detection step.

[0236] Typically, the techniques provided are applied to one or more samples to investigate the presence and / or levels of one or more target nucleic acids in the samples. In some embodiments, the sample is a biological sample, in some embodiments, the sample is an environmental sample. In some embodiments, the sample is a crude sample (e.g., a primary sample or a sample that has undergone minimal processing).

[0237] In some embodiments, the sample is processed (e.g., nucleic acids are partially or substantially isolated or purified from the primary sample). In some embodiments, only minimal processing is performed (i.e., the sample is a crude sample).

[0238] Typically, the collateral activity assays described herein are in vitro assays. In some embodiments, the collateral activity assays may be cell-free assays (e.g., substantially free of intact cells, or, in some embodiments, cell fragments).

[0239] In some embodiments, the collateral activity assays described herein are performed on samples that are or are prepared from primary biological samples (e.g., blood, saliva, tears, urine, etc.) or primary environmental samples (e.g., soil, water, etc.).

[0240] In some embodiments, the nucleic acid detection step and the target binding step are performed in a single vessel. In some embodiments, the target binding step and the signal emission step are performed in a single vessel. In some embodiments, the steps of (1) the target copying and / or amplification step, (2) the target binding step, and (3) the signal emission and / or detection step are performed in a single vessel. In some embodiments, all steps are performed in a single vessel, i.e., the improved assay provided is a one-pot assay.

[0241] In some embodiments, the improved collateral activity assays described herein are in vitro assays. In some embodiments, the collateral activity assays may be cell-free assays (e.g., substantially free of intact cells, or, in some embodiments, cell fragments).

[0242] In some embodiments, the improved collateral activity assays described herein are performed on samples that are or are prepared from primary biological samples (e.g., blood, saliva, tears, urine, etc.) or primary environmental samples (e.g., soil, water, etc.).

[0243] Pharmaceutical Compositions In some embodiments, the disclosure provides, inter alia, a pharmaceutical composition comprising the CRISPR-Cas technology of the disclosure. In some embodiments, the pharmaceutical composition comprises a Cas protein having collateral cleavage activity that is thermostable at least at a temperature above 60-65° C., and at least one guide sequence capable of forming a complex with the thermostable Cas protein and directing binding of the complex to at least one target nucleic acid. In some embodiments, the pharmaceutical composition further comprises a donor template nucleic acid.

[0244] In some embodiments, the pharmaceutical composition comprises a vector or vector system capable of expressing and / or providing the CRISPR-Cas technology of the present disclosure.

[0245] In some embodiments, the CRISPR-Cas technology of the present disclosure is formulated into a pharmaceutical composition by combining with a suitable pharma- ceutically acceptable carrier or diluent.

[0246] In some embodiments, the CRISPR-Cas technology of the present disclosure is formulated into a pharmaceutical composition in a pharma- ceutically acceptable vehicle. In some such embodiments, for example, the pharma- ceutically acceptable vehicle can be a vehicle approved by a regulatory agency.

[0247] In some embodiments, vehicle refers to a diluent, adjuvant, excipient, or carrier that the compound of the present invention is formulated for administration to a subject.In some such embodiments, pharmaceutical vehicles can be lipids, such as liposomes, such as liposomal dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.), saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In some embodiments, auxiliary agents, stabilizers, thickeners, lubricants, and colorants are used.In some embodiments, pharmaceutical compositions are formulated into preparations in solid, semi-solid, liquid, or gas form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microparticles, and aerosols.

[0248] In some embodiments, administration of the CRISPR-Cas technology described herein can be accomplished in a variety of ways. In some embodiments, administration includes oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, intraocular, etc. In some embodiments, the CRISPR-Cas technology can be systemic after administration or can be localized by using topical administration, intramural administration, or by using an implant that acts to retain an active dose at the implantation site. In some embodiments, the CRISPR-Cas technology is formulated for immediate activity or formulated for sustained release.

[0249] In some embodiments, the treatment method includes treating a disease or disorder of the central nervous system. In some such embodiments, the CRISPR-Cas technology may need to be formulated into a pharmaceutical composition that crosses the blood-brain barrier (BBB). For example, in some embodiments, drug delivery across the blood-brain barrier (BBB) ​​involves disruption of the BBB, either by permeation methods such as mannitol or leukotrienes, or by biochemical use of vasoactive substances such as bradykinin. In some embodiments, opening of the BBB is used to target the CRISPR-Cas technology to the brain. In some embodiments, a BBB disrupting agent is co-administered with the therapeutic composition of the invention when the composition is administered by intravascular injection. In some embodiments, other methods of crossing the BBB are used, including, for example, caveolin-1 mediated transcytosis, carrier-mediated transporters such as glucose and amino acid carriers, receptor-mediated transcytosis for insulin or transferrin, and the use of endogenous transport systems, including active efflux transporters such as p-glycoprotein.

[0250] In some embodiments, the CRISPR-Cas technology is delivered across the BBB by local delivery, for example, by intrathecal delivery.

[0251] In some embodiments, an effective amount of the preparation containing CRISPR-Cas technology is provided. In some embodiments, the calculation of the effective amount or effective dose of the pharmaceutical composition described herein to be administered is within the skill of the art and would be routine for the artisan. In some such embodiments, the final amount administered will depend on the route of administration and the nature of the disorder or condition to be treated.

[0252] In some embodiments, the effective amount given to a particular patient will depend on a variety of factors, some of which will vary from patient to patient. A competent clinician will be able to determine the effective amount of the pharmaceutical composition to administer to a patient and, if necessary, halt or reverse the progression of the disease state. For example, in some embodiments, using LD50 animal data and other information available about the agent, the clinician can determine the maximum safe dose for an individual, depending on the route of administration. In some embodiments, the dose administered intravenously may exceed the dose administered intrathecally, for example, given the larger body of fluid into which the therapeutic composition is administered. In some embodiments, the pharmaceutical composition is administered at higher doses or repeated doses to maintain therapeutic concentrations. Using conventional techniques, a competent clinician will be able to optimize the dose of a particular pharmaceutical composition during the course of routine clinical trials.

[0253] In some embodiments, the pharmaceutical composition includes a pharma- ceutically acceptable, non-toxic, diluent carrier, depending on the desired formulation, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. In some embodiments, the diluent is selected so as not to affect the biological activity of the combination. In some such embodiments, for example, the diluent is distilled water, buffered water, saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In some embodiments, the pharmaceutical composition includes other carriers, adjuvants, or non-toxic non-therapeutic agents, non-immunogenic stabilizers, excipients, etc. In some embodiments, the pharmaceutical composition includes additional substances that approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.

[0254] In some embodiments, the pharmaceutical composition includes any of a variety of stabilizing agents, such as, for example, antioxidants. In some embodiments, where the pharmaceutical composition includes a polypeptide, the polypeptide is complexed with a variety of well-known compounds that improve the in vivo stability of the polypeptide or otherwise improve its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, and improve solubility or uptake compared to an appropriate reference standard). Non-limiting examples of such modifiers or complexing agents include sulfates, gluconates, citrates, and phosphates. In some embodiments, the pharmaceutical composition, including the nucleic acid or polypeptide of the composition, can be complexed with a molecule that improves its in vivo properties compared to an appropriate reference standard. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0255] In some embodiments, the components used to formulate a high purity pharmaceutical composition are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). In some embodiments, compositions intended for in vivo use are sterile. In some embodiments, to the extent that a given pharmaceutical composition requires synthesis prior to use, the resulting product will typically be free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process.

[0256] In some embodiments, the pharmaceutical compositions are administered for prophylactic and / or therapeutic treatment. In some embodiments, the toxicity and therapeutic efficacy of the pharmaceutical compositions are determined according to standard pharmaceutical procedures, such as in cell cultures and / or experimental animals, including, for example, determining the LD50 (the dose lethal to 50% of the population) and / or the ED50 (the dose therapeutically effective in 50% of the population).

[0257] kit In another aspect, the disclosure provides a kit containing any one or more of the elements disclosed in the compositions and methods above. In some embodiments, the kit comprises a vector and / or vector system described herein. In some embodiments, the kit comprises one or more of the components of the CRISPR-Cas technology described herein, such as Cas protein(s), guide(s), donor template nucleic acid(s), and / or polynucleotides, vectors, and / or vector systems (e.g., DNA or RNA) encoding or providing same. In some embodiments, the kit comprises instructions in one or more languages ​​for using the kit. In some such embodiments, the instructions are for a particular application and / or method described herein. The elements can be provided individually or in combination. The kit can be provided in any suitable container. In some embodiments, suitable containers are, for example, vials, bottles, or tubes.

[0258] In some embodiments, the kit includes one or more reagents for use in a process that utilizes one or more of the elements described herein. The reagents can be provided in any suitable container. For example, in some embodiments, the kit provides one or more reaction or storage buffers. The reagents can be provided in a form that is usable in a particular assay or that requires the addition of one or more other components prior to use (e.g., in a concentrated or lyophilized form). In some embodiments, the buffer is not limited to a particular buffer. In some embodiments, the buffer can be any buffer, including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into a vector to operably link the guide sequence and the control element. In some embodiments, the kit includes a homologous recombination template polynucleotide. In some embodiments, the kit includes one or more of the vectors and / or one or more of the polynucleotides described herein. The kit may advantageously make it possible to provide all of the elements of the system of the invention. EXAMPLES

[0259] Example 1: Exemplary Thermostable Cas Protein Candidates This example describes specific thermostable Cas protein candidates. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

[0260] Example 2: Exemplary characterization of additional candidate thermostable Cas proteins This example shows the characterization of exemplary thermostable Cas proteins Pal1 (SEQ ID NO: 1), Pal2 low MW, Pal2 high MW (SEQ ID NO: 2), and Pal3 (SEQ ID NO: 3). Each enzyme was tested with four guides (labeled 342-353) at both 37°C and 56°C in Cas-only reactions using DnaseAlert as a reporter. Fluorescence signal was plotted against time for each reaction (Figure 1). Pal1 showed low activity towards two guides at 56°C. While no activity was observed towards Pal2 low MW or Pal3, activity was observed towards two guides at 56°C for Pal2 high MW. Pal1 and Pal2 activity at 56°C is shown in Figure 2. Further results of studies with these enzymes are shown in Figures 3-8. As can be seen, Pal1 showed activity towards two guides at 56°C and 70°C. Pal1 showed maximum activity at 57°C and significant activity at least to 67°C. Pal2 high MW showed activity towards both guides at 56°C. Pal2 high MW also showed maximum activity at 47-52°C, with significant activity at least below 57°C. No significant activity was observed at 37°C, 56°C, or 70°C for either Pal2 low MW or Pal3-6. Thus, one of skill in the art will appreciate that these enzymes are thermostable at least at about 56°C and / or within the range of 56°C-70°C. These exemplified specific enzymes may be described as thermoactive because their associated activity(ies) are dramatically reduced and / or undetectable at lower temperatures, such as 37°C. Without wishing to be bound by any particular theory, it is noted that enzymes from thermophilic organisms may often show reduced activity (or undetectable activity) at such temperatures.

[0261] To further characterize the exemplary thermostable Cas proteins Pal1 (SEQ ID NO:1), Pal2 (SEQ ID NO:2), Pal3 (SEQ ID NO:3), Pal4 (SEQ ID NO:4), Pal5 (SEQ ID NO:5), Pal6 (SEQ ID NO:6), Pal8 (SEQ ID NO:8), Pal9 (SEQ ID NO:9), and Pal10 (SEQ ID NO:10), enzymatic denaturation was investigated using an exemplary protein melting method. The Cas proteins were mixed with buffer and dye, and melting curves were generated. As the temperature increases, the Cas proteins unfold. As the exemplary Cas proteins unfold, hydrophobic regions are exposed, resulting in binding of the dye to the Cas proteins. Upon binding, the dye fluoresces. The change in fluorescence with temperature change is plotted against the temperature of the melting curve. The melting temperatures of the Cas proteins were calculated and compared to the melting temperatures of appropriate reference standards (e.g., Cas proteins with known thermostability, e.g., Aac and / or RS9). Changes in melting temperature correlate with changes in protein stability (e.g., thermostability) and activity (Figure 9). Figure 9 shows that Cas proteins (PAL1-10) have thermostable activity compared to the reference thermostable proteins Aac and RS9, i.e., Cas proteins PAL1-10 are thermostable.

[0262] Example 3: Collateral activity signals of thermostable Cas proteins in complex with guide-RNA

[0263] This example further demonstrates the collateral activity of the Cas proteins described herein. The collateral activity of thermostable Cas proteins (PAL5, PAL8, PAL9, and PAL10) complexed to different recombinant guide RNAs was tested in two different assays: an assay without target amplification (cas only) and an assay with target amplification (RT-SLK). Guide RNAs with different targets and lengths were engineered (SEQ ID NOs: 11-19). The recombinant guide RNAs are shown in Table 2.

[0264] [Table 2-1] [Table 2-2]

[0265] Cas-only reactions (Figures 10, 12, 14, and 16): Cas-only reactions contain the relevant Cas enzyme complexed with recombinant guide-RNA hybridized to the intended target, supplied as pure gBlock DNA. In detail, for Cas-only reactions, target amplification was performed separately from Cas detection (Pal5 only). In some experiments, single-stranded DNA targets (oligos) were added directly to the Cas reaction at a concentration of 100 nM. For Pal5, LAMP-amplified targets were amplified starting from SARS-CoV-2 genomic RNA at 100 cp / uL (200 cp / reaction) using N- or O-gene specific LAMP primers in a 20 uL reaction (1X Warmstart RT-LAMP mix (NEB), 1X primer mix). Reactions were incubated at 60°C for 40 min. For all Cas-only reactions, 5 uL of either ssDNA target or LAMP amplified product was added to 250 nM Pal enzyme, 250 nM of the corresponding guide, 8 mM MgCl2, and 250 nM DNAse Alert. Cas enzyme activation was monitored in a QS5 PCR machine at 60 °C, and fluorescence was measured every minute.

[0266] Real-time SHERLOCK (RT-SLK) reactions (Figures 11, 13, 15, and 17): For RT-SLK reaction, the target nucleic acid is first exponentially amplified using LAMP, while the amplified material is simultaneously detected in complex with its guide RNA using Cas enzyme. In detail, for RT-SLK reaction, LAMP-based amplification was combined with Cas readout in a single tube. The final concentrations for 20uL RT-SLK reaction are as follows: 1x Warmstart RT-LAMP mix (NEB) was combined with 1x LAMP primers (primer set N, or primer set O), 0.01U / uL TIPP (thermostable inorganic phosphatase (NEB)), 125nM C7-FAM reporter or DNAse Alert (250nM), 250nM Cas enzyme, and 250nM of the corresponding guide RNA. SARS-CoV-2 genomic RNA at 100 cp / uL (200 cp / reaction) was used as the starting target material, and the reactions were placed in a QS5 PCR machine and incubated at 60°C, with fluorescence monitored and measured every minute.

[0267] Figures 10-17 show that the thermostable Cas proteins tested (PAL5, PAL8, PAL9, and PAL10) are functional in a "one-pot" detection assay, enabling facile molecular diagnostics. Furthermore, Figures 11, 13, 15, and 17 show that the thermostable Cas proteins PAL5, PAL8, PAL9, and PAL10 are compatible with chemically enabled amplification (RT-SLK reaction), such as LAMP. The ability to combine amplification and detection in one pot reduces the complexity of the overall diagnostic or therapeutic assay.

[0268] Figures 10 and 11 show that by simply exchanging the variable domains of the guides (shown in bold in Table 2), the guide-RNAs allow the detection of multiple targets (here SCoV2-N or SCV2-O). Thus, PAL5 complexed to either guide-RNA crEF82 or guide-RNA crEF88 was able to detect its targets. The two guide-RNAs had different performance levels, but both showed clear signals above background.

[0269] Figures 14-17 show that guide-RNAs of various lengths complexed to PAL9 or PAL10 all retained full functionality (all showed clear signals above background), indicating that smaller guide-RNAs (e.g., crJP105 or crJP109) do not compromise functional activity in diagnostic or therapeutic assays compared to longer guide RNAs (e.g., crJP103, crJP104, or crJP107).

[0270] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above Description, but is as set forth in the following claims.

Claims

1. A Cas protein having collateral cleavage activity that is thermostable at a temperature above at least 55°C, and A guide RNA selected or recombined to be complementary to a target nucleic acid sequence, and A detection method comprising contacting a CRISPR-Cas complex containing the same with a sample potentially containing the target nucleic acid sequence.

2. The method according to claim 1, wherein the contacting step comprises contacting the CRISPR-Cas complex and the sample with a reporter susceptible to cleavage by the collateral activity of the Cas protein.

3. The method according to claim 1 or 2, wherein the contacting step comprises incubating at a temperature above the temperature for a certain period of time.

4. The method according to claim 1, further comprising the step of amplifying the nucleic acid present in the sample.

5. The method according to claim 4, wherein the amplifying step utilizes a thermostable nucleic acid polymerase.

6. The method according to claim 4 or 5, wherein the amplifying step and the contacting step are performed in a single container.

7. The method according to claim 1, wherein the Cas protein is a Cas12 protein.

8. The method according to claim 7, wherein the Cas protein has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8-10, SEQ ID NO: 7, or SEQ ID NO: 1-4.

9. In a method for performing a detection assay using a Cas protein having collateral cleavage activity, the improvement comprises using a Cas protein having thermostable collateral cleavage activity.

10. The improvement according to claim 9, wherein the Cas protein is a Cas12 protein.

11. The method for performing the detection assay is performed in a single reaction vessel, the improvement according to claim 9.

12. The improvement according to claim 9, wherein the thermostable collateral cleavage activity is thermostable at a temperature above about 55°C.

13. The improvement according to claim 9, wherein the thermostable collateral cleavage activity is thermostable at a temperature above about 60°C.

14. (a) A Cas protein having collateral cleavage activity that is thermostable at a temperature above at least 55°C, and (b) at least one guide capable of forming a complex with the thermostable Cas protein and capable of directing the binding of the complex to a target nucleic acid sequence, A non-naturally occurring or recombinant composition comprising. **Claim 15** The composition according to claim 14, wherein the at least one guide comprises two guide sequences capable of hybridizing to two different target nucleic acid sequences or to different regions of one target nucleic acid sequence. **Claim 16** The composition according to claim 14, wherein the at least one guide comprises a plurality of guide sequences capable of hybridizing to a plurality of different target nucleic acid sequences or to a plurality of different regions of one target nucleic acid sequence. **Claim 17** The composition according to claim 14, wherein the guide sequence is capable of hybridizing to one or more target nucleic acid sequences in a prokaryotic or eukaryotic cell. **Claim 18** A method of cleaving at least one target nucleic acid in a cell, comprising contacting the cell with a Cas protein having collateral cleavage activity that is thermostable at a temperature above at least 55° C. and at least one guide capable of hybridizing to the at least one target nucleic acid, wherein the Cas protein is capable of forming a complex with the at least one guide and capable of causing disruption in the at least one target nucleic acid, said method.

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