Patent Application
20220411771
The contents of the electronic sequence listing (300694.xml; Size: 58,914 bytes; and Date of Creation: Aug. 24, 2022) is herein incorporated by reference in its entirety.
Human cells encode a large number of RNAs, including many non-coding RNAs. These RNAs are expressed differentially in various cells and physiological conditions. However, the functions and regulatory mechanisms of the majority of these transcripts remain unknown. One potential key to understanding is the RNA-binding protein, which is a feature throughout the entire life cycle of RNA (including mRNA, lncRNA, etc.), indicating the importance of the study of detailed RNA-protein interactions.
RNA-binding proteins (RBPs) play important roles in various biological processes such as regulation, splicing, modification, localization, translation, and stabilization of RNAs. Many RNA-binding proteins, including some proteins that lack the classical RNA-binding domains, have distinct spatial and temporal distributions in cells and tissues. The malfunction of RBPs is responsible for many human diseases.
In order to gain insight into the function of RBPs, it is necessary to identify detailed interactions between an RNA and its binding proteins. Initially, the RNA immunoprecipitation (RIP) assay has been used to identify RNA-protein interactions, which was adapted from the chromatin immunoprecipitation assay (ChIP). However, because the RIP assay retains protein-protein interactions, it is not well suitable for studying direct RNA-protein contacts. To exploit zero-length covalent RNA-protein cross-linking and RNA fragmentation, a method named crosslinking and immunoprecipitation (CLIP) has been developed. By directly illuminating cells or tissues with UV-B light, it catalyzes the formation of covalent bonds between RNA and proteins that within the direct contact. Later, Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) was developed to further improve the cross-linking efficiency of CLIP.
Another class of highly regarded methods named RNA antisense purification-mass spectrometry (RAP-MS) and comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) have been developed recently. Biotin-labeled DNA fragments complementary to the target RNA sequences were used to capture the target RNAs. RNA-protein complexes bind to the biotin-tagged DNA fragments, which were captured by streptavidin magnetic beads. The advantage of these mass spectrometry-based techniques is to capture RNA-protein interactions under natural conditions. However, it is difficult to design DNA fragments suitable for those experiments. Therefore, the desires for widely applicable detecting the RNA-protein interaction of specific RNAs for in vivo labeling without in vitro manipulation remain unfulfilled.
Moreover, it is also valuable to detect DNA-protein interactions as such interactions can impact the transcription and other activities of DNA fragments.
The present technology enables study of interactions between nucleic acids and nucleic acid-binding molecules. A Cas protein (e.g., a catalytically dead Cas13) is fused to a proximity tagging enzyme (e.g., a Pup ligase) and thus brings the proximity tagging enzyme to a nucleic acid, when the Cas protein recognizes the nucleic acid, e.g., with a guide RNA. The proximity tagging enzyme then tags the molecule enabling it to be identified as one that interacts with the nucleic acid.
In accordance with one embodiment of the present disclosure, therefore, provided is a non-human transgenic organism, comprising a recombinant polynucleotide in at least one cell of the organism, wherein the polynucleotide encodes a fusion protein comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein Cas13 and a proximity tagging enzyme.
In some embodiments, the polynucleotide further comprises an inducible promoter or a tissue-specific promoter that is operably linked to and regulates the expression of the fusion protein.
In another embodiment, provided is a method of identifying a protein that binds to a target RNA, comprising contacting activating the inducible promoter in the non-human transgenic organism in the presence of a guide RNA that is specific to the target RNA, under conditions to allow the Cas13 protein to bind to the target RNA and the proximity tagging enzyme to tag proteins bound to the target RNA.
Also provided a fusion protein comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein Cas13 and a proximity tagging enzyme. In some embodiments, the Cas13 is selected from the group consisting of Cas13a, Cas13b, Cas13c, and Cas13d. Examples include LshCas13a, LwaCas13a, LseCas13a, LbmCas13a, LbnCas13a, CamCas13a, CgaCas13a, Cga2Cas13a, Pprcas13a, LweCas13a, LbfCas13a, Lwa2cas13a, RcsCas13a, RcrCas13a, RcdCas13a, LbuCas13a, HheCas13a, EreCas13a, EbaCas13a, BmaCas13a, LspCas13a, BzoCas13b, PinCas13b, PbuCas13b, AspCas13b, PsmCas13b, RanCas13b, PauCas13b, PsaCas13b, Pin2Cas13b, CcaCas13b, PguCas13b, PspCas13b, FbrCas13b, PgiCas13b, Pin3Cas13b, FnsCas13c, FndCas13c, FnbCas13c, FnfCas13c, FpeCas13c, FulCas13c, AspCas13c, UrCas13d, RffCas13d, RaCas13d, AdmCas13d, PIE0Cas13d, EsCas13d, and RfxCas13d. In some embodiments, the Cas13 is catalytically dead, such as dLwCas13a with an R474A or R1046A mutation.
In some embodiments, the proximity tagging enzyme is selected from the group consisting of a Pup ligase, a biotin ligase, and an ascorbate peroxidase. In some embodiments, the proximity tagging enzyme is PafA, TurboID, or MiniTurbo.
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
The term “isolated” as used herein with respect to cells, nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term “isolated” as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides is meant to encompass both purified and recombinant polypeptides.
As used herein, the term “recombinant” as it pertains to polypeptides or polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.
The experimental example has tested a system for detecting RNA-protein interactions, which is referred to as CRISPR-based RNA-United Interacting System (CRUIS), which uses the CRISPR-based RNA-target Cas nuclease as an RNA tracker to bring the proximity-labeling system to a designated target RNA. CRUIS can capture RNA-protein interactions of specific RNA sequences effectively. In CRUIS, a dead RNA-guided RNA targeting nuclease, e.g., LwaCas13a (dLwaCas13a), is used as a tracker to target specific RNA sequences, while a proximity enzyme, e.g., PafA, is fused to the nuclease to label any surrounding RNA-binding proteins. The labeled proteins can then be enriched and identified.
Using this technology, proteins that interact with specific RNAs can be labeled in living cells, which avoids the risk of RNA degradation introduced by processing RNA-protein complexes in vitro. In addition, this technology can avoid over-expressing the target RNA with the MS2-tag sequence in the cell, so the abundance of the target RNA in the cell is in a natural state and the acquired RNA is closer to the real situation.
In comparison to the conventional methods, CRUIS shows quite a few advantages. First, it provides a simple and effective way to obtain potential RNA-binding proteins of target RNA. Second, CRUIS can identify RNA-protein interactions in a natural state. Finally, CRUIS can label potential RNA-binding proteins in living cells, thereby avoiding the manipulation of RNA in vitro and decreasing the impact of RNA degradation. CRUIS can be universally used for different types of RNA, including lncRNA and mRNA, indicating that CRUIS has broad applicability. Furthermore, when using a DNA-targeting Cas protein, such as Cas9 and Cas12a/b, the technology can be useful for detecting DNA-protein interactions.
One embodiment of the present disclosure provides compositions and methods for detecting nucleic acid-molecule interactions. The present technology, in some embodiments, employs a fusion protein that includes a Cas protein and a proximity tagging enzyme. The Cas protein, through the use of an appropriate guide RNA, can selectively bind a nucleic acid molecule. Once bound, the proximity tagging enzyme can, under suitable conditions and with suitable substrates, tag molecules that interact with the nucleic acid and thus identifying those molecules with mass spectrometry.
In one embodiment, the present disclosure provides a fusion protein comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein and a proximity tagging enzyme.
The term “Cas protein” or “clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein” refers to RNA-guided DNA/RNA endonuclease enzymes associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, as well as other bacteria. Cas proteins include Cas9 proteins, Cas12a (Cpf1) proteins, Cas12b (formerly known as C2c1) proteins, Cas13 proteins and various engineered counterparts.
Example DNA-targeting Cas proteins include SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpf1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKH SaCas9, NmeCas9, StCas9, CjCas9, AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, LsCas12b and those provided in Table A below.
In some embodiments, the Cas protein is a DNA-targeting Cas protein, such as Cas9, Cas12a and Cas12b. In some embodiments, the Cas protein is a RNA-targeting Cas protein, such as Cas13.
Cas13 targets RNA. The Cas13 family contains at least four known subtypes, including Cas13a (formerly C2c2), Cas13b, Cas13c, and Cas13d, classified based on the identity of the Cas13 protein and additional locus features. All known Cas13 family members contain two HEPN domains, which confer RNase activity. Cas13 can be reprogrammed to cleave a targeted ssRNA molecule through a short guide RNA with complementarity to the target sequence.
Cas13s function similarly to Cas9, using a ˜64-nt guide RNA to encode target specificity. The Cas13 protein complexes with the guide RNA via recognition of a short hairpin in the crRNA, and target specificity is encoded by a 28-30-nt spacer that is complementary to the target region. In addition to programmable RNase activity, Cas13s can also exhibit collateral activity after recognition and cleavage of a target transcript, leading to non-specific degradation of any nearby transcripts regardless of complementarity to the spacer.
Non-limiting examples of Cas13 proteins are listed in the table below.
Leptotrichia shahii
Leptotrichia wadei
Listeria seeligeri
Lachnospiraceae bacterium MA2020
Lachnospiraceae bacterium NK4A179
Carnobacterium gallinarum DSM 4847
Carnobacterium gallinarum DSM 4847
Paludibacter propionicigenes WB4
Listeria weihenstephanensis FSL R9-0317
Listeriaceae bacterium FSL M6-635
Leptotrichia wadei F0279
Rhodobacter capsulatus SB 1003
Rhodobacter capsulatus R121
Rhodobacter capsulatus DE442
Leptotrichia buccalis C-1013-b
Herbinix hemicellulosilytica
Eubacteriaceae bacterium CHKCI004
Blautia sp. Marseille-P2398
Leptotrichia sp. oral taxon 879 str.F0557
Bergeyella zoohelcum
Prevotella intermedia
Prevotella buccae
Alistipes sp. ZOR0009
Prevotella sp. MA2016
Riemerella anatipestifer
Prevotella aurantiaca
Prevotella saccharolytica
Prevotella intermedia
Capnocytophaga canimorsus
Porphyromonas gulae
Prevotella sp. P5-125
Flavobacterium branchiophilum
Porphyromonas gingivalis
Prevotella intermedia
Fusobacterium necrophorum subsp.
funduliforme ATCC 51357
Fusobacterium necrophorum DJ-2
Fusobacterium necrophorum BFTR-1
Fusobacterium necrophorum subsp.
funduliforme 1_1_36S contl.14
Fusobacterium perfoetens ATCC
Fusobacterium ulcerans ATCC
Anaerosalibacter sp. ND1 genome
massiliensis ND1
Ruminoccocus flavefaciens FDI
Ruminoccocus albus
Anaerobic digester metagenome 15706
Eubacterium siraeum DSM15702
Ruminoccocus flavefaciens XPD3002
The Cas protein, in some embodiments, is catalytically inactive/dead. Catalytically dead Cas proteins can be readily prepared by mutating one or more amino acid residues in the Cas protein's catalytic domain. Dead Cas9, Cas12a, and Cas12b proteins are commercially available, commonly referred to as dCas9, dCas12a (dCpf1) and dCas12b (dC2c1).
The catalytic domain of the Cas13 protein includes two HEPN domains (higher eukaryotes and prokaryotes nucleotide-binding domain) which confer RNase activity. Examples of mutations that inactivate Cas13 include R474A and R1046A (located at the HEPN domain) for dLwCas13a.
A “proximity tagging enzyme” refers to an enzyme in a proximity tagging system. A proximity tagging system typically includes an enzyme (e.g., Pup ligase, biotin ligase, ascorbate peroxidase) and a substrate (e.g., Pup, biotin, ascorbate). The enzyme can perform the enzymatic reaction on the substrate when the enzyme is in proximity with another required substrate. For instance, a Pup ligase can conjugate a Pup protein to a target protein when the Pup ligase is close to the target protein, thereby tagging the target protein with the Pup protein. Non-limiting examples of proximity tagging systems are provided in the table below.
E. Coli
Corynebacterium glutamicum
E. Coli
E. Coli
A. Aeolicus
B. Subtilis
In a PUP-IT (Puplyation-based Interacting Tagging) system, the tagging enzyme is a prokaryotic ubiquitin-like protein (Pup) ligase in the Pup bacteria protein-conjugating system, PafA. Pup is a small bacteria protein that carries about 64 amino acids with Gly-Gly-Gln at the C-terminus. When the C-terminus Gln is deaminated to Glu (this form of Pup will be referred to as Pup(E)), in the presence of ATP, Pup ligase PafA can catalyze the phosphorylation of the Pup(E) C-terminus Glu, which in turn conjugates the C-terminus Glu to a lysine residue side chain on the target protein.
“Prokaryotic ubiquitin-like protein” or “Pup” is a functional analog of ubiquitin found in the prokaryote Mycobacterium tuberculosis. It serves the same function as ubiquitin, although the enzymology of ubiquitylation and pupylation is different. In contrast to the three-step reaction of ubiquitylation, pupylation requires two steps, therefore only two enzymes are involved in pupylation. Similar to ubiquitin, Pup attaches to specific lysine residues of substrate proteins by forming isopeptide bonds. It is then recognized by Mycobacterium proteasomal ATPase (Mpa) by a binding-induced folding mechanism that forms a unique alpha-helix. Mpa then delivers the Pup-substrate to the 20S proteasome by coupling of ATP hydrolysis for proteasomal degradation.
There are an abundance of known Pup proteins, which have well reserved amino acid sequences. For instance, a known Pup protein Superfamily (ID: pfam05639) includes 28 Pup proteins. In addition, the table below lists a number of Pup proteins as well as a truncated one (named “Truncated”) which was derived from BAV23336.1 and tested in the experimental examples.
A Pup protein suitable for use with the present technology, therefore, can be any of the Pup proteins disclosed herein, or their truncated forms that includes, e.g., the C-terminal 28 amino acid residues (e.g., SEQ ID NO: 2). In some embodiments, the C-terminal residue can be Glu or modified from another, natural amino acid to Glu.
The fusion protein, in some embodiments, may include one or more nuclear localization sequences (NLS).
A “nuclear localization signal or sequence” (NLS) is an amino acid sequence that tags a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal (NES), which targets proteins out of the nucleus. A non-limiting example of NLS is the internal SV40 nuclear localization sequence (iNLS). Some examples are PKKKRKV (SV40 Large T-antigen; SEQ ID NO:20), KRPAATKKAGQAKKKK (nucleoplasmin; SEQ ID NO:21), AVKRPAATKKAGQAKKKKLD (nucleoplasmin; SEQ ID NO:22), MSRRRKANPTKLSENAKKLAKEVEN (EGL-13; SEQ ID NO:23), PAAKRVKLD (c-Myc; SEQ ID NO:24) and KLKIKRPVK (TUS-protein; SEQ ID NO:25).
Suitable Cas proteins, Pup ligase, and Pup proteins can also include biological equivalents of those specifically known or described herein. The term “biological equivalent” of a protein or polypeptide refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference protein or polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four or five addition, deletion, substitution and their combinations thereof as compared to the reference protein or polypeptide. In some aspects, the equivalent sequence retains the activity (e.g., RNase, or conjugating to a lysine) or structure of the reference sequence.
In some embodiments, the amino acid substitution is a conservative amino acid substitution. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
The term “Pup ligase” or “Pup-protein ligase” refers to a group of proteins which, in the presence of ATP, catalyzes the phosphorylation of the C-terminus Glu of a Pup protein, which in turn conjugates the C-terminus Glu to a lysine residue side chain on a target protein. Pup ligases have well reserved amino acid sequences. Some of the Pup ligases are classified into a GenBank Superfamily (ID: TIGR03686). An example Pup ligase is “Pup-protein ligase [Corynebacterium glutamicum]” (Access No: OKX85684.1), the amino acid sequence of which is listed in the table below.
As noted above, once the molecule binds to the protein, the molecule will bring its coupled Pup ligase to the protein. Given that Pup is available in the sample, its C-terminus Glu can be phosphorylated by the Pup ligase which will also conjugate the C-terminus Glu to a lysine residue side chain on the protein.
In some embodiments, the Cas protein is placed at the N-terminal side of the proximity tagging enzyme. In some embodiments, the Cas protein is placed at the C-terminal side of the proximity tagging enzyme. It is demonstrated in the example that such fusion between the Cas protein and the proximity tagging enzyme still allows both of the proteins to be active.
In some embodiments, a linker is placed between the Cas protein and the proximity tagging enzyme. The linker may have a length that is at least 1, 2, 5, 10, 15, 20, 25, 30, 40 or 50 amino acid residues, in some embodiments. In some embodiments, the linker has a length that is not longer than 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, or 20 amino acid residues. In some embodiments, the fusion protein further includes a market protein such as GFP, YFP, and RFP.
The fusion protein can be used to study RNA-molecule interactions. In some embodiments, a method is provided for identifying a molecule that binds to a target nucleic acid. The method may entail contacting a biological sample that includes the target nucleic acid with a fusion protein of the present disclosure, in the presence of a guide RNA that is specific to the target nucleic acid, under conditions to allow the Cas protein to bind to the target nucleic acid and the proximity tagging enzyme to tag molecules bound to the target nucleic acid. Once the molecule is so tagged, it can be isolated and identified.
The proximity tagging enzyme, for instance, can be a Pup ligase, such as PafA. Accordingly then, the contacting is made in the presence of a Pup ligase substrate, PupE. If the proximity tagging enzyme is a biotin ligase, then the contacting can occur in the present of biotin.
The guide RNA can be any that allows the Cas protein to selectively bind to the target nucleic acid. In some embodiments, the guide RNA is a single guide RNA (sgRNA). Methods for designing suitable sgRNA for nucleic acid targeting are well known in the art.
In some embodiments, the contacting is in vitro, in vivo, ex vivo, without limitation. As discussed herein, the present technology allows study of nucleic acid -molecule interactions in their natural state, including in vivo.
Transgenic organisms can be used for detecting nucleic acid-molecule interactions in the organisms. For instance, Example 2 prepared transgenic mouse and Drosophila models the contained recombinant polynucleotide encoding the fusion protein regulated by an inducible promoter. The fusion protein can be expressed at the desired cells and/or at the desired stage.
The guide RNA, e.g., sgRNA, can be provided either by a recombinant DNA which can be constantly expressed (as no toxicity is expected), induced, or introduced by viral vector (e.g., AVV). Some of the proximity tagging enzymes can required another factor to function. For instance, when the PufA is used as the proximity tagging enzyme, the PupE cDNA can be introduced into the model with an AAV vector.
In one embodiment, therefore, provided is a non-human transgenic organism, comprising a recombinant polynucleotide in at least one cell of the organism, wherein the polynucleotide encodes a fusion protein comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein Cas13 and a proximity tagging enzyme. In some embodiments, the proximity tagging enzyme is selected from the group consisting of a Pup ligase, a biotin ligase, and an ascorbate peroxidase. Examples of proximity tagging enzyme are provided herein.
In a preferred embodiment, the proximity tagging enzyme is PafA. In another preferred embodiment, the proximity tagging enzyme is TurboID or miniTurbo. The PUP-IT system is herein shown as an efficient proximity tagging system for the intended purpose. The TurboID/miniTurbo enzymes, on the other hand, offer the simplicity of not requiring an additional protein for their tagging activities.
In some embodiments, the polynucleotide further comprises an inducible promoter or a tissue-specific promoter that is operably linked and regulates the expression of the fusion protein.
Inducible promoters may be inducible by Cu2+, Zn2+, tetracycline, tetracycline analog, ecdysone, glucocorticoid, tamoxifen, or an inducer of the lac operon. The promoter may be inducible by ecdysone, glucocorticoid, or tamoxifen. In specific embodiments, the inducible promoter is a phage inducible promoter, nutrient inducible promoter, temperature inducible promoter, radiation inducible promoter, metal inducible promoter, hormone inducible promoter, steroid inducible promoter, or combination thereof. Examples of radiation inducible promoters include fos promoter, jun promoter, or erg promoter. An example of heat inducible promoter is UAS.
A tissue specific promoter can be a liver fatty acid binding (FAB) protein gene promoter, insulin gene promoter, transphyretin promoter, α1-antitrypsin promoter, plasminogen activator inhibitor type 1 (PAI-1) promoter, apolipoprotein AI promoter, LDL receptor gene promoter, myelin basic protein (MBP) gene promoter, glial fibrillary acidic protein (GFAP) gene promoter, opsin promoter, LCK promoter, CD4 promoter, keratin promoter, myoglobulin promoter, or neural-specific enolase (NSE) promoter.
The induction can also be achieved with the Cre-LoxP system, in which the Cre protein can be activated by tamoxifen which then removes the LoxP sequence from the regulated gene.
Methods of using the transgenic organisms are also provided for identifying a protein that binds to a target RNA The method can entail contacting activating the inducible promoter in the non-human transgenic organism in the presence of a guide RNA that is specific to the target RNA, under conditions to allow the Cas13 protein to bind to the target RNA and the proximity tagging enzyme to tag proteins bound to the target RNA.
The guide RNA may be introduced with a viral vector such as AAV, or expressed from a recombinant polynucleotide in the non-human transgenic organism, without limitation.
Fusion proteins, conjugates, compositions and kits are also provided which are useful for carrying out certain embodiments of the present technology.
In some embodiments, a kit or package is provided comprising a fusion protein of the present disclosure and a substrate for the proximity tagging enzyme to tag a molecule with. In some embodiments, the proximity tagging enzyme is PafA and the substrate is a Pup protein. In some embodiments, the kit or package further include a suitable guide RNA.
Polynucleotides are also provided that encode any of the proteins disclosed herein. In some embodiments, cells are provided that contain a polynucleotide or protein of the present disclosure.
This example demonstrates the development of a new tool, CRISPR-based RNA-United Interacting System (CRUIS), which uses the CRISPR-based RNA-target Cas nuclease as an RNA tracker to bring the proximity-labeling system to a designated target RNA. CRUIS can capture RNA-protein interactions of specific RNA sequences effectively. In CRUIS, a dead RNA-guided RNA targeting nuclease LwaCas13a (dLwaCas13a) was used as a tracker to target specific RNA sequences, while proximity enzyme PafA was fused to dLwaCas13a to label surrounding RNA-binding proteins. Subsequently, the labeled proteins were enriched and identified by mass spectrometry.
HEK293T cells were grown in DMEM (Hyclone) supplemented with 10% FBS (Biological Industries) in a humidified incubator at 37° C. with 5% CO2. All constructs were prepared using E.Z.N.A.® Endo-free Plasmid DNA Mini Kit (Omega, cat. #D6950-01B) and transfected with Lipofectamine 2000 (Thermo, cat. #11668019). The sequence of CRUIS is available in Table 1. Stable cell lines were generated with the piggyBac transposon system, which is widely applicable to various cell lines including non-mammalian cell lines. GFP-positive cells were enriched by flow sorting after transfection. Single colonies were picked, expanded, and tested via PCR, western blot, and enzyme activity identification for PafA. The HEK293T cell line with the best inducibility (referred to as 293T-CRUIS) was expanded and used for all subsequent experiments.
The CRUIS construct (dLwaCas13a-PafA-P2A-EGFP) was generated by subcloning dLwaCas13a fused with PafA at the C-terminus and a self-cleaving P2A peptide-linked EGFP (enhanced green fluorescent protein) into a piggyBac transposon backbone. dLwaCas13a was obtained by introducing two point mutations (R474A and R1046A) in the LwaCas13a (Addgene plasmid #90097) HEPN domains. The PafA was obtained from pEF6a-CD28-PafA (Addgene plasmid #113400). ClonExpress MultiS One Step Cloning Kit (Vazyme, cat. #C113-01) and Mut Express II Fast Mutagenesis Kit V2 (Vazyme, cat. #C214-01) were used for construct generation. The CRUIS plasmid will be deposited to the open-access platform Addgene.
293T-CRUIS cells were plated in 24-well tissue culture plates on poly-d-lysine coverslips and transfected with 500 ng ACTB-sgRNA, and then 100 mM sodium malonate was applied for 1.5 h before fixing and permeabilizing the cells. For immunofluorescence of G3BP1, cells were blocked with 5% BSA and incubated overnight at 4° C. with anti-G3BP1 primary antibody (Proteintech, cat. #13057-2-AP), and anti-myc primary antibody (Cell Signaling, cat. #9B11). Cells were then incubated for 2 h at room temperature with secondary antibody and mounted using the anti-fade mounting medium.
Total RNAs from 5×105 293T cells were extracted with Trizol (Invitrogen, Cat. #15596026) and RNA concentration were determined by NanoDrop 2000c (Thermo Fisher). cDNA was synthesized using 1 μg RNA by the reverse transcription kit PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Cat. #6210A) according to the manufacturer's instructions. Each qRT-PCR reaction was performed with cDNA transcribed from 25 ng RNA in a final volume of 20 μl with ChamQ™ SYBR Color qPCR Master Mix (Vazyme Cat. #Q431-03), assayed by QuantStudio™ 7 Flex (Life Technologies). The qPCR data were normalized to GAPDH expressions by relative quantification (ΔΔCt) method. The primers used were: CXCR4 (forward primer, 5′-ACTACACCGAGGAAATGGGCT-3′, SEQ ID NO:26; reverse primer, 5′-CCCACAATGCCAGTTAAGAAGA-3′, SEQ ID NO:27), p21 (forward primer, 5′-TGTCCGTCAGAACCCATGC-3′, SEQ ID NO:28; reverse primer, 5′-AAAGTCGAAGTTCCATCGCTC-3′, SEQ ID NO:29); NORAD (forward primer, 5′-CAGAGGAGGTATGCAGGGAG-3′, SEQ ID NO:30; reverse primer, 5′-GGATGTCTAGCTCCAAGGGG-3′, SEQ ID NO:31), β-actin (forward primer, 5′-CATGTACGTTGCTATCCAGGC-3′, SEQ ID NO:32; reverse primer, 5′-CTCCTTAATGTCACGCACGAT-3′, SEQ ID NO:33). GAPDH (forward primer, 5′-AGATCCCTCCAAAATCAAGTGG-3′, SEQ ID NO:34; reverse primer, 5′-GGCAGAGATGATGACCCTTTT-3′, SEQ ID NO:35).
293T-CRUIS cell lines transfected with or without pCMV-bio-pupE were analyzed by western blot. About 2 million cells were harvested and washed with cold PBS. Lysis buffer (1% Triton, 50 mM Tris 7.5, 150 mM NaCl) with 100× protease inhibitor was added to the pellet. Cells were resuspended and incubated on ice for 1 h. Then the lysate was spun down and the supernatant collected with the addition of protein loading buffer. The samples were boiled at 100° C. for 10 min and loaded on 4-20% SDS-PAGE gels, followed by immune-bolting with anti-myc antibody and streptavidin-HRP (Cell Signaling, cat. #3999s) to identify the expression of dCas13a-PafA fusion protein and the activity of PafA ligase.
For the enrichment of Bio-PupE modified proteins by streptavidin magnetic beads. Thirty-six hours after transfection with sgRNA or non-target sgRNA into the 293T-CRUIS cell line, the treated cells were harvested and lysed using cell lysate buffer. 20 μl streptavidin magnetic beads used for capturing labeled proteins from cell lysate supernatant and washed 3 times by wash buffer (8 M urea, 50 mM Tris 8.0, 200 mM NaCl). The obtained proteins were boiled at 100° C. for 20 min and used for western blot to analyze whether HNRNPK was modified by Bio-PupE, HNRNPK was identified by specific antibody (Proteintech, cat. #11426-1-AP).
About 30 million cells transfected with pCMV-bio-pupE and sgRNA were used for the mass spectrum. Cells were harvested and washed with cold PBS, then incubated with 2 ml lysis buffer at 4° C. After shaking for 1 h, the lysate was spun down at 4° C. for 10 min The supernatant was transferred into new tubes, with the addition of urea and DTT to a final concentration of 8 M and 10 mM. The lysate was incubated at 56° C. for 1 hour, then treated with 25 mM iodoacetamide in the dark for 45 min to aminocarbonyl modify the Cys site of proteins. 25 mM DTT was added to terminate the modification. Streptavidin-biotin magnetic beads were washed with 500 μl PBS three times and then resuspended in lysis buffer with an equal volume of beads. The lysate was then added 50 μl beads and it was incubated on a rotator at 4° C. overnight. The beads were washed with the following buffers: twice with buffer 1 (50 mM Tris 8.0, 8 M urea, 200 mM NaCl, 0.2% SDS), once with buffer 2 (50 mM Tris 8.0, 200 mM NaCl, 8 M urea), twice with buffer 3 (50 mM Tris 8.0, 0.5 mM EDTA, 1 mM DTT), three times with buffer 4 (100 mM ammonium carboxylate), and finally the beads were resuspended in 100 μl buffer 4. Trypsin, 4 μg (Promega, cat. #v5113) was added to digest overnight at 37° C. The peptides were collected with ziptip by the addition of 1% formic acid, then washed with 0.1% TFA (Sigmal, cat. #14264) and eluted in 50 μl of 70% ACN (Merck Chemicals, cat. #100030) −0.1% TFA. The peptides were analyzed on an Orbitrap Fusion.
For statistical analysis, the R package Limma was applied for the analysis of LFQ intensity data. The target RNA binding proteins were determined by a moderated t-test (p. value<0.05) and fold change (fold change>3). Previously reported RNA binding proteins were obtained from StarBase v2.0 (starbase.sysu.edu.cn). The R package clusterProfiler was used to identify significantly enriched biological processes in the RNA interactome (p-value cutoff=0.01, q-value cutoff=0.05, p. adjust method=Benjamini & Hochberg). The subcellular localization of the identified RBPs was analyzed by an online gene annotation & analysis resource “Metascape” (www.metascape.org). All data visualization was implemented in R using the ggplot2 package.
For RNA immunoprecipitation experiments, HEK293T cells were plated in a 6-cm dish and transfected with target protein expression plasmid (labeled with HA-tag at the C-terminus). Thirty-six hours after transfection, proteins were crosslinked to RNA by adding formaldehyde drop-wise directly to the medium to a final concentration of 0.75% and rotating gently at room temperature for 15 min. After crosslinking, 125 mM glycine in PBS was used for quenching, and the cells were incubated for 10 min at room temperature. Cells were washed with ice-cold PBS, harvested by scraping, and the cell suspension was centrifuged at 800 g for 4 min to pellet the cells. Cells were lysed with RIPA buffer supplemented with Protease Inhibitor Cocktail, EDTA-free and Recombinant RNasin® Ribonuclease Inhibitor (Promega cat. #N2515). Cells were allowed to lyse on a rotator for 20 min at 4° C. and then sonicated for 2 min with a 30 s on/30 s off cycle at low intensity on a Bioruptor sonicator (Diagenode) at 4° C. Insoluble material was pelleted by centrifugation at 16,000 g for 10 min at 4° C., and the supernatant containing the clarified lysate was split into two portions for pulling down with anti-HA magnetic beads (bimake cat. #B26202) or mouse IgG-conjugated magnetic beads overnight in a rotator at 4° C. After incubation with sample lysate, beads were pelleted, washed three times with RIPA buffer, and then washed with 1×DNase buffer (RNase-free). Beads were resuspended in 100 μl DNase buffer (RNase-free). DNase I (RNase-free) was added, followed by incubation at 37° C. for 30 min on a rotator. Proteins were then digested by the addition of Proteinase K (Takara cat. #9034) for about 2 hours at 37° C. with rotation. After that, MicroElute RNA Clean Up Kit (Omega cat. #R6247-01) was used for RNA purification. Purified RNA was reverse transcribed to cDNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, cat. #6210A), and pulldown was quantified with qPCR using ChamQ™ SYBR Color qPCR Master Mix (Vazyme cat. #Q431-03) and the Life Technologies QuantStudio™ 7 Flex. Enrichment was quantified for samples compared with their matched IgG antibody controls. The primers used for RIP-qPCR were: forward primer, 5′-GACAGGCCGAGCCCTCTGC-3′; reverse primer, 5′-GGCTTCAAGGTCTGGGCACAGC-3′.
To implement CRUIS in cells, this example first constructed a transfection vector which fused dLwaCas13a and PafA, and then cloned the fused dLwaCas13a-PafA gene in-frame with the self-cleaving P2A peptide sequence and EGFP, and the fusion gene driven by a CAG promoter (
In order to express dLwaCas13a-PafA at certain levels, this example created a monoclonal HEK293T cell line with stably integrated dLwaCas13a-PafA (referred to as 293T-CRUIS) by the piggyBac transposon system. For 293T-CRUIS cells, it is only necessary to transfect an expression vector of sgRNA and PupE to achieve the labeling of the RNA-binding proteins of target RNAs (
To determine whether CRUIS can bind to the target RNA, retain normal catalytic activity, and label surrounding proteins, this example first selected several 293T-CRUIS cell lines and determined the proximity targeting activity. It was confirmed that PafA retained the ability to label adjacent proteins in 293T-CRUIS cells (
To further confirm whether CRUIS would be able to recognize target RNA with a specific sgRNA, this example used ACTB-targeted sgRNA to determine whether CRUIS colocalizes with ACTB-containing stress granules under conditions induced by sodium malonate. Twenty-four hours after transfecting ACTB-targeting sgRNA into the 293T-CRUIS cell line, stress granules were induced by adding 100 mM sodium malonate into the culture medium Immunochemical labeling with an antibody against the stress granule marker G3BP1 demonstrated that CRUIS had been recruited specifically into the stress granules (
To prove the concept, this example applied CRUIS to study the RBPs of NORAD, a long non-coding RNA. NORAD plays an important role in genomic stability. Moreover, previous studies have suggested that RBPs are critical for the function of NORAD. To this end, this example transfected the NORAD-target sgRNA into the 293T-CRUIS. Biotin was added to the medium at 12 hours after the transfection. Twenty-four hours later, the cells were collected and lysed (
It was found that 51 candidates were significantly enriched in the NORAD targeting sgRNA group (p value<0.05) compared with the non-targeting sgRNA control group (
The candidate NORAD-binding proteins identified by CRUIS are involved in biological processes that are distinct from those of the control sample (
Using CRUIS, this example verified some NORAD-binding proteins identified previously (
Capturing RBPs of p21 mRNA
To determine whether CRUIS is able to identify RBPs for mRNAs, this example designed sgRNAs to target p21 mRNA and applied CRUIS. The data from mass spectrometry retrieved putative RBPs for p21 mRNA, some of them are known RBPs of p21 mRNA (marked in red) (
This example tested transgenic mouse and Drosophila models useful for implementing the CRUIS technology.
dCas13-PafA in Mouse
A construct was prepared that included CRUIS (dCas13-PafA) with LoxP sequences: pCAG-loxp-STOP-loxp-CRUIS. A transgenic mouse was obtained that had the construct integrated at the Rosa26 locus, and through mating with a mouse with CreER.
To activate the CRUIS, an AAV carry a polynucleotide encoding a sgRNA and PupE was injected to the tail of the mouse. The sgRNA and PupE were expressed in the liver of the mouse.
Expression of the CRUIS was triggered by injection of Tamoxifen. After the tagging, additional biotin was supplied with food. The mouse was sacrificed and liver obtained for mass spectrum analysis of the tagged proteins. This process is illustrated in
dCas13-PafA in Drosophila
The Drosophila model was prepared similar to the mouse model (see illustration in
dCas13-TurboID/miniTurbo in Mouse
In this example, the CRUIS used TurboID and miniTurbo as the proximity tagging enzyme. The construct for expression in the mouse (with CreER) included pCAG-loxp-STOP-loxp-CRUIS. The sgRNA was also introduced through an AAV vector, and the expression of the CRUIS was triggered by injected Tamoxifen. This process is illustrated in
dCas13-TurboID/miniTurbo in Drosophila
The construct was dU6-sgRNA-UAS-CRUIS, and the process is similar to the Drosophila model above (illustrated in
The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/076562 | Feb 2020 | CN | national |
This application is a continuation of International Patent Application No. PCT/CN2021/077602, filed Feb. 24, 2021, which claims the benefit of International Patent Application No. PCT/CN2020/076562, filed Feb. 25, 2020, the content of each of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/077602 | Feb 2021 | US |
| Child | 17822418 | US |
