# Patent

## Title
Endoribonuclease and methods of use thereof

## Abstract

The present disclosure provides variant Cas endoribonucleases, nucleic acids encoding the variant Cas endoribonucleases, and host cells genetically modified with the nucleic acids. The variant Cas endoribonucleases find use in a variety of applications, which are also provided. The present disclosure also provides methods of detecting a specific sequence in a target polyribonucleotide; and methods of regulating production of a target RNA in a eukaryotic cell.

## Background
CROSS-REFERENCE
This application claims the benefit of U.S. Provisional Patent Application No. 61/660,414, filed Jun. 15, 2012, which application is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under Grant No. GM007232 awarded by the National Institutes of Health. The government has certain rights in the invention.


INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE
A Sequence Listing is provided herewith as a text file, “BERK-190WO-SeqList_ST25.txt” created on Jun. 3, 2013 and having a size of 65 KB. The contents of the text file are incorporated by reference herein in their entirety.
BACKGROUND
Bacteria and archaea possess adaptive immune systems that rely on small RNAs for defense against invasive genetic elements. CRISPR (clustered regularly interspaced short palindromic repeats) genomic loci are transcribed as long precursor RNAs, which must be enzymatically cleaved to generate mature CRISPR-derived RNAs (crRNAs) that serve as guides for foreign nucleic acid targeting and degradation. This processing occurs within the repetitive sequence and is catalyzed by a dedicated CRISPR-associated (Cas) family member in many CRISPR systems.
Endoribonucleases that process CRISPR transcripts are bacterial or archaeal enzymes capable of catalyzing sequence- and structure-specific cleavage of a single-stranded RNA. These enzymes cleave a specific phosphodiester bond within a specific RNA sequence.
SUMMARY
The present disclosure provides variant Cas endoribonucleases, nucleic acids encoding the variant Cas endoribonucleases, and host cells genetically modified with the nucleic acids. The variant Cas endoribonucleases find use in a variety of applications, which are also provided. The present disclosure also provides methods of detecting a specific sequence in a target polyribonucleotide; and methods of regulating production of a target RNA in a eukaryotic cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts Cas6 protein TTHA0078 bound to nucleotides 15-30 of Tt_R1 containing a 2′ deoxy at position G28, with the catalytic residue H37 highlighted in red.
FIG. 2 depicts a close up of catalytic residue H37 in close proximity to the RNA backbone between G28 and A29.
FIG. 3 depicts involvement of R129 in multiple base specific interactions with nucleotides G26, G27 and G28.
FIG. 4 depicts the impact of mutations at different parts of the stem of the RNA substrate on binding affinity of TTHA0078 to its substrate. (SEQ ID NO: 14).
FIG. 5 depicts the effect of RNA substrate length on binding affinity to TTHA0078. (SEQ ID NO: 15).
FIGS. 6A and 6B are graphs showing cleavage assay in multiple turnover conditions and gradually increased stoichiometric excess of substrate. FIG. 6A shows the data for TTHB231 with Tt_Repeat2 substrate; FIG. 6B shows the data for TTHA0078 with Tt_Repeat 1 substrate.
FIGS. 7A and 7B depict results of a cleavage assay of TTHA0078 (FIG. 7A) and TTHB231 (FIG. 7B) with their respective Histidine mutants and Tt_R1 as a substrate under single turnover conditions.
FIGS. 8A and 8B depict binding affinities of TTHA0078 wild type (WT) and TTHA0078 mutants at residues H37 and R129 for substrate binding to Tt_Repeat1 RNA (FIG. 8A) and Tt_Repeat2 RNA (FIG. 8B).
FIGS. 9A and 9B depict an alignment of Cas6 amino acid sequences. (Top to bottom, SEQ ID NOs: 16-23).
FIG. 10 depicts an alignment of Cas6 amino acid sequences. (Top to bottom, SEQ ID NOs: 24-27).
FIGS. 11A-F depict Cas6 amino acid sequences and RNA sequences and structures recognized by the Cas6 polypeptides. (Top to bottom: FIG. 11A, SEQ ID NOs: 19, 24, 1, 2, 28, 29; FIG. 11B, SEQ ID NOs: 3, 30, 31; FIG. 11C, SEQ ID NOs: 4, 32, 33; FIG. 11D, SEQ ID NOs: 5, 34, 35; FIG. 11E, SEQ ID NOs: 6, 36, 37; FIG. 11F, SEQ ID NOs: 7, 38, 39).
FIG. 12 provides an amino acid sequence alignment of the Cas6 amino acid sequences depicted in FIGS. 11A-F. (Top to bottom, SEQ ID NOs: 19, 24, 33, 31, 35, 37, 39).
FIG. 13 depicts cleavage of pre-crRNA by Cas5.
FIGS. 14A and 14B present data showing that Cas5 cleaves pre-crRNA at base G21. (FIG. 14B: SEQ ID NO: 40).
FIG. 15 provides an amino acid sequence alignment of Cas5 polypeptides. (Top to bottom, SEQ ID NOs: 13, 41, 42, 43, 44, 45).
FIGS. 16A-E depict Cas5 amino acid sequences and RNA sequences and structures recognized by the Cas5 polypeptides. (Top to bottom: FIG. 16A, SEQ ID NOs: 13, 8, 8; FIG. 16B, SEQ ID NOs: 46, 9, 9; FIG. 16C, SEQ ID NOs: 47, 10, 10; FIG. 16D, SEQ ID NOs: 48, 11, 11; FIG. 16E, SEQ ID NOs: 49, 12, 50).
FIG. 17 provides an amino acid sequence alignment of the Cas5 amino acid sequences depicted in FIGS. 16A-E. (Top to bottom: SEQ ID NOs: 13, 46, 47, 48, 49).
FIG. 18 depicts an example of a method for detecting a specific sequence in a target polyribonucleotide.
FIG. 19 depicts an exemplary method of isolating a target RNA. A Cas5 target stem-loop is shown. (SEQ ID NO: 51).
FIG. 20 depicts an exemplary method of regulating expression of a target RNA in a eukaryotic cell. A Cas5 RNA substrate sequence is shown. (SEQ ID NO: 52).
FIG. 21 depicts cleavage of target RNAs by Thermus thermophilus Cas6 proteins at a position immediately downstream of the stem loop structure. (Top to bottom: SEQ ID NOs: 53, 54).
FIGS. 22A-C depict cleavage of repeats R1 and R3 by TtCas6A and TtCas6B, and retention of the cleaved products. (Top to bottom: FIG. 22A, SEQ ID NOs: 1, 2, 55).
FIGS. 23A-D depict various structures of TtCas6A and TtCas6B enzymes bound to substrate mimic and product RNAs. (SEQ ID NO: 53). FIG. 23C shows the results from endonuclease activity assays of wild-type (WT) and active site mutant proteins. (SEQ ID NO: 53).
FIGS. 24A-D depict features of RNA recognition by TtCas6A and TtCas6B.
FIGS. 25A-F depict recognition of the 5′ segment of the repeat RNA. (FIG. 25D: left, SEQ ID NO: 56) (FIG. 25D: right bottom, SEQ ID NO: 57).
FIGS. 26A-B depict cleavage of repeats R1 and R3, but not R2, by TtCas6A and TtCas6B.
FIGS. 27A-C depict dimeric structures of TtCas6A and TtCas6B enzymes bound to substrate mimic and product RNAs.
FIGS. 28A-B depict sequence and structural alignments of TtCas6A and TtCas6B enzymes. (FIG. 28A: Top to bottom; amino acids 1-238 of SEQ ID NO: 19, amino acids 1-262 of SEQ ID NO: 24, all amino acids of SEQ ID NO: 58, all amino acids of SEQ ID NO: 59, and all amino acids of SEQ ID NO: 60).
FIGS. 29A-C depict various structural views of TtCas6A and TtCas6B complexed with substrate and/or product.
FIG. 30 depicts a table of X-ray data collection and refinement statistics.



DEFINITIONS
As used herein, “polyribonucleotide” refers to a polymeric form of ribonucleotides, and includes RNA, RNA containing deoxyribonucleotide(s), and DNA containing ribonucleotide(s). A polyribonucleotide can in some cases include one or more modified nucleotides (e.g., deoxyinosine, deoxyuridine or hydroxymethyldeoxyuridine). In some cases, a polyribonucleotide consists of a ribonucleotides only (i.e., does not include any deoxyribonucleotides). In some cases, a polyribonucleotide comprises ribonucleotides, and one or more modified ribonucleotides, but does not include any deoxyribonucleotides. In other cases, a polyribonucleotide comprises ribonucleotides, and may comprise one or more modified ribonucleotides, and one or more deoxyribonucleotides (including modified deoxyribonucleotides). In some cases, where a polyribonucleotide comprises one or more deoxyribonucleotides, the deoxyribonucleotides comprise from about 50% to about 40%, from about 40% to about 30%, from about 30% to about 20%, from about 20% to about 10%, from about 10% to about 1%, or less than 1%, of the total nucleotides in the polyribonucleotide.
The terms “nucleic acid” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, and primers.
A “biological sample” encompasses a variety of sample types obtained from a cell, extracellular matter, a tissue, or a multicellular organism. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term “biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid (e.g., cerebrospinal fluid, bronchoalveolar lavage fluid, urine, blood, a blood fraction (e.g., plasma; serum), sputum, and the like), and tissue samples. In some cases, a biological sample comprises cells. In other cases, a biological sample is cell free.
The term “operably linked” refers to functional linkage between molecules to provide a desired function. For example, “operably linked” in the context of nucleic acids refers to a functional linkage between nucleic acids to provide a desired function such as transcription, translation, and the like, e.g., a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second polynucleotide, wherein the expression control sequence affects transcription and/or translation of the second polynucleotide. “Operably linked” in the context of a polypeptide refers to a functional linkage between amino acid sequences (e.g., of different domains) to provide for a described activity of the polypeptide.
“Isolated” refers to a protein or nucleic acid that, if naturally occurring, is in an environment different from that in which it may naturally occur. “Isolated” is meant to include proteins or nucleic acids that are within samples that are substantially enriched for the protein or nucleic acid of interest and/or in which the protein or nucleic acid of interest is partially or substantially purified. Where the protein or nucleic acid is not naturally occurring, “isolated” indicates the protein or nucleic acid has been separated from an environment in which it was made by either synthetic or recombinant means.
“Substantially pure” indicates that an entity (e.g., polypeptide or a nucleic acid) makes up greater than about 50% of the total content of the composition (e.g., total protein of the composition) and typically, greater than about 60% of the total protein content. In some embodiments, “substantially pure” refers to compositions in which at least 75%, at least 85%, at least 90% or more of the total composition is the entity of interest (e.g. 95%, of the total protein). In some embodiments, the protein or nucleic acid of interest will make up greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99%, of the total protein or nucleic acid in the composition.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a site-specific endoribonuclease” includes a plurality of such site-specific endoribonucleases and reference to “the target nucleic acid” includes reference to one or more target nucleic acids and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION
The present disclosure provides variant Cas endoribonucleases, nucleic acids encoding the variant Cas endoribonucleases, and host cells genetically modified with the nucleic acids. The variant Cas endoribonucleases find use in a variety of applications, which are also provided. The present disclosure also provides methods of detecting a specific sequence in a target polyribonucleotide; and methods of regulating production of a target RNA in a eukaryotic cell.
Endoribonucleases
The present disclosure provides a sequence-specific endoribonuclease. In some embodiments, the present disclosure provides a sequence-specific endoribonuclease that binds to a recognition sequence in a target polyribonucleotide, but that does not cleave the target polyribonucleotide, i.e., the sequence-specific endoribonuclease is enzymatically inactive in hydrolyzing the target polyribonucleotide. In some embodiments, the present disclosure provides a sequence-specific endoribonuclease that binds to a recognition sequence in a target polyribonucleotide, and cleaves the target polyribonucleotide within or near the recognition sequence, i.e., the sequence-specific endoribonuclease is enzymatically active in hydrolyzing the target polyribonucleotide.
In some embodiments, a subject sequence-specific endoribonuclease is immobilized on an insoluble substrate. Suitable insoluble substrates include, but are not limited to agarose beads, magnetic beads, a test strip, a multi-well dish, and the like. The insoluble substrate can comprise a variety of substances (glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agaroses, and magnetite) and can be provided in a variety of forms, including, e.g., agarose beads, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, wells of reaction trays (e.g., multi-well plates), plastic tubes, etc.
Enzymatically Inactive Sequence-Specific Endoribonuclease
The present disclosure provides an enzymatically inactive, sequence-specific endoribonuclease, wherein the enzymatically inactive sequence-specific endoribonuclease binds to a target sequence in a polyribonucleotide in a sequence-specific manner. A subject enzymatically inactive, sequence-specific endoribonuclease binds a target polyribonucleotide in a sequence-specific manner, but does not cleave the target polyribonucleotide. A subject enzymatically inactive, sequence-specific endoribonuclease is useful for isolating a target RNA from a mixed population of nucleic acids, as described herein.
A subject enzymatically inactive, sequence-specific endoribonuclease is “conditionally” enzymatically inactive, e.g., a subject enzymatically inactive, sequence-specific endoribonuclease (e.g., a subject variant Cas6 endoribonuclease; a variant Cas5 endoribonuclease) is enzymatically inactive in the absence of imidazole; and the enzymatically inactive, sequence-specific endoribonuclease (e.g., subject variant Cas6 endoribonuclease; a variant Cas5 endoribonuclease) is activatable by imidazole. For example, the enzymatically inactive, sequence-specific endoribonuclease (e.g., subject variant Cas6 endoribonuclease; a variant Cas5 endoribonuclease) can be enzymatically activated by contacting the endoribonuclease with imidazole at a concentration of from about 100 mM to about 500 mM.
The presence of imidazole (e.g., in a concentration range of from about 100 mM to about 500 mM) reactivates the sequence-specific, enzymatically inactive endoribonuclease such that the endoribonuclease becomes enzymatically active, e.g., the endoribonuclease exhibits at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more than 95%, of wild-type sequence-specific endoribonuclease (e.g., an amino acid sequence as set forth in one of SEQ ID NOs: 16-27 31, 33, 35, 37, and 39; an amino acid sequence as set forth in one of SEQ ID NOs: 13 and 41-49; and the like).
Variant Cas6 Polypeptides
In some cases, a subject enzymatically inactive, sequence-specific endoribonuclease is a variant Cas6 polypeptide.
FIGS. 9, 10, and 11A-F depict non-limiting examples of amino acid sequences that can be modified to provide an enzymatically inactive, sequence-specific endoribonuclease. In some embodiments, a subject enzymatically inactive, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence set forth in one of SEQ ID NOs: 16-23, where the amino acid sequence includes a substitution at His-37, Arg-129 (or Arg-128 in the case of YP_005654445; or Arg-130 in the case of YP_004367049), or both His-37 and Arg-129 (or Arg-128 in the case of YP_005654445; or Arg-130 in the case of YP_004367049). For example, the variant Cas6 endoribonuclease can include a H37A (His-37 to Ala-37) substitution, an R129A (Arg-129 to Ala-129) substitution, or both a H37A and an R129A substitution.
In some embodiments, a subject enzymatically inactive, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 250 amino acids, or from about 250 amino acids to 264 amino acids, of an amino acid sequence set forth in one of SEQ ID NOs: 24-27, where the amino acid sequence includes a substitution at His-42 (e.g., a His-to-Ala substitution at amino acid 42).
In some embodiments, a subject enzymatically inactive, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids (aa) to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 240 amino acids (e.g., 239 aa, 240 aa, 241 aa, 242 aa, 243 aa, 244 aa), from about 240 amino acids to about 250 amino acids, from about 250 amino acids to about 260 amino acids (e.g., 264 amino acids), from about 260 amino acids to about 275 amino acids (e.g., 277 amino acids), or from about 275 amino acids to 314 amino acids, of an amino acid sequence set forth in one of SEQ ID NOs: 19, 24, 31, 33, 35, 37, and 39, where the amino acid sequence includes a substitution at His-37 (e.g., a His-to-Ala substitution at amino acid 37) of the sequence set forth in one of SEQ ID NOs: 19 and 24 or a corresponding amino acid in a sequence set forth in one of SEQ ID NOs: 31, 33, 35, 37, and 39, where the His corresponding to His-37 of the sequence set forth in one of SEQ ID NOs: 19 and 24 is bolded and underlined in FIGS. 11B-F.
In some cases, the substrate recognized by a variant Cas6 polypeptide comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, nucleotide sequence identity to one of the following sequences:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUGAGGAUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



and



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′.






In some cases, the substrate recognized by a variant Cas6 polypeptide comprises one of the following sequences:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′,






or a “minimal” sequence depicted in FIGS. 11A-F (SEQ ID NOs: 28-29, 30, 32, 34, 36, and 38).
An RNA substrate recognized by a variant Cas6 polypeptide of the present disclosure can have a length of from about 15 nucleotides (nt) to about 20 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt), from about 20 nt to about 25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt), from about 25 nt to about 30 nt (e.g., 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt), from about 30 nt to about 35 nt (e.g., 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt), from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, or more than 50 nt.
Variant Cas5 Polypeptides
In some cases, a subject enzymatically inactive, sequence-specific endoribonuclease is a variant Cas5 polypeptide.
FIGS. 15 and FIGS. 16A-E depict non-limiting examples of amino acid sequences that can be modified to provide an enzymatically inactive, sequence-specific endoribonuclease. In some embodiments, a subject enzymatically inactive, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence set forth in one of SEQ ID NOs: 13 and 41-49, where the amino acid sequence includes a substitution at His-141 of the sequence depicted in FIG. 15 as YP_009170 (SEQ ID NO: 13), or a corresponding histidine residue (see the His residue in bold in FIG. 15 or FIGS. 16A-E) in any of the amino acid sequences depicted in FIG. 15 (SEQ ID NOs: 13 and 41-45) or FIGS. 16A-E (SEQ ID NOs: 13 and 46-49).
In some cases, the substrate recognized by a variant Cas5 polypeptide comprises one of the following sequences:









(SEQ ID NO: 8)



5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;



 



(SEQ ID NO: 9)



5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;



 



(SEQ ID NO: 10)



5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;



 



(SEQ ID NO: 11)



5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;



and



 



(SEQ ID NO: 12)



5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′,






or a “minimal” sequence depicted in FIGS. 16A-E (SEQ ID NOs: 8-12).
An RNA substrate recognized by a variant Cas5 polypeptide of the present disclosure can have a length of from about 15 nucleotides (nt) to about 20 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt), from about 20 nt to about 25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt), from about 25 nt to about 30 nt (e.g., 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt), from about 30 nt to about 35 nt (e.g., 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt), from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, or more than 50 nt.
Modifications
In some embodiments, a subject enzymatically inactive, sequence-specific endoribonuclease (e.g., a subject variant Cas6 endoribonuclease; a subject variant Cas5 endoribonuclease) comprises a detectable label, including a moiety that provides a detectable signal. Suitable detectable labels and/or moieties that provide a detectable signal include, but are not limited to, an enzyme, a radioisotope, a member of a FRET pair, a member of a specific binding pair; a fluorophore; a fluorescent protein; a quantum dot; and the like.
FRET pairs (donor/acceptor) suitable for use include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL, fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5 and fluorescein/LC Red 705. In addition, a fluorophore/quantum dot donor/acceptor pair can be used.
Suitable fluorophores (“fluorescent label”) include any molecule that may be detected via its inherent fluorescent properties, which include fluorescence detectable upon excitation. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 2002 Molecular Probes Handbook, 9th Ed., by Richard P. Haugland, hereby expressly incorporated by reference.
Suitable enzymes include, but are not limited to, horse radish peroxidase, luciferase, β-galactosidase, alkaline phosphatase, and the like.
Suitable fluorescent proteins include, but are not limited to, a green fluorescent protein (GFP), e.g., a GFP from Aequoria victoria or a mutant or derivative thereof e.g., as described in U.S. Pat. Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; 5,874,304; a red fluorescent protein; a yellow fluorescent protein; any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and the like.
Suitable nanoparticles include, e.g., quantum dots (QDs), fluorescent or luminescent nanoparticles, and magnetic nanoparticles. Any optical or magnetic property or characteristic of the nanoparticle(s) can be detected.
QDs and methods for their synthesis are well known in the art (see, e.g., U.S. Pat. Nos. 6,322,901; 6,576,291; and 6,815,064). QDs can be rendered water soluble by applying coating layers comprising a variety of different materials (see, e.g., U.S. Pat. Nos. 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; and 6,649,138). For example, QDs can be solubilized using amphiphilic polymers. Exemplary polymers that have been employed include octylamine-modified low molecular weight polyacrylic acid, polyethylene-glycol (PEG)-derivatized phospholipids, polyanhydrides, block copolymers, etc. QDs can be conjugated to a polypeptide via any of a number of different functional groups or linking agents that can be directly or indirectly linked to a coating layer (see, e.g., U.S. Pat. Nos. 5,990,479; 6,207,392; 6,251,303; 6,306,610; 6,325,144; and 6,423,551).
QDs with a wide variety of absorption and emission spectra are commercially available, e.g., from Quantum Dot Corp. (Hayward Calif.; now owned by Invitrogen) or from Evident Technologies (Troy, N.Y.). For example, QDs having peak emission wavelengths of approximately 525, 535, 545, 565, 585, 605, 655, 705, and 800 nm are available. Thus the QDs can have a range of different colors across the visible portion of the spectrum and in some cases even beyond.
Suitable radioisotopes include, but are not limited to 14C, 3H, 32P, 33P, 35S, 125I, and 131I. The use of radioisotopes as labels is well known in the art.
In some embodiments, a subject enzymatically inactive, sequence-specific endoribonuclease (e.g., a subject variant Cas6 endoribonuclease) is immobilized on an insoluble substrate. Suitable insoluble substrates include, but are not limited to agarose beads, magnetic beads, a test strip, a multi-well dish, and the like. The insoluble substrate can comprise a variety of substances (glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agaroses, and magnetite) and can be provided in a variety of forms, including, e.g., agarose beads, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, wells of reaction trays (e.g., multi-well plates), plastic tubes, etc.
In some embodiments, a subject enzymatically inactive, sequence-specific endoribonuclease (e.g., a subject variant Cas6 endoribonuclease, a subject variant Cas5 endoribonuclease) is purified, e.g., is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, or greater than 99% pure.
Compositions
The present disclosure provides compositions comprising a subject sequence-specific, enzymatically inactive, endoribonuclease. A subject composition can comprise, in addition to a subject sequence-specific, enzymatically inactive, endoribonuclease, one or more of: a salt, e.g., NaCl, MgCl2, KCl, MgSO4, etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a reducing agent, e.g., dithiothreitol; a protease inhibitor; and the like.
Enzymatically Active Sequence-Specific Endoribonuclease
Endoribonucleases suitable for use in a subject method will in some embodiments be an enzymatically active sequence-specific endoribonuclease (e.g., a Cas6 polypeptide or a Cas5 polypeptide).
Suitable Cas6 polypeptide amino acid sequences are provided in, e.g., GenBank Accession No. YP_143344 (Thermus thermophilus HB8); GenBank Accession No. YP_145470 (Thermus thermophilus HB8); GenBank Accession No. YP_005869 (Thermus thermophilus HB27); GenBank Accession No. YP_006059433 (Thermus thermophilus JL-18); GenBank Accession No. YP_005654445 (Thermus sp. CCB_US3_UF1); GenBank Accession No. ZP_03497188 (Thermus aquaticus); GenBank Accession No. YP_003684129 (Meiothermus silvanus DSM 9946); GenBank Accession No. YP_004367049 (Marinithermus hydrothermalis); GenBank Accession No. YP_005641609 (Thermus thermophilus SG0.5JP17-16); GenBank Accession No. YP_006185 (Thermus thermophilus HB27); GenBank Accession No. YP_006059769 (Thermus thermophilus JL-18); YP_003506022 Meiothermus ruber DSM 1279).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence depicted in FIGS. 9A and 9B (SEQ ID NOs: 16-23).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 250 amino acids, or from about 250 amino acids to 264 amino acids, of an amino acid sequence depicted in FIG. 10 (SEQ ID NOs: 24-27).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids (aa) to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 240 amino acids (e.g., 239 aa, 240 aa, 241 aa, 242 aa, 243 aa, 244 aa), from about 240 amino acids to about 250 amino acids, from about 250 amino acids to about 260 amino acids (e.g., 264 amino acids), from about 260 amino acids to about 275 amino acids (e.g., 277 amino acids), or from about 275 amino acids to 314 amino acids, of an amino acid sequence depicted in FIGS. 11A-F or FIG. 12 (SEQ ID NOs: 19, 24, 31, 33, 35, 37, and 39).
In some cases, the substrate recognized by a Cas6 polypeptide comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, nucleotide sequence identity to one of the following sequences:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



and



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′.






In some cases, the substrate recognized by a Cas6 polypeptide comprises one of the following sequences:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′,






or a “minimal” sequence depicted in FIGS. 11A-F (SEQ ID NOs: 28-29, 30, 32, 34, 36, and 38).
An RNA substrate recognized by a Cas6 polypeptide can have a length of from about 15 nucleotides (nt) to about 20 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt), from about 20 nt to about 25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt), from about 25 nt to about 30 nt (e.g., 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt), from about 30 nt to about 35 nt (e.g., 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt), from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, or more than 50 nt.
Suitable Cas5 polypeptides are provided in, e.g., GenBank Accession No. YP_009170 (Desulfovibrio vulgaris Cas5); GenBank Accession No. YP_004513910 (Methylomonas methanica MC09 Cas5); GenBank Accession No. YP_004496339 (Desulfotomaculum carboxydivorans Cas5); GenBank Accession No. ZP_05883174 (Vibrio metchnikovii Cas5); GenBank Accession No. YP_001174211 (Pseudomonas stutzeri Cas5); etc.
In some embodiments, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence depicted in FIG. 15 (SEQ ID NOs: 13 and 41-45), or in FIGS. 16A-E (SEQ ID NOs: 13 and 46-49).
In some cases, the substrate recognized by a variant Cas5 polypeptide comprises one of the following sequences:









(SEQ ID NO: 8)



5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;



 



(SEQ ID NO: 9)



5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;



 



(SEQ ID NO: 10)



5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;



 



(SEQ ID NO: 11)



5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;



and



 



(SEQ ID NO: 12)



5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′,






or a “minimal” sequence depicted in FIGS. 16A-E (SEQ ID NOs: 8-12).
An RNA substrate recognized by a Cas5 polypeptide can have a length of from about 15 nucleotides (nt) to about 20 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt), from about 20 nt to about 25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt), from about 25 nt to about 30 nt (e.g., 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt), from about 30 nt to about 35 nt (e.g., 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt), from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, or more than 50 nt.
In some instance, an enzymatically active sequence-specific endoribonuclease comprises a moiety that provides for detection. For example, a subject enzymatically active sequence-specific endoribonuclease can comprise a covalently or non-covalently linked moiety that provides for detection.
Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Moieties that provide for detection include, but are not limited to, a fluorescent molecule; a quantum dot; an enzyme (other than the endoribonuclease), where the enzyme catalyzes conversion of a substrate to a detectable product, where the product is directly detectable; a nanoparticle; and the like.
Suitable fluorescent proteins that can be linked to a subject enzymatically active sequence-specific endoribonuclease include, but are not limited to, a green fluorescent protein (GFP), e.g., a GFP from Aequoria victoria or a mutant or derivative thereof e.g., as described in U.S. Pat. Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; 5,874,304; a red fluorescent protein; a yellow fluorescent protein; any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and the like.
Suitable nanoparticles include, e.g., quantum dots (QDs), fluorescent or luminescent nanoparticles, and magnetic nanoparticles. Any optical or magnetic property or characteristic of the nanoparticle(s) can be detected.
QDs and methods for their synthesis are well known in the art (see, e.g., U.S. Pat. Nos. 6,322,901; 6,576,291; and 6,815,064). QDs can be rendered water soluble by applying coating layers comprising a variety of different materials (see, e.g., U.S. Pat. Nos. 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; and 6,649,138). For example, QDs can be solubilized using amphiphilic polymers. Exemplary polymers that have been employed include octylamine-modified low molecular weight polyacrylic acid, polyethylene-glycol (PEG)-derivatized phospholipids, polyanhydrides, block copolymers, etc. QDs can be conjugated to a polypeptide via any of a number of different functional groups or linking agents that can be directly or indirectly linked to a coating layer (see, e.g., U.S. Pat. Nos. 5,990,479; 6,207,392; 6,251,303; 6,306,610; 6,325,144; and 6,423,551).
QDs with a wide variety of absorption and emission spectra are commercially available, e.g., from Quantum Dot Corp. (Hayward Calif.; now owned by Invitrogen) or from Evident Technologies (Troy, N.Y.). For example, QDs having peak emission wavelengths of approximately 525, 535, 545, 565, 585, 605, 655, 705, and 800 nm are available. Thus the QDs can have a range of different colors across the visible portion of the spectrum and in some cases even beyond.
In some embodiments, a subject enzymatically active, sequence-specific endoribonuclease is purified, e.g., is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, or greater than 99% pure.
Compositions
The present disclosure provides compositions comprising a subject sequence-specific, enzymatically active endoribonuclease. A subject composition can comprise, in addition to a subject sequence-specific enzymatically active, endoribonuclease, one or more of: a salt, e.g., NaCl, MgCl2, KCl, MgSO4, etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a protease inhibitor; and the like.
Methods of Producing a Sequence-Specific Endoribonuclease
A subject sequence-specific endoribonuclease (e.g., a subject sequence-specific enzymatically active, endoribonuclease; a subject sequence-specific enzymatically inactive, endoribonuclease) can be produced by any known method, e.g., conventional synthetic methods for protein synthesis; recombinant DNA methods; etc.
Where a subject sequence-specific endoribonuclease is chemically synthesized, the synthesis may proceed via liquid-phase or solid-phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence, is an example of a suitable method for the chemical synthesis of a subject sequence-specific endoribonuclease. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing a subject sequence-specific endoribonuclease. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett. 12:723-8.
Standard recombinant methods can be used for production of a subject sequence-specific endoribonuclease. For example, nucleic acids encoding a subject sequence-specific endoribonuclease are inserted into expression vectors. The DNA segments encoding a subject sequence-specific endoribonuclease are operably linked to control sequences in the expression vector(s) that ensure the expression of the encoded polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. The expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the endoribonuclease.
Nucleic Acids and Host Cells
The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a subject sequence-specific endoribonuclease (e.g., a subject sequence-specific, enzymatically active endoribonuclease; a subject sequence-specific, enzymatically inactive endoribonuclease). In some embodiments, the nucleic acid is an expression vector, where the expression vector can provide for production of the sequence-specific endoribonuclease, e.g., in a cell.
A nucleotide sequence encoding a subject sequence-specific endoribonuclease (e.g., a subject sequence-specific, enzymatically active endoribonuclease; a subject sequence-specific, enzymatically inactive endoribonuclease) can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in the intended target cells (e.g., a cell that is genetically modified to synthesize the encoded endoribonuclease).
In some embodiments, a subject nucleic acid comprises a nucleotide sequence encoding a polypeptide having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, with an amino acid sequence set forth in any one of FIGS. 9A, 9B, 10, 11A-F, and 12 (SEQ ID NOs: 16-27, 31, 33, 35, 37, and 39). In some embodiments, a subject nucleic acid comprises a nucleotide sequence encoding a variant Cas6 polypeptide, as described above.
In some embodiments, a subject nucleic acid comprises a nucleotide sequence encoding a polypeptide having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, with an amino acid sequence set forth in FIG. 15 (SEQ ID NOs: 13 and 41-45) or FIGS. 16A-E (SEQ ID NOs: 13 and 46-49). In some embodiments, a subject nucleic acid comprises a nucleotide sequence encoding a variant Cas5 polypeptide, as described above.
A nucleotide sequence encoding a subject sequence-specific endoribonuclease (e.g., a subject sequence-specific, enzymatically active endoribonuclease; a subject sequence-specific, enzymatically inactive endoribonuclease) can be operably linked to a transcription control element (e.g., a promoter, an enhancer, etc.). Suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters.
In some embodiments, e.g., for expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93; Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter (Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see, e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol. 10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spy promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g., Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein—Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. Acids Res. 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLambda. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (LacI repressor protein changes conformation when contacted with lactose, thereby preventing the LacI repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25).
A nucleotide sequence encoding a subject sequence-specific endoribonuclease (e.g., a subject sequence-specific, enzymatically active endoribonuclease; a subject sequence-specific, enzymatically inactive endoribonuclease) can be present in an expression vector and/or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector.
Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).
Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.
The present disclosure provides isolated genetically modified host cells (e.g., in vitro cells) that are genetically modified with a subject nucleic acid. In some embodiments, a subject isolated genetically modified host cell can produce a subject sequence-specific endoribonuclease (e.g., a subject sequence-specific, enzymatically active endoribonuclease; a subject sequence-specific, enzymatically inactive endoribonuclease).
Suitable host cells include eukaryotic host cells, such as a mammalian cell, an insect host cell, a yeast cell; and prokaryotic cells, such as a bacterial cell. Introduction of a subject nucleic acid into the host cell can be effected, for example by calcium phosphate precipitation, DEAE dextran mediated transfection, liposome-mediated transfection, electroporation, or other known method.
Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.
Suitable yeast cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, and the like.
Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al. (1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains which can be employed in the present invention include, but are not limited to, Salmonella typhi and S. typhimurium. Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, the laboratory strain is one that is non-pathogenic. Non-limiting examples of other suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like. In some embodiments, the host cell is Escherichia coli. 
Methods of Directly Sequencing a Target Polyribonucleotide
The present disclosure provides a method of directly determining the nucleotide sequence of a target polyribonucleotide. Thus, for example, the method does not require synthesis of a polydeoxyribonucleotide counterpart of a target polyribonucleotide in order to determine the nucleotide sequence of the target polyribonucleotide.
Viral diagnostics, personalized medicine, single-cell transcript analysis, and translational profiling are all fields in which direct RNA detection and sequencing find use. A subject polyribonucleotide sequencing method, and a subject method of detecting a specific sequence in a polyribonucleotide, find use in these various fields.
A subject polyribonucleotide sequencing method generally involves: a) contacting a target polyribonucleotide with an oligonucleotide probe comprising a specific known sequence and an enzymatically inactive sequence-specific endoribonuclease under conditions that favor duplex formation between the oligonucleotide probe and the target polyribonucleotide, wherein the enzymatically inactive sequence-specific endoribonuclease binds the specific sequence in the duplex; and b) detecting specific binding between the oligonucleotide probe and the target polyribonucleotide, where specific binding of the enzymatically inactive sequence-specific endoribonuclease to the duplex indicates the presence of the specific sequence in the target polyribonucleotide.
In some cases, the enzymatically inactive sequence-specific endoribonuclease is linked (covalently or non-covalently) to an emissive label. By “emissive label” is meant any molecule that may be detected via its inherent emission properties, which include emission detectable upon excitation. Suitable emissive labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 2002 Molecular Probes Handbook, 9th Ed., by Richard P. Haugland.
In some instances, the oligonucleotide probe used in a subject polyribonucleotide sequencing method is linked to a donor molecule, the enzymatically inactive sequence-specific endoribonuclease is linked to an acceptor molecule, and detection of duplex formation is by fluorescence resonance energy transfer (also referred to as “Förster resonance energy transfer” or “FRET”).
Förster resonance energy transfer (FRET) is phenomenon known in the art wherein excitation of one emissive dye is transferred to another without emission of a photon. A FRET pair consists of a donor chromophore and an acceptor chromophore (where the acceptor chromophore may be a quencher molecule). The emission spectrum of the donor and the absorption spectrum of the acceptor must overlap, and the two molecules must be in close proximity. The distance between donor and acceptor at which 50% of donors are deactivated (transfer energy to the acceptor) is defined by the Förster radius, which is typically 10-100 angstroms. Changes in the emission spectrum comprising FRET pairs can be detected, indicating changes in the number of that are in close proximity (i.e., within 100 angstroms of each other). This will typically result from the binding or dissociation of two molecules, one of which is labeled with a FRET donor and the other of which is labeled with a FRET acceptor, wherein such binding brings the FRET pair in close proximity.
Binding of such molecules will result in an increased emission of the acceptor and/or quenching of the fluorescence emission of the donor. FRET pairs (donor/acceptor) suitable for use include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL, fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5 and fluorescein/LC Red 705. In addition, a fluorophore/quantum dot donor/acceptor pair can be used. EDANS is (5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid); DABCYL is 4-(4-dimethylaminophenyl)diazenylbenzoic acid.
Cy3, Cy5, Cy 5.5, and the like, are cyanines. For example, Cy3 and Cy5 are reactive water-soluble fluorescent dyes of the cyanine dye family. Cy3 dyes are red ( ̃550 nm excitation,  ̃570 nm emission and therefore appear green), while Cy5 is fluorescent in the red region ( ̃650/670 nm) but absorbs in the orange region ( ̃649 nm). Alexa Fluor dyes, Dylight, IRIS Dyes, Seta dyes, SeTau dyes, SRfluor dyes and Square dyes can also be used.
In another aspect of FRET, an emissive donor molecule and a nonemissive acceptor molecule (“quencher”) may be employed. In this application, emission of the donor will increase when quencher is displaced from close proximity to the donor and emission will decrease when the quencher is brought into close proximity to the donor. Useful quenchers include, but are not limited to, DABCYL, QSY 7 and QSY 33. Useful fluorescent donor/quencher pairs include, but are not limited to EDANS/DABCYL, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL and fluorescein/QSY 7 dye.
In some cases, the enzymatically inactive sequence-specific endoribonuclease is linked (covalently or non-covalently) to a label enzyme. By “label enzyme” is meant an enzyme which may be reacted in the presence of a label enzyme substrate which produces a detectable product. Suitable label enzymes also include optically detectable labels (e.g., in the case of horse radish peroxidase (HRP)). Suitable label enzymes include but are not limited to, HRP, alkaline phosphatase, luciferase, β-galactosidase, and glucose oxidase. Methods for the use of such substrates are well known in the art. The presence of the label enzyme is generally revealed through the enzyme's catalysis of a reaction with a label enzyme substrate, producing an identifiable product. Such products may be opaque, such as the reaction of horseradish peroxidase with tetramethyl benzedine, and may have a variety of colors. Other label enzyme substrates, such as Luminol (available from Pierce Chemical Co.), have been developed that produce fluorescent reaction products. Methods for identifying label enzymes with label enzyme substrates are well known in the art and many commercial kits are available. Examples and methods for the use of various label enzymes are described in Savage et al., Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989).
In some cases, the enzymatically inactive sequence-specific endoribonuclease comprises a radioisotope. By “radioisotope” is meant any radioactive molecule. Suitable radioisotopes for use in the invention include, but are not limited to 14C, 3H, 32P, 33P, 35S, 125I, and 131I. The use of radioisotopes as labels is well known in the art.
In some cases, the enzymatically inactive sequence-specific endoribonuclease is linked (covalently or non-covalently) to a member of a specific binding pair (“partner of a binding pair”). By “partner of a binding pair” or “member of a binding pair” is meant one of a first and a second moiety, wherein the first and the second moiety have a specific binding affinity for each other. Suitable binding pairs include, but are not limited to, antigen/antibodies (for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, and rhodamine anti-rhodamine), biotin/avidin (or biotin/streptavidin) and calmodulin binding protein (CBP)/calmodulin.
In some embodiments, the oligonucleotide probe comprises a modification that provides for increased resistance to non-specific hydrolysis. Such modifications are well known in the art and include, e.g., nuclease-resistant internucleosidic linkages, modified backbones, base modifications, base substitutions, sugar modifications, and the like.
Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included.
A modified oligonucleotide can comprise one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— (known as a methylene (methylimino) or MMI backbone), —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— (wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH2—). MMI type internucleoside linkages are disclosed in U.S. Pat. No. 5,489,677. Suitable amide internucleoside linkages are disclosed in U.S. Pat. No. 5,602,240.
A modified oligonucleotide can comprise one or more morpholino backbone structures as described in, e.g., U.S. Pat. No. 5,034,506. For example, in some embodiments, a modified oligonucleotide comprises a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage. Morpholino nucleic acids (“morpholinos”) include bases bound to morpholine rings instead of deoxyribose rings; in addition, the phosphate backbone can include a non-phosphate group, e.g., a phosphorodiamidate group instead of phosphates. Summerton (1999) Biochim. Biophys. Acta 1489:141; Heasman (2002) Dev. Biol. 243:209; Summerton and Weller (1997) Antisense & Nucl. Acid Drug Dev. 7:187; Hudziak et al. (1996) Antisense & Nucl. Acid Drug Dev. 6:267; Partridge et al. (1996) Antisense & Nucl. Acid Drug Dev. 6:169; Amantana et al. (2007) Bioconj. Chem. 18:1325; Morcos et al. (2008) BioTechniques 45:616.
A modified oligonucleotide can comprise a modified backbone. Modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
A modified oligonucleotide can comprise one or more substituted sugar moieties. Suitable oligonucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Also suitable are O((CH2)nO)mCH3, O(CH2)—OCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other suitable oligonucleotides comprise a sugar substituent group selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, and the like. A suitable modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further suitable modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH3)2.
A modified oligonucleotide can comprise one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).
Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
A suitable enzymatically inactive sequence-specific endoribonuclease includes an enzymatically inactive sequence-specific endoribonuclease described hereinbelow. For example, an enzymatically inactive sequence-specific endoribonuclease that is a variant of an amino acid sequence as depicted in any one of FIGS. 9A, 9B, 10, 11A-F, 12, 15, and 16A-E (SEQ ID NOs: 16-27, 31, 33, 35, 37, and 39 or SEQ ID NOs: 13, and 41-49), where the variant is as described above (e.g., with a substitution of a histidine that renders the endoribonuclease conditionally inactive) can be used.
In some embodiments, the target polyribonucleotide is linked (covalently or non-covalently) to a solid support (an insoluble support). Suitable insoluble supports include, but are not limited to, beads, plates (e.g., multi-well plates), strips, etc., where the insoluble support can comprise various materials including, but not limited to, polystyrene, polypropylene, agarose, and the like.
Oligonucleotide probes (“detection oligonucleotide”) can be RNA, DNA, or any chemically modified version of an RNA or DNA, e.g., peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and the like.
A subject polyribonucleotide sequencing method can include one or more washing steps, e.g., to remove non-specifically bound components such as non-specifically bound oligonucleotide probes, any non-specifically bound detectable moieties, and the like.
A non-limiting example of how to carry out a subject polyribonucleotide sequencing method is as follows. A target polyribonucleotide bound to a solid support. The target polyribonucleotide is of unknown sequence and is the “RNA to be sequenced.” Four oligonucleotide probes of four different known nucleotide sequences each comprise a different fluorophore (fluorophores 1-4). The fluorophores are members of FRET pairs. The counterpart members of the FRET pairs are quantum dots. The quantum dot is linked to an enzymatically inactive sequence-specific endoribonuclease. The enzymatically inactive sequence-specific endoribonuclease binds, but does not cleave, the duplex formed between an oligonucleotide probe and the target polyribonucleotide. Only one of the four oligonucleotide probes binds to and forms a duplex with the target polyribonucleotide. A washing step removes any unbound oligonucleotide probes. Binding of oligonucleotide probe-fluorophore2 results in duplex formation with the target polyribonucleotide. Fluorophore2 is thus brought into proximity to the quantum dot linked to the enzymatically inactive sequence-specific endoribonuclease, and fluorescence is quenched.
Methods of Cleaving a Polyribonucleotide
The present disclosure provides a method of cleaving a polyribonucleotide in a sequence-specific manner. The method generally involves contacting a substrate polyribonucleotide with an enzymatically active sequence-specific endoribonuclease (e.g., a Cas5 endoribonuclease; a Cas6 endoribonuclease) under conditions that favor sequence-specific cleavage of the polyribonucleotide substrate. A subject method of cleaving a polyribonucleotide in a sequence-specific manner can be used to: 1) remove an affinity tag from a substrate polyribonucleotide; 2) to generate a population of product polyribonucleotides having homogeneity at the 5′ end, e.g., where the substrate polyribonucleotides are in vitro transcribed mRNAs; and 3) to regulate gene expression in a cell in vitro or in vivo.
Substrate Polyribonucleotides
The terms “substrate polyribonucleotide” and “target polyribonucleotide” are used interchangeably herein to refer to a polyribonucleotide that is bound by a sequence-specific endoribonuclease in a sequence-specific manner. A substrate polyribonucleotide can be single stranded. In some instances, a substrate polyribonucleotide is double stranded.
An endoribonuclease binds to and cleaves a substrate polyribonucleotide in a sequence-specific manner. Thus, for example, an endoribonuclease binds to and cleaves a substrate polyribonucleotide at a specific sequence, referred to herein as a “recognition sequence” or a “recognition site.”
A recognition sequence can be a tetranucleotide sequence, a pentanucleotide sequence, a hexanucleotide sequence, a heptanucleotide sequence, an octanucleotide sequence, or longer than an octanucleotide. For example, in some embodiments, the recognition sequence is 9 ribonucleotides, 10 ribonucleotides, 11 ribonucleotides, 12 ribonucleotides, 13 ribonucleotides, 14 ribonucleotides, 15 ribonucleotides, 16 ribonucleotides, 17 ribonucleotides, 18 ribonucleotides, 19 ribonucleotides, or 20 ribonucleotides in length. In some embodiments, a sequence-specific endoribonuclease cleaves immediately 5′ of a recognition sequence. In some embodiments, a sequence-specific endoribonuclease cleaves immediately 3′ of a recognition sequence. In some embodiments, a sequence-specific endoribonuclease cleaves within a recognition sequence. In some cases, a recognition sequence is immediately 5′ of a secondary structure. In some cases, a recognition sequence is located 5′ of a secondary structure and within 1 nucleotide (nt), 2 nt, 3 nt, 4 nt, 5 nt, or 5 nt to 10 nt of the secondary structure. In some cases, a recognition sequence is immediately 3′ of a secondary structure. In some cases, a recognition sequence is located 3′ of a secondary structure and within 1 nucleotide (nt), 2 nt, 3 nt, 4 nt, 5 nt, or 5 nt to 10 nt of the secondary structure.
In some embodiments, a substrate polyribonucleotide comprises the structure XxX2X3X4X5X6X7X8X9X10X11X12X13X14X15, where nucleotides X1-X5 base pair with X11-X15 such that X1 and X15 form the base of a stem structure, and such that X6, X7, X8, X9, and X10 form a loop; the structure is a regular A-form helical structure.
In some embodiments, the substrate polyribonucleotide comprises an affinity tag; and a subject method provides for removal of the affinity tag from the substrate polyribonucleotide.
Sequence-Specific Endoribonucleases
Endoribonucleases that bind to and cleave a substrate polyribonucleotide in a sequence-specific manner include enzymatically active polypeptides that cleave (hydrolyze) a substrate polyribonucleotide in a metal ion-independent fashion.
Suitable endoribonucleases are sequence-specific, enzymatically active endoribonucleases (e.g., Cas5 endoribonucleases; Cas6 endoribonucleases) as described above.
Reaction Conditions
A sequence-specific endoribonuclease can hydrolyze a substrate polyribonucleotide in a sequence-specific manner at a temperature in a range from about 15° C. to about 100° C., e.g., in a range of from about 15° C. to about 17° C., from about 17° C. to about 20° C., from about 20° C. to about 25° C., from about 25° C. to about 30° C., from about 30° C. to about 40° C., from about 40° C. to about 50° C., from about 50° C. to about 60° C., from about 60° C. to about 70° C., from about 70° C. to about 80° C., from about 80° C. to about 90° C., or from about 90° C. to about 100° C.
A sequence-specific endoribonuclease can hydrolyze a substrate polyribonucleotide in a sequence-specific manner in a pH range of from about 4.0 to about 8.0, e.g., from about pH 4.0 to about 4.5, from about pH 4.5 to about 5.0, from about pH 5.0 to about 5.5, from about pH 5.5 to about 6.0, from about pH 6.0 to about 6.5, from about pH 6.5 to about 7.0, from about pH 7.0 to about 7.5, from about pH 6.5 to about 7.5, from about pH 7.5 to about 8.0, from about pH 6.5 to about 8.0, or from about pH 5.5 to about 7.5.
Methods of Detecting a Sequence in a Target Polyribonucleotide
The present disclosure provides a method of detecting a sequence in a target polyribonucleotide. The methods are useful for detecting the presence of a particular sequence in a polyribonucleotide, and can therefore be used to detect a polyribonucleotide comprising a particular sequence. For example, the method can be used to detect the presence of a polyribonucleotide of a pathogen in a sample (e.g., in a biological sample).
A subject method can detect as few as 100 copies, down to a single copy, of a target polyribonucleotide. Thus, e.g., a subject method can detect from 1 to about 5, from about 5 to about 10, from about 10 to about 50, or from about 50 to about 100, or more than 100, copies of a target polyribonucleotide in a sample (e.g., in a single cell, in a single embryo, or other biological sample). A subject method is thus useful for various forensic, research, and diagnostic applications.
In some embodiments, a subject method of detecting a specific sequence in a target polyribonucleotide comprises: a) contacting the target polyribonucleotide with a oligonucleotide probe comprising the specific sequence and an enzymatically active sequence-specific Cas5 endoribonuclease under conditions that favor duplex formation between the oligonucleotide probe and the target polyribonucleotide, wherein the duplex is cleaved by the Cas5 endoribonuclease; and b) detecting specific binding between the oligonucleotide probe and the target polyribonucleotide, wherein detection of duplex formation between the oligonucleotide probe and the target polyribonucleotide indicates the presence of the specific sequence in the target polyribonucleotide.
In other embodiments, a subject method of detecting a specific sequence in a target polyribonucleotide comprises: a) contacting the target polyribonucleotide with a oligonucleotide probe comprising the specific sequence and an enzymatically active sequence-specific Cas6 endoribonuclease under conditions that favor duplex formation between the oligonucleotide probe and the target polyribonucleotide, wherein the duplex is cleaved by the Cas6 endoribonuclease; and b) detecting specific binding between the oligonucleotide probe and the target polyribonucleotide, wherein detection of duplex formation between the oligonucleotide probe and the target polyribonucleotide indicates the presence of the specific sequence in the target polyribonucleotide.
In some cases, the oligonucleotide probe is linked to a peptide, and the peptide is released upon cleavage of the duplex by the Cas5 endoribonuclease or the Cas6 endoribonuclease; in these cases, the detection step involves detection of the released peptide. For example, the released peptide is detected by binding to an antibody specific for the peptide, e.g., where the antibody is immobilized. In some embodiments, the target polyribonucleotide is immobilized on a solid support. Target polyribonucleotides include any of a variety of polynucleotides, e.g., the target polyribonucleotide can be a polyribonucleotide of a pathogen.
As noted above, in some embodiments, the antibody or the target polynucleotide is immobilized on a solid support (insoluble support). Suitable insoluble supports include, but are not limited to agarose beads, magnetic beads, a test strip, a multi-well dish, and the like. The insoluble support can comprise a variety of substances (glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agaroses, and magnetite) and can be provided in a variety of forms, including, e.g., agarose beads, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, wells of reaction trays (e.g., multi-well plates), plastic tubes, etc.
In some embodiments, the method generally involves: a) contacting a target polyribonucleotide with a sequence-specific endoribonuclease; and b) detecting cleavage fragments produced by site-specific cleavage of the target polyribonucleotide, where production of cleavage fragments expected upon cleavage at a specific sequence in the polyribonucleotide indicates the presence of the specific sequence.
In other embodiments, a subject method of detecting a sequence in a target polyribonucleotide involves: a) contacting a target polyribonucleotide with: i) a sequence-specific endoribonuclease; and ii) an oligonucleotide probe comprising a linked detection moiety, where the oligonucleotide probe comprises a specific, known nucleotide sequence; wherein the oligonucleotide probe forms a duplex with a complementary sequence in the target polyribonucleotide based on binding of the known nucleotide sequence present in the oligonucleotide probe to a complementary sequence in the target polyribonucleotide, and where the sequence-specific endoribonuclease cleaves the duplex in a sequence-specific manner, thereby releasing the detection moiety from the oligonucleotide probe; and b) detecting the released detection moiety, where release of the detection moiety indicates the presence of the specific sequence. In some embodiments, two or more different oligonucleotide probes are used, each comprising a different specific, known nucleotide sequence.
In some embodiments, the detection moiety is a polypeptide. The polypeptide can be detected using an immunological assay (e.g., an enzyme-linked immunosorbent assay (ELISA); a radioimmunoassay (RIA); etc.), using an antibody specific for the polypeptide detection moiety. The antibody specific for the polypeptide detection moiety can comprise a detectable label. The immunological assay can be carried out on a test strip (e.g., in a lateral flow assay) or other suitable medium such as a multi-well plate.
In some embodiments, the detection moiety is a fluorescent protein, where suitable fluorescent proteins are as described herein. In other embodiments, the detection moiety is luciferin or other substrate for luciferase. Suitable luciferins or other luciferase substrates include, e.g., luciferin (e.g., a firefly luciferin); an aminoluciferin; coelenterazine; a modified coelenterazine as described in U.S. Pat. No. 7,537,912; a coelenterazine analog as described in U.S. Patent Publication No. 2009/0081129 (e.g., a membrane permeant coelenterazine analog as described in U.S. Patent Publication No. 2009/0081129, e.g., one of Structures II, III, IV, V, and VI of U.S. Patent Publication No. 2009/0081129); aminoluciferin; dihydroluciferin; luciferin 6′ methylether; or luciferin 6′ chloroethylether. See, e.g., Branchini, B. R. et al. Anal. Biochem. 2010, 396, 290-296; and Mezzanotte, L. et al., In vivo bioluminescence imaging of murine xenograft cancer models with a red-shifted thermostable luciferase. Mol. Imaging Biol. (2009, Nov. 9, online; PubMed ID: 19937390).
A non-limiting example of a subject detection method is illustrated schematically in FIG. 18. In the example depicted in FIG. 18, small oligonucleotides that bind discrete regions of a target polynucleotide (e.g., a viral RNA) are contacted with the target polynucleotide, where the oligonucleotides comprise detectable moieties (e.g., ligands; peptides; etc.). An enzymatically active, sequence-specific restriction endonuclease (RRE) that targets the oligonucleotide/viral RNA duplex is added. The enzyme cleaves the oligonucleotide/viral RNA duplex; and ligands are released for detection. The enzyme cleaves further duplexes, thereby amplifying the signal. Released ligands are detected using a lateral flow (e.g., test strip) or an immunological based assay (e.g., ELISA).
Endoribonucleases suitable for use in a subject method include an enzymatically active sequence-specific endoribonuclease (e.g., a Cas6 polypeptide or a Cas5 polypeptide).
Suitable Cas6 polypeptide amino acid sequences are provided in, e.g., GenBank Accession No. YP_143344 (Thermus thermophilus HB8); GenBank Accession No. YP_145470 (Thermus thermophilus HB8); GenBank Accession No. YP_005869 (Thermus thermophilus HB27); GenBank Accession No. YP_006059433 (Thermus thermophilus JL-18); GenBank Accession No. YP_005654445 (Thermus sp. CCB_US3_UF1); GenBank Accession No. ZP_03497188 (Thermus aquaticus); GenBank Accession No. YP_003684129 (Meiothermus silvanus DSM 9946); GenBank Accession No. YP_004367049 (Marinithermus hydrothermalis); GenBank Accession No. YP_005641609 (Thermus thermophilus SG0.5JP17-16); GenBank Accession No. YP_006185 (Thermus thermophilus HB27); GenBank Accession No. YP_006059769 (Thermus thermophilus JL-18); YP_003506022 Meiothermus ruber DSM 1279).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence depicted in FIGS. 9A and 9B (SEQ ID NOs: 16-23).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 250 amino acids, or from about 250 amino acids to 264 amino acids, of an amino acid sequence depicted in FIG. 10 (SEQ ID NOs: 24-27).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids (aa) to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 240 amino acids (e.g., 239 aa, 240 aa, 241 aa, 242 aa, 243 aa, 244 aa), from about 240 amino acids to about 250 amino acids, from about 250 amino acids to about 260 amino acids (e.g., 264 amino acids), from about 260 amino acids to about 275 amino acids (e.g., 277 amino acids), or from about 275 amino acids to 314 amino acids, of an amino acid sequence depicted in FIGS. 11A-F or FIG. 12 (SEQ ID NOs: 19, 24, 31, 33, 35, 37, and 39).
In some cases, the substrate recognized by a Cas6 polypeptide comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, nucleotide sequence identity to one of the following sequences:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



and



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′.






In some cases, the substrate recognized by a Cas6 polypeptide comprises one of the following sequences:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′,






or a “minimal” sequence depicted in FIGS. 11A-F (SEQ ID NOs: 28-29, 30, 32, 34, 36, and 38).
An RNA substrate recognized by a Cas6 polypeptide can have a length of from about 15 nucleotides (nt) to about 20 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt), from about 20 nt to about 25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt), from about 25 nt to about 30 nt (e.g., 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt), from about 30 nt to about 35 nt (e.g., 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt), from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, or more than 50 nt.
Suitable Cas5 polypeptides are provided in, e.g., GenBank Accession No. YP_009170 (Desulfovibrio vulgaris Cas5); GenBank Accession No. YP_004513910 (Methylomonas methanica MC09 Cas5); GenBank Accession No. YP_004496339 (Desulfotomaculum carboxydivorans Cas5); GenBank Accession No. ZP_05883174 (Vibrio metchnikovii Cas5); GenBank Accession No. YP_001174211 (Pseudomonas stutzeri Cas5); etc.
In some embodiments, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence depicted in FIG. 15 (SEQ ID NOs: 13 and 41-45), or in FIGS. 16A-E (SEQ ID NOs: 13 and 46-49).
In some cases, the substrate recognized by a variant Cas5 polypeptide comprises one of the following sequences:









(SEQ ID NO: 8)



5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;



 



(SEQ ID NO: 9)



5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;



 



(SEQ ID NO: 10)



5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;



 



(SEQ ID NO: 11)



5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;



and



 



(SEQ ID NO: 12)



5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′,






or a “minimal” sequence depicted in FIGS. 16A-E (SEQ ID NOs: 8-12).
An RNA substrate recognized by a Cas5 polypeptide can have a length of from about 15 nucleotides (nt) to about 20 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt), from about 20 nt to about 25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt), from about 25 nt to about 30 nt (e.g., 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt), from about 30 nt to about 35 nt (e.g., 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt), from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, or more than 50 nt.
The target polyribonucleotide to be detected can be present in a sample, e.g., a biological sample such as blood, a blood product (e.g., plasma), urine, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, a tissue, cells, etc. The target polyribonucleotide can be isolated or purified. The target polyribonucleotide can be a messenger RNA (mRNA), a viral RNA, bacterial RNA, parasite RNA, or other RNA species. Viral RNAs include, but are not limited to, any member of the Flaviviridae, e.g., hepatitis C virus, Dengue virus, Yellow Fever Virus, West Nile Virus, etc.; any member of Retroviridae; an immunodeficiency virus (e.g., human immunodeficiency virus); etc.
The target polyribonucleotide to be detected can be present in a cell of a multicellular organism (or can be obtained from a cell of a multicellular organism).
The target polyribonucleotide to be detected can be present in or obtained from a cell or organism of any of the six kingdoms, e.g., Bacteria (e.g., Eubacteria); Archaebacteria; Protista; Fungi; Plantae; and Animalia. Suitable sources of target polyribonucleotides include plant-like members of the kingdom Protista, including, but not limited to, algae (e.g., green algae, red algae, glaucophytes, cyanobacteria); fungus-like members of Protista, e.g., slime molds, water molds, etc.; animal-like members of Protista, e.g., flagellates (e.g., Euglena), amoeboids (e.g., amoeba), sporozoans (e.g, Apicomplexa, Myxozoa, Microsporidia), and ciliates (e.g., Paramecium). Suitable sources of target polyribonucleotides include members of the kingdom Fungi, including, but not limited to, members of any of the phyla: Basidiomycota (club fungi; e.g., members of Agaricus, Amanita, Boletus, Cantherellus, etc.); Ascomycota (sac fungi, including, e.g., Saccharomyces); Mycophycophyta (lichens); Zygomycota (conjugation fungi); and Deuteromycota. Suitable sources of target polyribonucleotides include members of the kingdom Plantae, including, but not limited to, members of any of the following divisions: Bryophyta (e.g., mosses), Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g., liverworts), Lycophyta (e.g., club mosses), Sphenophyta (e.g., horsetails), Psilophyta (e.g., whisk ferns), Ophioglossophyta, Pterophyta (e.g., ferns), Cycadophyta, Gingkophyta, Pinophyta, Gnetophyta, and Magnoliophyta (e.g., flowering plants). Suitable sources of target polyribonucleotides include members of the kingdom Animalia, including, but not limited to, members of any of the following phyla: Porifera (sponges); Placozoa; Orthonectida (parasites of marine invertebrates); Rhombozoa; Cnidaria (corals, anemones, jellyfish, sea pens, sea pansies, sea wasps); Ctenophora (comb jellies); Platyhelminthes (flatworms); Nemertina (ribbon worms); Ngathostomulida (jawed worms); Gastrotricha; Rotifera; Priapulida; Kinorhyncha; Loricifera; Acanthocephala; Entoprocta; Nemotoda; Nematomorpha; Cycliophora; Mollusca (mollusks); Sipuncula (peanut worms); Annelida (segmented worms); Tardigrada (water bears); Onychophora (velvet worms); Arthropoda (including the subphyla: Chelicerata, Myriapoda, Hexapoda, and Crustacea, where the Chelicerata include, e.g., arachnids, Merostomata, and Pycnogonida, where the Myriapoda include, e.g., Chilopoda (centipedes), Diplopoda (millipedes), Paropoda, and Symphyla, where the Hexapoda include insects, and where the Crustacea include shrimp, hill, barnacles, etc.; Phoronida; Ectoprocta (moss animals); Brachiopoda; Echinodermata (e.g. starfish, sea daisies, feather stars, sea urchins, sea cucumbers, brittle stars, brittle baskets, etc.); Chaetognatha (arrow worms); Hemichordata (acorn worms); and Chordata. Suitable members of Chordata include any member of the following subphyla: Urochordata (sea squirts; including Ascidiacea, Thaliacea, and Larvacea); Cephalochordata (lancelets); Myxini (hagfish); and Vertebrata, where members of Vertebrata include, e.g., members of Petromyzontida (lampreys), Chondrichthyces (cartilaginous fish), Actinopterygii (ray-finned fish), Actinista (coelocanths), Dipnoi (lungfish), Reptilia (reptiles, e.g., snakes, alligators, crocodiles, lizards, etc.), Ayes (birds); and Mammalian (mammals). Suitable plants include any monocotyledon and any dicotyledon.
Thus, e.g., a target polyribonucleotide can be present in or obtained from cells from organisms that include, but are not limited to, a protozoan, a plant, a fungus, an algal cell, a yeast cell, a reptile, an amphibian, a mammal, a marine microorganism, a marine invertebrate, an arthropod, an isopod, an insect, an arachnid, an archaebacterium, and a eubacterium.
A target polyribonucleotide can be present in or obtained from a non-human embryo, e.g., a Drosophila embryo; a zebrafish embryo; a mouse embryo; etc.
A target polyribonucleotide can be present in or obtained from a stem cell, e.g., an in vitro stem cell; a non-human stem cell; etc. Suitable stem cells include embryonic stem cells, adult stem cells, and induced pluripotent stem (iPS) cells.
In some embodiments, target polyribonucleotide will be isolated from a tissue taken from an organism; from a particular cell or group of cells isolated from an organism; etc. For example, where the organism is a plant, the target polyribonucleotide will in some embodiments be isolated from the xylem, the phloem, the cambium layer, leaves, roots, etc. Where the organism is an animal, the target polyribonucleotide will in some embodiments be isolated from a particular tissue (e.g., lung, liver, heart, kidney, brain, spleen, skin, fetal tissue, etc.), or a particular cell type (e.g., neuronal cells, epithelial cells, endothelial cells, astrocytes, macrophages, glial cells, islet cells, T lymphocytes, B lymphocytes, etc.).
Methods of Regulating Production of a Target RNA
The present disclosure provides a method of regulating production of a target RNA in a cell. The method generally involves contacting a genetically modified host cell with an agent that activates an inducible promoter, where the genetically modified host cell is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding an enzyme that catalyzes cleavage at a sequence-specific cleavage site in a substrate polyribonucleotide, where the enzyme-encoding nucleotide sequence is operably linked to the inducible promoter, and where, upon activation of the inducible promoter, the enzyme is produced in the cell and cleaves said target RNA from a precursor RNA.
FIG. 20 provides a schematic depiction of an exemplary method of regulating production of a target RNA. In FIG. 20, an endogenous target RNA is modified to include a Cas5 RNA substrate in the 3′ untranslated region (3′ UTR). Cas5 expression in the host cell leads to binding and cleavage of the RNA substrate. The cleaved RNA now lacks its polyA tail and will be degraded.
For example, in some embodiments, the present disclosure provides a method of regulating production of a target RNA in a eukaryotic cell, where the method involves contacting a genetically modified host cell with an agent that activates an inducible promoter, where the genetically modified host cell is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding an enzymatically active sequence-specific Cas5 endoribonuclease or a Cas6 endoribonuclease that catalyzes cleavage at a sequence-specific cleavage site in a substrate polyribonucleotide, where the enzyme-encoding nucleotide sequence is operably linked to the inducible promoter, and where, upon activation of the inducible promoter, the enzyme is produced in the cell and cleaves said target RNA from a precursor RNA. In some cases, the target RNA species is a regulatory RNA. In some cases, cleavage of said target RNA from a precursor RNA inactivates the precursor RNA.
Endoribonucleases suitable for use in a subject method include an enzymatically active sequence-specific endoribonuclease (e.g., a Cas6 polypeptide or a Cas5 polypeptide).
Suitable Cas6 polypeptide amino acid sequences are provided in, e.g., GenBank Accession No. YP_143344 (Thermus thermophilus HB8); GenBank Accession No. YP_145470 (Thermus thermophilus HB8); GenBank Accession No. YP_005869 (Thermus thermophilus HB27); GenBank Accession No. YP_006059433 (Thermus thermophilus JL-18); GenBank Accession No. YP_005654445 (Thermus sp. CCB_US3_UF1); GenBank Accession No. ZP_03497188 (Thermus aquaticus); GenBank Accession No. YP_003684129 (Meiothermus silvanus DSM 9946); GenBank Accession No. YP_004367049 (Marinithermus hydrothermalis); GenBank Accession No. YP_005641609 (Thermus thermophilus SG0.5JP17-16); GenBank Accession No. YP_006185 (Thermus thermophilus HB27); GenBank Accession No. YP_006059769 (Thermus thermophilus JL-18); YP_003506022 Meiothermus ruber DSM 1279).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence depicted in FIGS. 9A and 9B (SEQ ID NOs: 16-23).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 250 amino acids, or from about 250 amino acids to 264 amino acids, of an amino acid sequence depicted in FIG. 10 (SEQ ID NOs: 24-27).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids (aa) to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 240 amino acids (e.g., 239 aa, 240 aa, 241 aa, 242 aa, 243 aa, 244 aa), from about 240 amino acids to about 250 amino acids, from about 250 amino acids to about 260 amino acids (e.g., 264 amino acids), from about 260 amino acids to about 275 amino acids (e.g., 277 amino acids), or from about 275 amino acids to 314 amino acids, of an amino acid sequence depicted in FIGS. 11A-F or FIG. 12 (SEQ ID NOs: 19, 24, 31, 33, 35, 37, and 39).
Suitable Cas5 polypeptides are provided in, e.g., GenBank Accession No. YP_009170 (Desulfovibrio vulgaris Cas5); GenBank Accession No. YP_004513910 (Methylomonas methanica MC09 Cas5); GenBank Accession No. YP_004496339 (Desulfotomaculum carboxydivorans Cas5); GenBank Accession No. ZP_05883174 (Vibrio metchnikovii Cas5); GenBank Accession No. YP_001174211 (Pseudomonas stutzeri Cas5); etc.
In some embodiments, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence depicted in FIG. 15 (SEQ ID NOs: 13 and 41-45), or in FIGS. 16A-E (SEQ ID NOs: 13 and 46-49).
A suitable inducible promoter can include a promoter that is functional in a eukaryotic cell. Suitable inducible promoters are known in the art. For example, suitable inducible promoters include, but are not limited to, a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1. Suitable inducible promoters include tetracycline-inducible promoters; a metallothionein promoter; tetracycline-inducible promoters, methionine-inducible promoters; and galactose-inducible promoters, which promoters are all well known in the art. Other suitable promoters include the ADH2 alcohol dehydrogenase promoter (repressed in glucose, induced when glucose is exhausted and ethanol is made) and the CUP1 metallothionein promoter (induced in the presence of Cu2+, Zn2+).
Agents that induce any given inducible promoter are known in art. For example, tetracycline-regulatable promoters can be regulated by tetracycline or doxycycline; carbohydrates can be used to induce a carbohydrate-inducible promoter (e.g., galactose for a galactose-inducible promoter); methionine can be used to induce a methionine-inducible promoter; metals can be used to induce a metallothionein promoter.
The target RNA can be a regulatory RNA. Regulator RNAs are well known in the art and include, e.g., micro-RNAs, short hairpin RNAs (shRNAs), and the like.
In some embodiments, cleavage of the target RNA from a precursor RNA inactivates the precursor RNA.
The genetically modified host cell can be an in vitro cell, e.g., a prokaryotic cell, or a eukaryotic cell (e.g., a mammalian cell, including primary cells, transformed cell lines, and the like). The genetically modified host cell can be an in vivo cell. In some embodiments, the in vivo cell is a non-human cell.
The genetically modified host cell can be a cell of a multicellular organism (or can be obtained from a cell of a multicellular organism).
The genetically modified host cell can be a cell obtained from or present in an organism of any of the six kingdoms, e.g., Bacteria (e.g., Eubacteria); Archaebacteria; Protista; Fungi; Plantae; and Animalia. Suitable organisms include plant-like members of the kingdom Protista, including, but not limited to, algae (e.g., green algae, red algae, glaucophytes, cyanobacteria); fungus-like members of Protista, e.g., slime molds, water molds, etc.; animal-like members of Protista, e.g., flagellates (e.g., Euglena), amoeboids (e.g., amoeba), sporozoans (e.g, Apicomplexa, Myxozoa, Microsporidia), and ciliates (e.g., Paramecium). Suitable organisms include members of the kingdom Fungi, including, but not limited to, members of any of the phyla: Basidiomycota (club fungi; e.g., members of Agaricus, Amanita, Boletus, Cantherellus, etc.); Ascomycota (sac fungi, including, e.g., Saccharomyces); Mycophycophyta (lichens); Zygomycota (conjugation fungi); and Deuteromycota. Suitable organisms include members of the kingdom Plantae, including, but not limited to, members of any of the following divisions: Bryophyta (e.g., mosses), Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g., liverworts), Lycophyta (e.g., club mosses), Sphenophyta (e.g., horsetails), Psilophyta (e.g., whisk ferns), Ophioglossophyta, Pterophyta (e.g., ferns), Cycadophyta, Gingkophyta, Pinophyta, Gnetophyta, and Magnoliophyta (e.g., flowering plants). Suitable organisms include members of the kingdom Animalia, including, but not limited to, members of any of the following phyla: Porifera (sponges); Placozoa; Orthonectida (parasites of marine invertebrates); Rhombozoa; Cnidaria (corals, anemones, jellyfish, sea pens, sea pansies, sea wasps); Ctenophora (comb jellies); Platyhelminthes (flatworms); Nemertina (ribbon worms); Ngathostomulida (jawed worms); Gastrotricha; Rotifera; Priapulida; Kinorhyncha; Loricifera; Acanthocephala; Entoprocta; Nemotoda; Nematomorpha; Cycliophora; Mollusca (mollusks); Sipuncula (peanut worms); Annelida (segmented worms); Tardigrada (water bears); Onychophora (velvet worms); Arthropoda (including the subphyla: Chelicerata, Myriapoda, Hexapoda, and Crustacea, where the Chelicerata include, e.g., arachnids, Merostomata, and Pycnogonida, where the Myriapoda include, e.g., Chilopoda (centipedes), Diplopoda (millipedes), Paropoda, and Symphyla, where the Hexapoda include insects, and where the Crustacea include shrimp, krill, barnacles, etc.; Phoronida; Ectoprocta (moss animals); Brachiopoda; Echinodermata (e.g. starfish, sea daisies, feather stars, sea urchins, sea cucumbers, brittle stars, brittle baskets, etc.); Chaetognatha (arrow worms); Hemichordata (acorn worms); and Chordata. Suitable members of Chordata include any member of the following subphyla: Urochordata (sea squirts; including Ascidiacea, Thaliacea, and Larvacea); Cephalochordata (lancelets); Myxini (hagfish); and Vertebrata, where members of Vertebrata include, e.g., members of Petromyzontida (lampreys), Chondrichthyces (cartilaginous fish), Actinopterygii (ray-finned fish), Actinista (coelocanths), Dipnoi (lungfish), Reptilia (reptiles, e.g., snakes, alligators, crocodiles, lizards, etc.), Ayes (birds); and Mammalian (mammals). Suitable plants include any monocotyledon and any dicotyledon.
Thus, e.g., a genetically modified host cell can be a cell obtained from or present in a protozoan, a plant, a fungus, an algal cell, a yeast, a reptile, an amphibian, a mammal, a marine microorganism, a marine invertebrate, an arthropod, an isopod, an insect, an arachnid, an archaebacterium, and a eubacterium.
Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.
The genetically modified host cell can be a cell obtained from or present in a non-human embryo, e.g., a Drosophila embryo; a zebrafish embryo; a mouse embryo; etc.
The genetically modified host cell can be a stem cell, e.g., an in vitro stem cell; a non-human stem cell; etc. Suitable stem cells include embryonic stem cells, adult stem cells, and induced pluripotent stem (iPS) cells.
Methods of Isolating a Target Nucleic Acid
The present disclosure provides methods of isolating a target nucleic acid from a mixed population of nucleic acids. The methods generally involve: a) contacting a mixed population of nucleic acids with an immobilized sequence-specific, enzymatically inactive endoribonuclease, wherein the mixed population of nucleic acids includes a target nucleic acid comprising a “tag” (or “recognition”) nucleotide sequence that is specifically bound by the immobilized sequence-specific, enzymatically inactive endoribonuclease, such that the target nucleic acid comprising the tag nucleotide sequence (“tagged target nucleic acid”) binds to the immobilized sequence-specific, enzymatically inactive endoribonuclease, forming a tagged target nucleic acid/immobilized sequence-specific enzymatically active endoribonuclease complex, wherein the contacting step takes place in a liquid solution (a “binding solution”); and b) adding imidazole to the liquid solution to a final concentration of from about 100 mM to about 500 mM (e.g., from about 100 mM to about 150 mM, from about 150 mM to about 200 mM, from about 200 mM to about 250 mM, from about 250 mM to about 300 mM, from about 300 mM to about 350 mM, from about 350 mM to about 400 mM, from about 400 mM to about 450 mM, or from about 450 mM to about 500 mM), thereby forming a reactivation solution that enzymatically reactivates the enzymatically inactive endoribonuclease such that the endoribonuclease becomes enzymatically active and cleaves the target nucleic acid from the “tag” nucleotide sequence, thereby releasing the target nucleic acid. FIG. 19 is a schematic representation of an exemplary embodiment of a subject method for isolating a target RNA.
The method can further include one or more washing steps. For example, after step (a) and before step (b), the immobilized sequence-specific, enzymatically inactive endoribonuclease that comprises a bound target nucleic acid comprising a “tag” nucleotide sequence can be washed one or more times with the binding solution, such that the target nucleic acid remains bound to the sequence-specific, enzymatically inactive endoribonuclease, and any unbound nucleic acids are washed away.
The mixed population of nucleic acids can include RNA and DNA. The target nucleic acid is an RNA that comprises a “tag” or “recognition” nucleotide sequence that is specifically bound by the sequence-specific endoribonuclease. In its enzymatically inactive state (“uninduced” state), the endoribonuclease can bind, but cannot cleave, the tagged target RNA. In its enzymatically active state (“induced” state) (e.g., in the presence of imidazole in a concentration of from about 100 mM to about 500 mM), the endoribonuclease can both bind and cleave the recognition nucleotide sequence in the tagged target nucleic acid, thereby releasing the target nucleic acid from the tag.
The binding solution can include a buffer and a salt; and lacks imidazole. The reactivation solution can include imidazole in a final concentration of from about 100 mM to about 500 mM, e.g., from about 100 mM to about 150 mM, from about 150 mM to about 200 mM, from about 250 mM to about 350 mM, from about 350 mM to about 400 mM, or from about 400 mM to about 500 mM. The presence of imidazole reactivates the sequence-specific, enzymatically inactive endoribonuclease such that the endoribonuclease becomes enzymatically active, e.g., the endoribonuclease exhibits at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more than 95%, of wild-type sequence-specific endoribonuclease (e.g., an amino acid sequence as depicted in any of FIGS. 9, 10, 11, 12, 15, and 16).
Endoribonucleases suitable for use in a subject method include an enzymatically active sequence-specific endoribonuclease (e.g., a Cas6 polypeptide or a Cas5 polypeptide).
Suitable Cas6 polypeptide amino acid sequences that can be modified to be enzymatically in active are provided in, e.g., GenBank Accession No. YP_143344 (Thermus thermophilus HB8); GenBank Accession No. YP_145470 (Thermus thermophilus HB8); GenBank Accession No. YP_005869 (Thermus thermophilus HB27); GenBank Accession No. YP_006059433 (Thermus thermophilus JL-18); GenBank Accession No. YP_005654445 (Thermus sp. CCB_US3_UF1); GenBank Accession No. ZP_03497188 (Thermus aquaticus); GenBank Accession No. YP_003684129 (Meiothermus silvanus DSM 9946); GenBank Accession No. YP_004367049 (Marinithermus hydrothermalis); GenBank Accession No. YP_005641609 (Thermus thermophilus SG0.5JP17-16); GenBank Accession No. YP_006185 (Thermus thermophilus HB27); GenBank Accession No. YP_006059769 (Thermus thermophilus JL-18); YP_003506022 Meiothermus ruber DSM 1279).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence depicted in FIGS. 9A and 9B (SEQ ID NOs: 16-23).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 250 amino acids, or from about 250 amino acids to 264 amino acids, of an amino acid sequence depicted in FIG. 10 (SEQ ID NOs: 24-27).
In some cases, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids (aa) to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, from about 225 amino acids to about 240 amino acids (e.g., 239 aa, 240 aa, 241 aa, 242 aa, 243 aa, 244 aa), from about 240 amino acids to about 250 amino acids, from about 250 amino acids to about 260 amino acids (e.g., 264 amino acids), from about 260 amino acids to about 275 amino acids (e.g., 277 amino acids), or from about 275 amino acids to 314 amino acids, of an amino acid sequence depicted in FIGS. 11A-F or FIG. 12 (SEQ ID NOs: 19, 24, 31, 33, 35, 37, and 39).
In some cases, the substrate (e.g., the “tag” or recognition sequence) recognized by a Cas6 polypeptide comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, nucleotide sequence identity to one of the following sequences:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



and



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′.






In some cases, the substrate (e.g., the “tag” or recognition sequence) recognized by a Cas6 polypeptide comprises one of the following sequences:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′,






or a “minimal” sequence depicted in FIGS. 11A-F (SEQ ID NOs: 28-29, 30, 32, 34, 36, and 38).
An RNA substrate recognized by a Cas6 polypeptide can have a length of from about 15 nucleotides (nt) to about 20 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt), from about 20 nt to about 25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt), from about 25 nt to about 30 nt (e.g., 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt), from about 30 nt to about 35 nt (e.g., 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt), from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, or more than 50 nt.
Suitable Cas5 polypeptides are provided in, e.g., GenBank Accession No. YP_009170 (Desulfovibrio vulgaris Cas5); GenBank Accession No. YP_004513910 (Methylomonas methanica MC09 Cas5); GenBank Accession No. YP_004496339 (Desulfotomaculum carboxydivorans Cas5); GenBank Accession No. ZP_05883174 (Vibrio metchnikovii Cas5); GenBank Accession No. YP_001174211 (Pseudomonas stutzeri Cas5); etc.
In some embodiments, an enzymatically active, sequence-specific endoribonuclease comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity with a contiguous stretch of from about 150 amino acids to about 175 amino acids, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 225 amino acids, or from about 225 amino acids to the full length (e.g., 235 amino acids, 236 amino acids, 237 amino acids, 238 amino acids, 239 amino acids, 247 amino acids, 251 amino acids, 262 amino acids, or 267 amino acids) of an amino acid sequence depicted in FIG. 15 (SEQ ID NOs: 13 and 41-45), or in FIGS. 16A-E (SEQ ID NOs: 13 and 46-49).
In some cases, the substrate (e.g., the “tag” or recognition sequence) recognized by a Cas5 polypeptide comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, nucleotide sequence identity to one of the following sequences:









(SEQ ID NO: 8)



5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;



 



(SEQ ID NO: 9)



5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;



 



(SEQ ID NO: 10)



5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;



 



(SEQ ID NO: 11)



5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;



and



 



(SEQ ID NO: 12)



5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′,






or a “minimal” sequence depicted in FIGS. 16A-E (SEQ ID NOs: 8-12).
In some cases, the substrate (e.g., the “tag” or recognition sequence) recognized by a Cas5 polypeptide comprises one of the following sequences:









(SEQ ID NO: 8)



5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;



 



(SEQ ID NO: 9)



5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;



 



(SEQ ID NO: 10)



5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;



 



(SEQ ID NO: 11)



5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;



and



 



(SEQ ID NO: 12)



5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′,






or a “minimal” sequence depicted in FIGS. 16A-E (SEQ ID NOs: 8-12).
An RNA substrate recognized by a variant Cas5 polypeptide of the present disclosure can have a length of from about 15 nucleotides (nt) to about 20 nt (e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt), from about 20 nt to about 25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt), from about 25 nt to about 30 nt (e.g., 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt), from about 30 nt to about 35 nt (e.g., 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt), from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, or more than 50 nt.
The “tag” or “recognition” nucleotide sequence can be introduced into a nucleic acid using standard recombinant methods. Thus, the tagged target nucleic acid will include a tag that is enzymatically cleaved, thereby releasing the target nucleic acid.
In some embodiments, the tagged target nucleic acid (RNA) will have one or more polypeptides bound thereto. A tagged target RNA that has one or more polypeptides bound thereto is referred to herein as a RNA protein complex. Thus, in some embodiments, the target RNA that is isolated using a subject method is an RNA protein complex. In some embodiments, a subject method can further comprise analyzing the polypeptide(s) bound to the isolated target RNA.
A subject method provides for isolation of a target RNA (or RNA protein complex). In some embodiments, a subject method provides for purification of a target RNA (or RNA protein complex) such that the target RNA (or RNA protein complex) is at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or greater than 98% pure.
In some embodiments, a protein bound to a target RNA in a target RNA/protein complex can be eluted from the RNA/protein complex. The eluted protein can be further characterized, e.g., by sequencing, enzymatic digestion, a functional assay, etc.
The mixed population of nucleic acids can be present in a cell lysate. For example, an expression vector comprising a nucleotide sequence encoding a tagged target RNA is introduced into a cell (e.g., in vitro or in vivo), such that the cell synthesizes the tagged target RNA. A lysate is made from the cell and the lysate (optionally subjected to one or more steps to enrich for nucleic acids) is applied to the immobilized sequence-specific enzymatically-inactive endoribonuclease.
The sequence-specific enzymatically-inactive endoribonuclease can be immobilized on any of a variety of insoluble support. Suitable insoluble supports include, but are not limited to agarose beads, magnetic beads, a test strip, a multi-well dish, and the like. The insoluble support can comprise a variety of substances (glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agaroses, and magnetite) and can be provided in a variety of forms, including, e.g., agarose beads, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, wells of reaction trays (e.g., multi-well plates), plastic tubes, etc.
The present disclosure also provides a method of isolating a polypeptide that binds a target RNA, where the method comprises: a) contacting an immobilized complex with a liquid solution comprising a polypeptide that binds the target RNA, where the immobilized complex comprises the variant Cas5 endoribonuclease or the variant Cas6 endoribonuclease and a tagged target RNA comprising a recognition nucleotide sequence that is specifically bound by the variant Cas5 or Cas6 endoribonuclease, where said contacting results in binding of the polypeptide to the target RNA, where said contacting is carried out in a binding solution lacking imidazole; and b) eluting the bound polypeptide.
Kits
The present disclosure also provides kits for determining the nucleotide sequence of a target polyribonucleotide. The present disclosure provides kits for carrying out sequence-specific cleavage of a substrate polyribonucleotide. The present disclosure provides kits for carrying out detection of an RNA sequence in a target polyribonucleotide. The present disclosure provides kits for carrying out isolation of a target RNA. The present disclosure provides kits for carrying out isolation of a polypeptide that binds a target RNA.
Kits for Carrying Out Direct Sequencing of a Polyribonucleotide
A subject kit for carrying out direct sequencing of a polyribonucleotide includes at least a subject sequence-specific, enzymatically inactive endoribonuclease, where the sequence-specific, enzymatically inactive endoribonuclease is purified. In some embodiments, the enzymatically inactive, sequence-specific endoribonuclease is linked to an acceptor molecule or a donor molecule, for FRET detection.
A subject kit for carrying out direct sequencing of a polyribonucleotide includes at least a subject sequence-specific, enzymatically inactive endoribonuclease; and can include one or more additional components, where the one or more additional components can be: 1) a buffer; 2) a probe oligonucleotide comprising a defined sequence; 3) a probe oligonucleotide comprising a defined sequence, where the probe oligonucleotide is linked to an acceptor molecule or a donor molecule, for FRET detection; 4) an insoluble support, for linking to a target polyribonucleotide; 5) a positive control polyribonucleotide, where the positive control polyribonucleotide comprises a known nucleotide sequence; 6) a positive control probe oligonucleotide that binds to and forms a duplex with the known sequence of the positive control polyribonucleotide.
In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
Kits for Carrying Out Sequence-Specific Cleavage of a Substrate Polyribonucleotide
A subject kit for carrying out sequence-specific cleavage of a substrate polyribonucleotide includes at least a purified sequence-specific endoribonuclease and/or a nucleic acid comprising a nucleotide sequence encoding the sequence-specific endoribonuclease. A subject kit for carrying out sequence-specific cleavage of a substrate polyribonucleotide can include, in addition to a purified sequence-specific endoribonuclease (and/or a nucleic acid comprising a nucleotide sequence encoding the sequence-specific endoribonuclease), one or more additional components. Suitable additional components include, e.g., a buffer; a polyribonucleotide substrate that serves as a positive control; polyribonucleotide size standards; a negative control substrate; and the like. The components can each be in separate containers. The kit can further include one or more positive and negative controls.
In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
Kits for Carrying Out Detection of a Sequence in a Target Polyribonucleotide
A subject kit for carrying out detection of a sequence in a target polyribonucleotide (e.g., for carrying out detection of a polyribonucleotide) can include an oligonucleotide probe comprising a known sequence. In some embodiments, the kit will include an oligonucleotide probe comprising a known sequence and comprising a detectable moiety, e.g., a polypeptide that can be detected using an immunological assay; a fluorescent protein; a luciferin; etc. The kit can further include a positive control polyribonucleotide that comprises a nucleotide sequence capable of forming a duplex with the oligonucleotide probe. The kit can further include an enzymatically active, sequence-specific endoribonuclease that specifically detects and cleaves a duplex formed by the oligonucleotide probe and a target polyribonucleotide. The kit can further include one or more of a buffer; components for detecting the detectable moiety; a test strip; and the like. The kit can further include one or more positive and negative controls.
In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
Kits for Carrying Out Isolation of a Target RNA
A subject kit for carrying out isolation (e.g., purification) of a target RNA can include one or more of: 1) a subject sequence-specific, enzymatically inactive endoribonuclease; 2) an expression construct comprising a “tag” nucleotide sequence, i.e., a nucleotide sequence that is specifically bound by the sequence-specific, enzymatically inactive endoribonuclease, where a nucleotide sequence encoding a target RNA of choice can be inserted 3′ of the “tag” nucleotide sequence; and 3) imidazole. The sequence-specific, enzymatically inactive endoribonuclease can be immobilized on an insoluble support. The kit can further include a liquid composition for contacting a mixed population of nucleic acids with the immobilized sequence-specific, enzymatically inactive endoribonuclease. The kit can further include a wash buffer. The kit can further include one or more positive and negative controls. A positive control could include an expression vector comprising a nucleotide sequence encoding a tagged target RNA, where the tag is specifically bound by the sequence-specific, enzymatically inactive endoribonuclease. The components can each be in separate containers.
For example, a subject kit can include a subject sequence-specific, enzymatically inactive endoribonuclease. A subject kit can further include a recombinant expression vector comprising, in order from 5′ to 3′ and in operable linkage: a) a nucleotide sequence encoding an RNA substrate that is specifically bound by a subject variant Cas5 or Cas6 endoribonuclease; and b) a multiple cloning site suitable for insertion of a nucleic acid encoding the target RNA. The nucleotide sequence encoding the RNA substrate can be operably linked to a promoter. In some instances, the promoter is an inducible promoter. The RNA substrate can comprise a substrate nucleotide sequence, as described above. In some cases, the recombinant expression vector comprises, inserted into the multiple cloning site, a nucleotide sequence encoding the target RNA. The kit can further include a buffer that lacks imidazole. The kit can further include imidazole or an imidazole solution. The kit can further include one or more wash buffers. In some cases, the kit will include a positive control expression vector. The variant Cas5 or Cas6 endoribonuclease can be immobilized on an insoluble support, where suitable insoluble supports include, but are not limited to agarose beads, magnetic beads, a test strip, a multi-well dish, and the like. The insoluble support can comprise a variety of substances (glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agaroses, and magnetite) and can be provided in a variety of forms, including, e.g., agarose beads, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, wells of reaction trays (e.g., multi-well plates), plastic tubes, etc.
In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
Example 1
Generation and Characterization of Cas6 Mutants
Cas6 proteins TTHA0078 (TtCas6A) (SEQ ID NO: 19) and TTHB231(TtCas6B) (SEQ ID NO: 24) were characterized.
Crystal Structure of TTHA0078 with an RNA Substrate
Protein TTHA0078 was grown under the following conditions: 0.1 M Bis_Tris-Propane pH 6.5; 14-18% poly(ethylene glycol) (PEG) 3350; and 0.2 M Sodium Sulfate.
On the gel sizing column, the crystal consisted of two copies of the protein. FIG. 1 shows Cas6 protein TTHA0078 bound to nucleotides 15-30 of Tt_R1 containing a 2′ deoxy at position G28. The putative catalytic residue H37 is highlighted in red. The unbound part of the homodimer does not show density for residues 32-40, which indicates that the loop becomes ordered only when it's bound to the substrate.
FIG. 2 provides a close-up of catalytic residue H37 in close proximity to the RNA backbone between G28 and A29. The RNA substrate contained a 2′ deoxy at position G28 to avoid cleavage.
FIG. 3 depicts involvement of R129 of TTHA0078 in multiple base specific interactions with nucleotides G26, G27 and G28.
FIG. 21 shows cleavage of target RNAs by Thermus thermophilus Cas6 proteins at a position immediately downstream of the stem loop structure. Cleavage positions are indicated with a red arrow. Synthetic RNAs (1 microM) were incubated with recombinant Cas6 proteins (10 microM) in 150 mM KCl and 20 mM Hepes pH 7.5 at 65° C. for 5 min. Reaction products were isolated by phenol-chloroform extraction, resolved by electrophoresis on a 15% denaturing polyacrylamide gel, and visualized by staining with ethidium bromide.
Characterization of TTHA0078 and TTHB231 Binding Affinity
It was found that TTHA0078 and TTHB231 bind their substrates with different affinities and discriminate between targets; and that the stem and the 5′ handle are both of major importance for binding of substrate.
Biochemical analyses were performed on TTHA0078 and TTHB231. TTHA0078 binds its substrates with affinities of  ̃60 pM (Tt_R1) and  ̃1.6 nM (Tt_R2) and therefore strongly favors binding of Tt_R1 over Tt_R2 while binding affinities of  ̃4 nM (Tt_R1) and  ̃15 nM (Tt_R2) indicate the opposite for TTHB231. Biochemical analysis focused on TTHA0078 and its interactions with Tt_R1. To determine the importance of the stem for Protein-RNA binding, a series of mutated RNAs, which had an A-U instead of a C-G at different positions of the 4 nt long stem, was created.
The data presented in FIG. 4 show that mutations at different parts of the stem have significant impact on binding affinity of TTHA0078 to its substrate.
In contrast to comparable previous studies, binding of TTHA0078 to the RNA in absence of its 5′ handle was not observed. To determine which parts of the 5′ handle have a major effect on binding affinity, a series of RNAs that are gradually truncated was created; and the binding affinities of the truncated forms was determined. In FIG. 5, the number at position n indicates that RNA of that length binds x-fold stronger than the next shorter RNA. For example, nts [7 . . . 29] bind 2.3 fold stronger to TTHA0078 than nts [8 . . . 29].
TTHA0078 and TTHB231 are Single Turnover Enzymes.
To determine if the two Cas6 proteins are capable of cleaving multiple substrates an assay was performed in which the enzyme was present in sub-stoichiometric amounts. Both enzymes seem to be single turnover enzymes, since the cleaved fractions greatly fit a single exponential decay curve that flattens out proportional to the stoichiometric excess of RNA. The data are shown in FIGS. 6A and 6B. FIGS. 6A and 6B are graphs showing the results of the cleavage assay in multiple turnover conditions and gradually increased stoichiometric excess of substrate. FIG. 6A shows the data for TTHB231 with Tt_Repeat2 substrate; FIG. 6B shows the data for TTHA0078 with Tt_Repeat 1 substrate. At higher excess of RNA the percentage of cleaved RNA flattens out significantly below 100% even after 120 minutes of incubation.
Example 2
Cas5 Mutant
The bacterium Desulfovibrio vulgaris (Dvu) possesses a CRISPR system of the subtype I-C. Each of its four uncharacterized Cas genes (Cas5, Cas8c, Cas7, and Cas4) was tagged with maltose-binding protein (MBP), purified, and tested for in vitro cleavage of the D. vulgaris CRISPR RNA hairpin. Cas5 was identified as the endoribonuclease of this subtype I-C system.
The amino acid sequence of Dvu Cas5 is as follows:







(SEQ ID NO: 13)


MTHGAVKTYGIRLRVWGDYACFTRPEMKVERVSYDVMPPSAARGILEA


 


IHWKPAIRWIVDRIHVLRPIVFDNVRRNEVSSKIPKPNPATAMRDRKP


 


LYFLVDDGSNRQQRAATLLRNVDYVIEAHFELTDKAGAEDNAGKHLDI


 


FRRRARAGQSFQQPCLGCREFPASFELLEGDVPLSCYAGEKRDLGYML


 


LDIDFERDMTPLFFKAVMEDGVITPPSRTSPEVRA,






where His-141 is bolded and underlined.
As shown from the cleavage assay data provided in FIG. 11, Cas5 is an endoribonuclease that cleaves pre-crRNA.
As shown in FIG. 12, comparison between the Cas5-processed RNA product and a hydrolysis ladder and T1 digest demonstrates that Cas5 cleaves at the bottom of the pre-crRNA stem loop, after base G21.
Electrostatic mapping of a known Cas5 family protein structure (see PDB entry 3KG4) revealed a positively charged region of the protein suggestive of a nucleic acid binding pocket. Conservation mapping based on BLAST results of the D. vulgaris Cas5 protein sequence and displayed on the 3KG4 protein structure showed high conservation of residues in the putative substrate-binding site.
Histidine 141 was chosen as a target for site-directed mutagenesis based on its location within the putative RNA binding region.
Data are shown in FIGS. 13 and 14 (FIGS. 14A and 14B). FIG. 13 depicts cleavage of pre-crRNA by Cas5. FIGS. 14A and 14B present data showing that Cas5 cleaves pre-crRNA at base G21.
Example 3
Evolution of CRISPR RNA Recognition and Processing by Cas6 Endonucleases
Results
TtCas6A and TtCas6B Bind and Cleave CRISPR Repeats R1 and R3 and Retain their Product RNAs after Cleavage
The genome of Thermus thermophilus HB8 harbors eleven CRISPR loci containing three distinct types of repeats, termed R1-3 herein (FIG. 22A; FIG. 26). All CRISPR loci are constitutively transcribed (Agari et al, 2010; Juranek et al, 2012). Irrespective of the CRISPR locus of origin, all crRNAs in T. thermophilus contain a 5′-terminal 8-nucleotide handle derived from the repeat sequence that results from sequence-specific cleavage at the 3′ end of the hairpin structure predicted in each crRNA repeat (Juranek et al, 2012). Three Cas6-superfamily genes have been identified in the T. thermophilus genome: TTHB231, TTHB192 and TTHA0078 (FIG. 26A). Previous structural and biochemical studies showed that the TTHB192 gene product, a member of the Cas6e family, cleaves the R2 repeat found in the two spacer/repeat arrays flanking the type I-E (E. coli subtype) Cas operon in the T. thermophilus genome. While TTHB231 is embedded in a hybrid type I operon flanked by R3 repeat loci, TTHA0078 is not part of any CRISPR locus.
To determine whether TTHA0078 and TTHB231 (hereafter referred to as TtCas6A and TtCas6B, respectively) are responsible for processing pre-crRNAs originating from R1 and/or R3 repeat loci, recombinant TtCas6A and TtCas6B proteins were expressed and purified from Escherichia coli and tested for endonucleolytic activity using in vitro transcribed RNAs. Both proteins cleaved R1 and R3 repeat RNAs efficiently, while neither was able to cleave R2 repeat RNA (FIG. 26B). To characterize the binding affinities of TtCas6A and TtCas6B to their cognate crRNA repeats, we performed electrophoretic mobility shift assays (EMSAs) using 5′-[32P]-radiolabeled R1 and R3 repeat RNAs. As endonucleolytic cleavage occurred to completion during the course of the binding reactions, the calculated equilibrium dissociation constants reflect product, rather than substrate, binding. TtCas6A bound to the R1 repeat cleavage product with an apparent Kd of 90±21 pM, while binding to the R3 repeat cleavage product was approximately nine-fold weaker (808±154 pM). TtCas6B bound to R1 and R3 repeats with comparable dissociation constants of 1.96±0.28 nM and 3.90±0.78 nM, respectively (FIG. 1B). The observed high-affinity product binding is consistent with the conclusion that, like many other Cas6-superfamily endonucleases, both TtCas6A and TtCas6B function as single-turnover enzymes. To test this hypothesis, we performed cleavage assays at a range of substrate:enzyme molar ratios, measured the rate of cleavage, and quantified the product yield (FIG. 22C). The cleavage reaction yields scaled proportionally to enzyme concentration at sub-stoichiometric enzyme concentrations, while the apparent first order rate constants remained essentially unchanged, indicating single-turnover catalysis. Collectively, these findings suggest that TtCas6A and TtCas6B are involved in processing precursor transcripts of repeat R1 and R3-containing CRISPR loci in T. thermophilus and that both enzymes likely remain bound to their products following cleavage.
FIG. 22 illustrates that TtCas6A and TtCas6B both cleave repeats R1 and R3 and retain their cleaved products. (A) Sequences and predicted secondary structures of T. thermophilus CRISPR repeats. Sites of cleavage are indicated with blue arrows. TtCas6e (TTHB192) cleaves repeat R2, while TtCas6A (TTHA0078) and TtCas6B (TTHB231) both cleave repeats R1 and R3. (B) Cleavage product binding affinities of TtCas6A and TtCas6B enzymes. Maltose binding protein (MBP)-fused TtCas6A or TtCas6B were bound to in 5′-radiolabelled, vitro transcribed R1 and R3 RNAs. Bound and unbound fractions were resolved by electrophoresis on a native polyacrylamide gel and visualized by phosphorimaging. The data for these and all subsequent binding assays were fit with standard binding isotherms (solid line). Error bars on each data point denote standard error of the mean (SEM) from three independent experiments. (C) Experiments to confirm single turnover. RNA cleavage assays were carried out at indicated protein:RNA ratios. RNA cleavage was monitored using denaturing polyacrylamide gel electrophoresis. The data from these and all subsequent endoribonuclease activity assays were fit with single exponential curves to yield first-order rate constants. (Top to bottom: FIG. 22A, SEQ ID NOs: 1, 2, 55).
FIG. 26 demonstrates that TtCas6A and TtCas6B cleave repeats R1 and R3, but not R2. (A) Diagram of CRISPR loci and cas genes in the genome of Thermus thermophilus HB8 based on the CRISPRdb database (Grissa et al, 2007). The diagram was modified after Agari et al, 2010 (Agari et al, 2010) and includes the present annotation of the T. thermophilus CRISPR loci. cas genes are depicted as white arrowheads and numerals in the arrowheads indicate gene numbers for TTHA (chromosomal) and TTHB (plasmid pTT27) genes, respectively. Gene names are shown above the arrowheads, and the three Cash-encoding genes are colored in teal (TTHA0078), blue (TTHB231) and light green (TTHB192), respectively. CRISPR arrays are depicted as patterned arrowheads and those that share a pattern have the same or similar repeat sequences (R1 to R3). (B) RNA cleavage by TtCas6A and TTCas6B enzymes. In vitro transcribed repeat R1-3 RNAs were incubated with purified recombinant TtCas6A or TtCas6B. Cleavage products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by staining with SYBR Gold.
Crystal Structures of RNA-Bound TtCas6A and TtCas6B Reveal the Mechanism of Substrate Recognition and Cleavage.
To determine how TtCas6A and TtCas6B bind and cleave their RNA substrates, we solved crystal structures of these proteins both alone and in complexes with their cognate RNAs (FIG. 23A, FIG. 30). For TtCas6A, we obtained a structure of the enzyme bound to a substrate mimic based on the R1 repeat sequence, consisting of the R1 stem-loop flanked by two additional nucleotides on either end of the stem. Cleavage of the substrate mimic RNA was prevented by introducing a 2′-deoxyribonucleotide at the G28 position, thereby removing the 2′-hydroxyl nucleophile required for the cleavage reaction. In addition to the substrate mimic complex, a crystal structure of a complex of TtCas6A and a cleaved RNA product was obtained when full-length R1 repeat was bound to wild-type TtCas6A and allowed to undergo cleavage during subsequent complex purification and crystallization. Finally, we determined the crystal structure of the TtCas6A H37A mutant, lacking a critical active site residue, in the absence of bound RNA. For TtCas6B, we determined crystal structures of the wild-type enzyme alone and in complex with a product of the R3 repeat cleavage reaction.
In all crystal structures, both TtCas6A and TtCas6B form crystallographic (RNA-free TtCas6B) or non-crystallographic (all TtCas6A structures and the TtCas6B-product complex) dimers (FIG. 27), consistent with size exclusion chromatography results indicating that both enzymes are dimers in solution. The buried surface area of the TtCas6A dimer is 924 Å2, while the TtCas6B dimer buries 1008 Å2. Strikingly, while the TtCas6B-R3 product crystal structure reveals a 2:2 stoichiometry, both substrate and product TtCas6A-R1 RNA complexes crystallized with an apparent 2:1 stoichiometry, with only one RNA molecule bound to the non-crystallographic TtCas6A dimer. Size exclusion chromatography of TtCas6A and TtCas6B-RNA complexes used for crystallization as well as their absorbance ratios at 280 and 260 nm were indicative of 2:2 stoichiometry. Additionally, both proteins behaved similarly in cleavage assays (FIG. 22C) and no negative cooperativity was observed for TtCas6A in binding assays. The apparent 2:1 stoichiometry of the TtCas6A-RNA complexes is therefore likely a crystallization-induced artifact.
FIG. 27 depicts dimeric structures of TtCas6A and TtCas6B enzymes bound to substrate mimic and product RNAs. Proteins are depicted in ribbon format and colored in teal (TtCas6A) or blue (TTCas6B). Bound RNAs are depicted in cartoon format and colored in yellow. In all cases, the two protein molecules form a non-crystallographic dimer in the asymmetric unit of the crystal. (A) Ribbon diagram showing the 2:1 TtCas6A-R1 substrate mimic complex. (B) Ribbon diagram showing the 2:1 TtCas6A-R1 product complex (C) Ribbon diagram showing the 2:2 TtCas6B-R3 product complex.
Overall, both TtCas6A and TtCas6B adopt double-ferredoxin folds similar to those observed for TtCas6e, PfCas6 and a non-catalytic Cas6 homolog from Pyrococcus horikoshii (FIG. 28A,B). The N-terminal ferredoxin domains of the two TtCas6 proteins also superimpose well with the single ferredoxin fold found in the structure of PaCas6f. The two TtCas6 enzymes are highly similar to each other and superimpose with a root-mean-square deviation (rmsd) of 2.1 Å over 227 Cα atoms, reflecting the high degree of sequence identity (32%) between the two proteins (FIG. 28A,B).
As anticipated, the R1 and R3 repeat RNAs form stem-loop structures. In both proteins, the RNAs bind in a positively charged cleft located between the two ferredoxin folds, as observed in the structures of TtCas6e-R2 repeat complexes. In further analogy with TtCas6e, TtCas6A and TtCas6B complexes also insert a beta-hairpin from their C-terminal ferredoxin domains into the major groove of the dsRNA stems (FIG. 23A). In the TtCas6A-substrate mimic complex, the two nucleotides downstream of the scissile phosphate are recognized in a sequence-specific manner through base-specific interactions (described in detail below). The structures of the RNA product complexes of both TtCas6A and TtCas6B reveal 2′-3′ cyclic phosphate groups in the respective active sites, consistent with a catalytic mechanism involving nucleophilic attack by the 2′-hydroxyl of the upstream nucleotide (G28) (FIG. 23A, B).
The active site of TtCas6A is located in a pocket surrounded by helix α1 and the α1-β2 and β10-β11 loops (FIG. 23B). The scissile phosphate group is contacted by Arg22 and His37, and positioned in an extended conformation that would permit an in-line attack by the 2′-hydroxyl of G28 (FIG. 23B). His37 is positioned to hydrogen bond with the 5′ or 3′ bridging oxygen atoms, and might therefore act as the general acid that protonates the leaving group during catalysis, in addition to charge-stabilizing the scissile phosphate. The active site of TtCas6B is composed of His23, His42 and Tyr256, whereby Tyr256 and His42 hydrogen bond to the 2′ and 3′ oxygens of the cyclic phosphate product. In a substrate complex, Tyr256 would likely be positioned to deprotonate the 2′-hydroxyl of G28 during nucleophilic attack, while His42, in analogy with His37 in TtCas6A, would stabilize the scissile phosphate and protonate the leaving group.
To shed light on the catalytic mechanism of TtCas6A and TtCas6B, we performed cleavage assays using wild type proteins as well as active site mutants TtCas6A H37A and TtCas6B H42A using repeat R1 as a substrate (FIG. 2C). The first order rate constant determined under single-turnover conditions for wild type TtCas6A (3.2 min−1) and TtCas6B (3.7 min−1) are in good agreement with first order rate constants previously determined for PaCas6f and TtCas6e. Strikingly, we found TtCas6A H37A to be almost inactive (17,000-fold cleavage defect), while TtCas6B H42A showed only a  ̃300-fold cleavage defect, indicating that despite considerable structural homology, the catalytic mechanisms of TtCas6A and TtCas6B might be substantially different. To confirm the role of the active site histidine His37 in TtCas6A, we sought to replace the histidine side chain by adding imidazole (a histidine mimic) to the cleavage reaction. This imidazole complementation strategy has been used recently to convert PaCsy4 into an inducible endoribonuclease. In the presence of 500 mM imidazole, the cleavage rate of TtCas6A H37A was enhanced  ̃360-fold, underscoring the importance of the active site histidine in the catalytic mechanism of TtCas6A.
The Active Site of TtCas6A Undergoes a Conformational Ordering Upon RNA Binding
The crystal structures of TtCas6A-RNA complexes allow comparisons of the RNA-free and RNA-bound states of the enzyme due to the presence of an RNA-free TtCas6 molecule in the crystallographic asymmetric unit. In the RNA-free TtCas6A, the loop connecting helix α1 and strand β2 (residues 33-40), which contains the active site histidine His37, is disordered (FIG. 23D). Upon substrate RNA binding, the loop becomes ordered and forms a short helical segment, as the backbone carbonyls of Pro40 and His37 form hydrogen bonds with the 2′ hydroxyl groups of G26 and G27, respectively, and the His37 side chain forms a hydrogen bond with the 3′-hydroxyl oxygen of G28. Additional interactions mediate substrate recognition downstream of the scissile phosphate; the 6-amino group of A29 forms a hydrogen bond with the amide carbonyl of Pro34, while the U30 is specifically recognized through hydrogen bonding interactions with the backbone amide of Gly85 and carbonyl of Arg83. The ordering of the His37-containing active site loop persists in the product complex, suggesting that scissile phosphate recognition by His37 and additional interactions with the ribose-phosphate backbone upstream of the cleavage site drive the conformational change upon substrate recognition.
FIG. 23 depicts various structures of TtCas6A and TtCas6B enzymes bound to substrate mimic and product RNAs. (A) Ribbon diagrams showing the overall views of Cash-RNA complexes: TtCas6A-R1 substrate mimic (left), TtCas6A-R1 product (middle) and TtCas6B-R3 product (right). Bound RNAs are depicted in cartoon format. The scissile phosphate groups are depicted as spheres. All cartoon molecular diagrams were generated using Pymol. (“www” followed by “.pymol.org”). (B) Zoomed-in views of the TtCas6 active sites, shown in the same orientation as in A. Hydrogen bonding interactions are denoted with dashed lines; numbers indicate interatomic distances in A. (C) Endonuclease activity assays of wild-type (WT) and active site mutant proteins. For the TtCas6A H37A mutant, the cleavage assay was also carried out in the presence of 500 mM imidazole. (D) Active site of TtCas6A undergoes conformational ordering upon substrate recognition. Left: zoomed-in view of the active site in the RNA-free TtCas6A molecule in the 2:1 protein-R1 substrate mimic complex. Right: zoomed-in view of the active site in the RNA-bound TtCas6A molecule. Hydrogen-bonding interactions are denoted with dashed lines. (SEQ ID NO: 53).
FIG. 28 depicts sequence and structural alignments of TtCas6A and TtCas6B enzymes. (A) Structure-based sequence alignment of TtCas6A and TtCas6B with Pyrococcus furiosus Cas6 (PfCas6, PDB code 3PKM), T. thermophilus Cas6e (TtCas6e, also known as Cse3 or CasE, PDB code 2Y8W) and Pseudomonas aeruginosa Cas6f (PaCas6f, also known as Csy4, PDB code 2XLK). The structures were aligned using the PDBeFold server (Velankar et al, 2011). The sequence alignment was generated using ESPript 2.2 (Gouet et al, 1999). Active site residues are highlighted in green. Secondary structure elements present in the TtCas6A protein are shown above the sequence. (B) Structural superpositions of Cas6-RNA complexes. Top; from left to right: Ribbon diagrams of TtCas6B-R3 product RNA complex (blue, this study), PfCas6-RNA complex (pink, PDB code 3PKM), TtCas6e-20 nt substrate mimic RNA complex (light green, PDB code 2Y8W) and PaCas6f-substrate mimic complex (light blue, PDB code 2XLK). The structures were superimposed using DALI (Holm & Sander, 1995) and are shown in identical orientations. Bottom: Structural superpositions of Cas6 enzymes with TtCas6A. Pairwise root mean square deviations (rmsds) and the number of Ca atoms in each superposition are indicated.
FIG. 30 depicts a table of X-ray data collection and refinement statistics.
Recognition of RNA Sequence and Geometry by TtCas6A and TtCas6B
In both TtCas6A and TtCas6B, extensive networks of ionic and hydrogen bonding interactions are involved in RNA recognition (FIG. 24A,B). In both proteins, the RNA-stem loop straddles the β10-β11 loop, and is positioned in a cleft between the active site loop and the beta-hairpin (β7-β8) that inserts into the major groove. In TtCas6A, the hairpin presents Arg129 for sequence-specific hydrogen bonding contacts with the lower three C-G base pairs in the stem (FIG. 24A). TtCas6B lacks an equivalent residue in the major groove-binding hairpin. Instead, the side chain of Ser147 hydrogen bonds to the base of G25 as the only sequence-specific contact with the RNA (FIG. 24A). In both Cas6-RNA complexes, the ribose-phosphate backbone in the 3′ half of the stem-loop is anchored through a series of hydrogen bonding contacts involving the phosphate groups of nucleotides 25-28 and the 2′-hydroxyl groups of nucleotide G26 in the TtCas6A-RNA complexes and nucleotide G27 in the TTCas6B-product complex, respectively (FIG. 24A,B). Thus, as in the structures of PaCas6f (PaCsy4) and TtCas63 (TtCse3), the RNAs are recognized both via their sequence and their shape.
To test the importance of the stem sequence for substrate RNA recognition by TtCas6A, a series of EMSAs was performed using R1 repeat-derived RNAs that carried single base pair substitutions (C-G→A-U). All mutant RNAs contained the complete 5′ segment and additional two nucleotides downstream of the cleavage site. Compared to the wild-type RNA, substitution of any of the four C-G base pairs in the stem led to about five-fold decrease in affinity (FIG. 24C). This is consistent with the observation that the lower three C-G base pairs are specifically read out by Arg129. The binding defect observed upon mutation of the closing (uppermost) base pair could be due to loss of stability of the stem-loop structure, which is determined by the closing base-pair (Serra et al, 1993). To further investigate the protein determinants of RNA binding, we tested the TtCas6A mutants H37A and R129A and performed binding assays using substrates R1 and R3 (FIG. 24D). Mutation of Arg129 resulted in a strong binding defect with  ̃260- and  ̃290-fold decrease in affinity for R1 and R3, respectively, when compared to wild-type TtCas6A, in agreement with the observed function of this residue in simultaneous recognition of the lower three C-G base pairs in the RNA stem-loop. A similar, but somewhat weaker effect was observed for TtCas6A H37A, which yielded  ̃70- and  ̃90-fold reduction in affinities for R1 and R3 repeat RNAs, respectively. The binding defects indicate that besides playing a key role in the catalysis of RNA cleavage, the His37 side-chain also contributes to substrate binding. This is consistent with the ordering of the active site loop observed upon substrate recognition, which appears to be driven in part by the interaction between the His37 side chain and the scissile phosphate group.
FIG. 24 depicts RNA recognition by TtCas6A and TtCas6B. (A) Detailed views of RNA binding by TtCas6A (left) and TtCas6B (right). Hydrogen bonding interactions are indicated with dashed lines. Spheres denote backbone amide nitrogen atoms of Lys226 in TtCas6A (left) and Ala145 and Lys253 in TtCas6B (right). (B) Schematic diagrams of protein-RNA contacts in the TtCas6A-R1 substrate mimic (left) and TtCas6B-R3 product complexes. Amino acid residues contacting the bound RNA via ionic or hydrogen-bonding interactions are highlighted. Arrows mark the scissile phosphates. Circles denote phosphodiester groups in the RNA backbone. Lines indicate base-pairing interactions. (C) Base-pair contributions to R1 repeat recognition by TtCas6A. A series of RNAs in which individual C-G base pairs were substituted with A-U were prepared and assayed for binding to TtCas6A using EMSAs. The data for each base pair substitution is expressed as Kd and as fold reduction in affinity relative to wild-type R1 RNA. (D) R1 (left) or R3 (right) product RNA binding by WT TtCas6A or R129A and H37A mutants was quantified using EMSAs. The data are plotted as in FIG. 22B.
Recognition of the 5′-Segment of the Repeat Suggests a Two-Site Model for RNA Binding
The structures of TtCas6A- and TtCas6B-product RNA complexes reveal that besides recognizing the stem-loop, the enzymes also make specific interactions with the upstream RNA sequence. In the TtCas6A-R1 product complex, two nucleotides upstream of the stem-loop are observed in 2Fo-Fc electron density maps. The remainder of the 5′ segment of the R1 repeat RNA is not ordered, although the RNA is intact in the crystal. The purine bases of the two ordered nucleotides in the 5′ segment are inserted into a crevice at the interface of the two TtCas6A molecules in the non-crystallographic dimer (FIG. 25A). G16 engages in hydrogen bonding with the side chain of His134 and the backbone carbonyl of Asp188. The base of A15 is hydrogen bonded to the backbone amide and carbonyl groups of His134. In the TtCas6B-R3 product complex, the two RNA molecules in the asymmetric unit adopt slightly different conformations at their 5′ ends. In one molecule, only one nucleotide (G17) upstream of the repeat stem-loop is ordered, forming hydrogen bonds with the side chains of Arg208 and Glu197, each contributed by one TtCas6B molecule in the non-crystallographic dimer (FIG. 25A). In the other RNA molecule, both G17 and A16 are ordered, and the base of A16 is tucked in and stacks below the terminal base pair of the R3 repeat stem-loop (FIG. 29A).
Inspection of the molecular surface of TtCas6A reveals a deep groove tracing the interface of the two ferredoxin folds. This groove extends from the A15-binding site towards a highly positively charged patch located on the reverse side of the protein from the active site (FIG. 25B). A similar groove is observed in TtCas6B (FIG. 29B). A sulfate ion is bound to the basic patch in the structures of both TtCas6A-R1 substrate and TtCas6A-R1 product complexes, and is contacted by the side chains of Arg121 and Arg223 (FIG. 29C). In the structure of PfCas6 bound to a fragment of its cognate repeat RNA, nucleotides 2-10 of the repeat bind to a positively charged groove located on the face of the protein opposite from the active site. Superposition of the PfCas6 and TtCas6A RNA complex structures reveals that the basic groove in TtCas6A overlaps with the PfCas6A RNA binding site such that the 3′ end of the bound PfCas6 RNA fragment (nucleotide A10) aligns with the 5′ end (nucleotide A15) of the R1 repeat RNA (FIG. 25C). This suggests that the basic groove in TtCas6A might constitute an additional RNA binding site that interacts with the unstructured 5′ segment of the R1 repeat RNA upstream of A15. Neither nucleotides G1-G14 of the R1 product RNA, nor nucleotides G1-U15 of the R3 RNA are ordered in the crystal structures of the TtCas6A and TtCas6B complex complexes. However, it is possible that in both cases, this is a consequence of the high ionic strength of the crystallization condition and the presence of sulfate in the TtCas6A crystals. We therefore hypothesized that the unstructured 5′ segment contributes to R1 RNA binding by TtCas6A. To test this, we measured the affinity of TtCas6A for a series of RNAs based on the R1 repeat in which nucleotides were progressively removed from the 5′ end (FIG. 25D). Deletion of nucleotides 1-8 had little effect on binding affinity. Truncation of the R1 repeat RNA beyond nucleotide G9 led to a gradual loss of binding affinity, with an approximately 900-fold increase in Kd upon deletion of residues 1-13 of the R1 repeat RNA. These results indicate that nucleotides 9-13 in the unstructured 5′ segment of the repeat RNA make a considerable contribution to binding, which would be consistent with the existence of a second RNA binding site in TtCas6A.
FIG. 25 demonstrates recognition of the 5′ segment of the repeat RNA. (A) Details of sequence-specific recognition of nucleotides upstream of the stem-loop in RNA repeats. Top: TtCas6A-R1 product complex. Nucleotides 1-14 of the R1 product RNA are disordered. Bottom: TtCas6B-R3 product complex. Nucleotides 1-15 of the R3 product RNA are disordered. (B) Surface electrostatic potential map of TtCas6A identifies a second RNA binding site. Top: Cartoon diagram of the 2:1 TtCas6A-R1 product RNA complex. RNA is shown. Bound sulfate ions are depicted in stick format. Bottom: Electrostatic surface potential map of TtCas6A, shown in the same orientations as above. Positively charged region and negatively charged regions are shown. The positively charged patch located on the surface opposite from the active site is highlighted with a black ellipse. (C) Structural superposition of the TtCas6A-R1 product RNA (TtR1) and PfCas6-repeat RNA (PfRNA) (PDB code: 3PKM) complexes. TtCas6A; PfCas6. T. thermophilus R1 repeat RNA. PfRNA is colored black. Nucleotide A15 of TtR1 aligns with G10 of PfRNA. (D) Nucleotides in the single-stranded 5′ segment of R1 repeat RNA contribute to binding. TtCas6A binding to a series of truncated RNAs based on the R1 repeat was quantified by EMSAs as in FIG. 22B. The data are expressed as Kd and as a fold binding defect relative to wild type R1 repeat. 5′-terminal G nucleotides resulting from in vitro transcription are shown in G. (E) Structural superposition of TtCas6A dimer with P. furiosus repeat RNA, based on the superposition shown in D. TtCas6A is colored according to surface electrostatic potential and shown in the same orientations as in B. TtR1 is colored orange; PfRNA is colored black. (F) Cartoon model of RNA recognition by TtCas6 enzymes. TtCas6A binds the stem-loop RNA (solid line) at the interface of the two ferrdoxin-like domains. Additionally, the 5′ segment of the repeat RNA (dashed line) is bound by a distal positively charged cleft on the opposite surface from the active site. (FIG. 25D: left, SEQ ID NO: 56)(FIG. 25D: right bottom, SEQ ID NO: 57).
FIG. 29 depicts various structural views of TtCas6A and TtCas6B complexed with substrate and/or product. (A) Details of sequence-specific recognition of nucleotides upstream of the stem-loop in TtCas6B-R3 product RNA complex. Nucleotides 1-14 of the R3 product RNA are disordered. (B). Top: Cartoon diagram of the 2:2 TtCas6B-R3 product RNA complex. RNA is shown in orange. Bottom: Electrostatic surface potential map of TtCas6A, shown in the same orientations as above. Blue, positively charged region; red, negatively charged region. The positively charged patch located on the surface opposite from the active site is highlighted with a black ellipse. (C) Detailed view of the sulfate binding site in TtCas6A-R1 product RNA complex. The sulfate ion and neighboring amino acid residues are shown in stick format.
REFERENCES
Adams P D, Afonine P V, Bunkóczi G, Chen V B, Davis I W, Echols N, Headd J J, Hung L-W, Kapral G J, Grosse-Kunstleve R W, McCoy A J, Moriarty N W, Oeffner R, Read R J, Richardson D C, Richardson J S, Terwilliger T C & Zwart P H (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66: 213-221
Afonine P V, Grosse-Kunstleve R W, Echols N, Headd J J, Moriarty N W, Mustyakimov M, Terwilliger T C, Urzhumtsev A, Zwart P H & Adams P D (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68: 352-367
Agari Y, Sakamoto K, Tamakoshi M, Oshima T, Kuramitsu S & Shinkai A (2010) Transcription profile of Thermus thermophilus CRISPR systems after phage infection. J Mol Biol 395: 270-281
Al-Attar S, Westra E R, van der Oost J & Brouns S J J (2011) Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol. Chem. 392: 277-289
Brouns S J J, Jore M M, Lundgren M, Westra E R, Slijkhuis R J H, Snijders A P L, Dickman M J, Makarova K S, Koonin E V & van der Oost J (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321: 960-964
Carte J, Wang R, Li H, Terns R M & Terns M P (2008) Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev 22: 3489-3496
Ebihara A, Yao M, Masui R, Tanaka I, Yokoyama S & Kuramitsu S (2006) Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain. Protein Sci 15: 1494-1499
Emsley P, Lohkamp B, Scott W G & Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66: 486-501
Gesner E M, Schellenberg M J, Garside E L, George M M & Macmillan A M (2011) Recognition and maturation of effector RNAs in a CRISPR interference pathway. Nat Struct Mol Biol 18: 688-692
Gouet P, Courcelle E, Stuart DI & Métoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15: 305-308
Grissa I, Vergnaud G & Pourcel C (2007) The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8: 172
Hale C R, Zhao P, Olson S, Duff M O, Graveley B R, Wells L, Terns R M & Terns M P (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139: 945-956
Haurwitz R E, Jínek M, Wiedenheft B, Zhou K & Doudna J A (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329: 1355-1358
Haurwitz R E, Sternberg S H & Doudna J A (2012) Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA. EMBO J: 1-9
Holm L & Sander C (1995) Dali: a network tool for protein structure comparison. Trends Biochem Sci 20: 478-480
Jore M M, Lundgren M, van Duijn E, Bultema J B, Westra E R, Waghmare S P, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer M R, Barendregt A, Zhou K, Snijders A P L, Dickman M J, Doudna J A, Boekema E J, Heck A J R, van der Oost J & Brouns S J J (2011) Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18: 529-536
Juranek S, Eban T, Altuvia Y, Brown M, Morozov P, Tuschl T & Margalit H (2012) A genome-wide view of the expression and processing patterns of Thermus thermophilus HB8 CRISPR RNAs. RNA 18: 783-794
Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66: 125-132
Kunin V, Sorek R & Hugenholtz P (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 8: R61
Lee H Y, Haurwitz R E, Apffel A, Zhou K, Smart B, Wenger C D, Laderman S, Bruhn L & Doudna J A (2013) RNA-protein analysis using a conditional CRISPR nuclease. Proc Natl Acad Sci USA
Lintner N G, Kerou M, Brumfield S K, Graham S, Liu H, Naismith J H, Sdano M, Peng N, She Q, Copie V, Young M J, White M F & Lawrence C M (2011) Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE). Journal of Biological Chemistry 286: 21643-21656
Makarova K S, Aravind L, Wolf Y I & Koonin E V (2011a) Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct 6: 38
Makarova K S, Haft D H, Barrangou R, Brouns S J J, Charpentier E, Horvath P, Moineau S, Mojica F J M, Wolf Y I, Yakunin A F, van der Oost J & Koonin E V (2011b) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9: 467-477
Marraffini L A & Sontheimer E J (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11: 181-190
McCoy A J, Grosse-Kunstleve R W, Adams P D, Winn M D, Storoni L C & Read R J (2007) Phasercrystallographic software. J Appl Crystallogr 40: 658-674
Morris R J, Perrakis A & Lamzin V S (2003) ARP/wARP and automatic interpretation of protein electron density maps. Meth Enzymol 374: 229-244
Sashital D G, Jínek M & Doudna J A (2011) An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3. Nat Struct Mol Biol 18: 680-687
Serra M J, Lyttle M H, Axenson T J, Schadt C A & Turner D H (1993) RNA hairpin loop stability depends on closing base pair. Nucleic Acids Res 21: 3845-3849
Shao Y & Li H (2013) Recognition and Cleavage of a Nonstructured CRISPR RNA by Its Processing Endoribonuclease Cas6. Structure 21: 385-393
Sternberg S H, Haurwitz R E & Doudna J A (2012) Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA 18: 661-672
Terns M P & Terns R M (2011) CRISPR-based adaptive immune systems. Curr Opin Microbiol 14: 321-327
Terwilliger T C (2004) Using prime-and-switch phasing to reduce model bias in molecular replacement. Acta Crystallogr D Biol Crystallogr 60: 2144-2149
Velankar S, Alhroub Y, Best C, Caboche S, Conroy M J, Dana J M, Fernandez Montecelo M A, van Ginkel G, Golovin A, Gore S P, Gutmanas A, Haslam P, Hendrickx P M S, Heuson E, Hirshberg M, John M, Lagerstedt I, Mir S, Newman L E, Oldfield T J, et al (2011) PDBe: Protein Data Bank in Europe. Nucleic Acids Res 40: D445-D452
Vonrhein C, Blanc E, Roversi P & Bricogne G (2007) Automated structure solution with autoSHARP. Methods Mol Biol 364: 215-230
Wang R, Preamplume G, Terns M P, Terns R M & Li H (2011) Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage. Structure 19: 257-264
Wang R, Zheng H, Preamplume G, Shao Y & Li H (2012) The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex. Protein Sci 21: 405-417
Wiedenheft B, Lander G C, Zhou K, Jore M M, Brouns S J J, van der Oost J, Doudna JA & Nogales E (2011a) Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477: 486-489
Wiedenheft B, Sternberg S H & Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482: 331-338
Wiedenheft B, van Duijn E, Bultema J B, Bultema J, Waghmare S P, Waghmare S, Zhou K, Barendregt A, Westphal W, Heck A J R, Heck A, Boekema E J, Boekema E, Dickman M J, Dickman M & Doudna JA (2011b) RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci USA 108: 10092-10097
Zwart P H, Afonine P V, Grosse-Kunstleve R W, Hung L-W, loerger T R, McCoy A J, McKee E, Moriarty N W, Read R J, Sacchettini J C, Sauter N K, Storoni L C, Terwilliger T C & Adams P D (2008) Automated structure solution with the PHENIX suite. Methods Mol Biol 426: 419-435
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

## Claims

1. A variant Cas endoribonuclease comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:19, wherein the endoribonuclease comprises an amino acid substitution at a histidine residue such that the variant Cas endoribonuclease is enzymatically inactive in the absence of imidazole, and wherein the variant Cas endoribonuclease is activatable in the presence of imidazole.

2. The variant Cas endoribonuclease of claim 1, wherein the amino acid substitution is a substitution at His37.

3. The variant Cas endoribonuclease of claim 1, wherein the variant Cas endoribonuclease comprises a moiety that provides a detectable signal.

4. The variant Cas endoribonuclease of claim 1, wherein the endoribonuclease is immobilized on an insoluble support.

5. The variant Cas endoribonuclease of claim 1, wherein the variant Cas endoribonuclease binds an RNA substrate comprising a nucleotide sequence selected from:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′;



 



(SEQ ID NO: 8)



5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;



 



(SEQ ID NO: 9)



5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;



 



(SEQ ID NO: 10)



5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;



 



(SEQ ID NO: 11)



5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;



and



 



(SEQ ID NO: 12)



5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′.

6. A kit for purifying a target RNA present in a mixed population of nucleic acids, the kit comprising:
the variant Cas endoribonuclease of claim 1.

7. The kit of claim 6, further comprising a recombinant expression vector comprising, in order from 5′ to 3′ and in operable linkage:
a) a nucleotide sequence encoding an RNA substrate that is specifically bound by the variant Cas endoribonuclease of claim 1; and
b) a multiple cloning site suitable for insertion of a nucleic acid encoding the target RNA.

8. The kit of claim 7, wherein the nucleotide sequence encoding the RNA substrate is operably linked to a promoter.

9. The kit of claim 7, wherein the RNA substrate comprises a nucleotide sequence selected from:









(SEQ ID NO: 1)



5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;



 



(SEQ ID NO: 2)



5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;



 



(SEQ ID NO: 3)



5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;



 



(SEQ ID NO: 4)



5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 5)



5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 6)



5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;



 



(SEQ ID NO: 7)



5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′;



 



(SEQ ID NO: 8)



5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;



 



(SEQ ID NO: 9)



5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;



 



(SEQ ID NO: 10)



5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;



 



(SEQ ID NO: 11)



5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;



and



 



(SEQ ID NO: 12)



5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′.

10. The kit of claim 7, wherein the recombinant expression vector comprises, inserted into the multiple cloning site, a nucleotide sequence encoding the target RNA.

11. A method of isolating a target RNA present in a mixed population of nucleic acids, the method comprising:
a) contacting a mixed population of nucleic acids with the variant Cas endoribonuclease of claim 1, where the variant Cas endoribonuclease is immobilized on an insoluble support, wherein the mixed population of nucleic acids comprises a tagged target RNA comprising a recognition nucleotide sequence that is specifically bound by the immobilized variant Cas endoribonuclease, forming a tagged target RNA-immobilized variant Cas endoribonuclease complex, wherein said contacting is carried out in a binding solution lacking imidazole;
b) adding imidazole to the binding solution to a final concentration of from about 100 mM to about 500 mM, thereby forming a reactivation solution that enzymatically reactivates the immobilized variant Cas endoribonuclease, wherein the reactivated immobilized variant Cas endoribonuclease cleaves the target RNA from the tag; and
c) collecting the released target RNA.

12. The method of claim 11, further comprising a wash step carried out after step (a) and before step (b).

13. A method of isolating a polypeptide that binds a target RNA, the method comprising:
a) contacting an immobilized complex with a liquid solution comprising a polypeptide that binds the target RNA, wherein the immobilized complex comprises the variant Cas endoribonuclease of claim 1 and a tagged target RNA comprising a recognition nucleotide sequence that is specifically bound by the variant Cas endoribonuclease, wherein said contacting results in binding of the polypeptide to the target RNA, wherein said contacting is carried out in a binding solution lacking imidazole; and
b) eluting the bound polypeptide.