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CN-121986161-A - Cas protein, CRISPR-Cas system containing same and application of CRISPR-Cas system

CN121986161ACN 121986161 ACN121986161 ACN 121986161ACN-121986161-A

Abstract

The present disclosure provides a Cas protein, a CRISPR-Cas system comprising the same, and applications thereof, in particular, the Cas protein of the present disclosure includes an OBD domain, REC domain, ruvC domain, helical domain, nuc domain, and has a structure shown in formula I or formula II. The Cas protein of the present disclosure has very good gene editing activity, can effectively edit or cleave a target gene, and can effectively treat a disorder or disease in a subject in need thereof.

Inventors

  • ZHANG HONGLING
  • FENG CHANGRUI

Assignees

  • 上海尧唐生物科技股份有限公司

Dates

Publication Date
20260505
Application Date
20241206
Priority Date
20231206

Claims (20)

  1. A Cas protein comprising an OBD domain, a REC domain, a RuvC domain, helical domain, and a Nuc domain; Optionally, the RuvC domain comprises RuvC-I, ruvC-II and RuvC-III domains; optionally, the Cas protein does not comprise an HNH domain and a PI domain; optionally, the RuvC-III domain is located between the Nuc-I domain and the Nuc-II domain; optionally, the OBD domain is a bi-split domain (bi-split domain) comprising an OBD-I domain, an OBD-II domain, said OBD-I domain being at the N-terminus and said Nuc-II domain being at the C-terminus; optionally, the Cas protein does not perform a nucleic acid cleavage function with the aid of tracrRNA.
  2. The Cas protein of claim 1, wherein the Cas protein has domains A1-A2-A3-A4-Z5 (I) in order from N-terminus to C-terminus, Wherein A1 is REC domain; A2 is an OBD domain; A3 is RuvC domain; A4 is Helical domain; Z5 contains a RuvC domain and a Nuc domain; and each "-" is independently a bond or a linker; Optionally, the OBD domain comprises an OBD-II domain; Optionally, the REC domain comprises a REC-I domain, a REC-II domain; Optionally, the RuvC domain comprises a RuvC-I domain, a RuvC-II domain, and a RuvC-III domain; optionally, Z5 has the structure shown in formula II, Y1-Y2-Y3-Y4 (II), Wherein Y1 is a RuvC-II domain; Y2 is a Nuc-I domain; y3 is a RuvC-III domain; Y4 is a Nuc-II domain; And each "-" is independently a bond or a linker.
  3. The Cas protein of claim 1, wherein the Cas protein has the structure from N-terminus to C-terminus of Z1-Z2-Z3-Z4-X-Z5 (III), Wherein Z1 is an absent or OBD-I domain; z2 is REC domain; Z3 is an OBD-II domain; Z4 is a RuvC-I domain; X is Helical domain; Z5 contains a RuvC domain and a Nuc domain; and each "-" is independently a bond or a linker; Optionally, the REC domain comprises a REC-I domain, a REC-II domain; Optionally, the RuvC domain is selected from the group consisting of a RuvC-II domain, a RuvC-III domain, or a combination thereof; optionally, the Nuc domain is selected from the group consisting of a Nuc-I domain, a Nuc-II domain, or a combination thereof; optionally, Z5 has the structure shown in formula II Y1-Y2-Y3-Y4 (II); Wherein Y1 is a RuvC-II domain; Y2 is a Nuc-I domain; y3 is a RuvC-III domain; Y4 is a Nuc-II domain; And each "-" is independently a bond or a linker.
  4. The Cas protein of claim 1, wherein the OBD domain, REC domain, ruvC domain, helical domain, nuc domain has an amino acid sequence that is at least about 80% (e.g., at least about 80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％ or 100%) sequence identical to the amino acid sequence of the OBD domain, REC domain, ruvC domain, helical domain, nuc domain in table 1; optionally, the Cas protein is about 500 to about 1200 amino acids in length, optionally, the Cas protein comprises about 700 and 1100 amino acids, more optionally, the Cas protein comprises about 900 and 1000 amino acids; Optionally, the Cas protein is a class 2V-type Cas endonuclease; Optionally, the Cas protein comprises a sequence having at least 70%, at least 75%, at least 80%, or at least 90% sequence identity to any one of SEQ ID NOs 1, 3, 5, 7-9.
  5. A fusion protein comprising the Cas protein of claim 1 and one or more functional domains; Optionally, the functional domain is selected from the group consisting of a localization signal, a reporter protein, a Cas protein targeting moiety, a DNA binding domain, an epitope tag, a transcriptional activation domain, a transcriptional repression domain, a nuclease, a deamination domain, a methylase, a demethylase, a transcriptional release factor, an HDAC, a lytic active polypeptide, a ligase, an integrase, a transposase, a recombinase, a polymerase, and a base excision repair inhibitor (e.g., uracil-DNA glycosylase inhibitor (UGI)).
  6. The fusion protein of claim 5, wherein the functional domain comprises enzymatic activity on a target sequence of one or more of the following: Methylase activity, demethylase activity, acetylase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMO activity, desumo activity, ribosylation activity, deglycosylation activity, myristoylation activity, glycosylation activity (e.g., from O-GlcNAc transferase) and deglycosylation activity; Optionally, the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain; optionally, the adenosine deaminase catalytic domain or cytidine deaminase catalytic domain comprises one or more of ADAR1, ADAR2, apodec, AID, or TAD; optionally, the adenosine deaminase catalytic domain comprises an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% or 100% identity to the amino acid sequence set forth in SEQ ID No. 30 and which retains deamination activity of the amino acid sequence set forth in SEQ ID No. 30; Optionally, the amino acid sequence of the adenosine deaminase catalytic domain exhibits amino acid additions, insertions, deletions and substitutions relative to the amino acid sequence set forth in SEQ ID NO. 30; Optionally, the catalytic domain of the adenosine deaminase comprises a mutant of the amino acid sequence shown in SEQ ID NO. 30 E18K+F19S+N20L, designated adenosine deaminase 004V14 (see WO2023193536A 1); Optionally, the adenosine deaminase catalytic domain comprises an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 31 (selected from 005V1 deaminase in CN114634923a, amino acid sequence of SEQ ID No.2 in this application) and which retains the deamination activity of the amino acid sequence shown in SEQ ID No. 31; Optionally, the amino acid sequence of the adenosine deaminase catalytic domain exhibits amino acid additions, insertions, deletions and substitutions relative to the amino acid sequence set forth in SEQ ID NO. 31; optionally, the adenosine deaminase catalytic domain comprises a mutant of the amino acid sequence shown in SEQ ID NO. 31, Q680G+Q149M+P150R, designated deaminase 005V1-10-3; Optionally, the functional domain is a full length or functional fragment of TadA e; Optionally, the localization signals comprise Nuclear Localization Signals (NLS) and/or nuclear output signals (NES); optionally, the sequence of the nuclear localization signal is shown in any one of SEQ ID NOs 22-29, 32-39, 41-45; Optionally, the sequence of the nuclear localization signal is located at, near or near the terminus (e.g., N-terminus or C-terminus) of the Cas protein of claim 1; optionally, the nuclear export signal comprises protein tyrosine kinase 2 (e.g., human protein tyrosine kinase 2); Optionally, the reporter protein comprises glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol Acetyl Transferase (CAT), beta-galactosidase, beta-glucuronidase, autofluorescent protein; Optionally, the autofluorescent proteins include green fluorescent proteins (e.g., GFP-2, tagGFP, turboGFP, eGFP, copGFP, aceGFP, etc.), hcRed, dsRed, cyan fluorescent proteins (e.g., eCFP, cerulean, cyPet, amCyanl, etc.), yellow fluorescent proteins (e.g., YFP, eYFP, citrine, venus, YPet, phiYFP, etc.), blue fluorescent proteins (e.g., eBFP2, azurite, mKalamal, GFPuv, sapphire, T-sapphire); Optionally, the DNA binding domain comprises a methylation binding protein, lexADBD, gal4DBD; optionally, the epitope tag comprises a histidine tag, a V5 tag, a FLAG tag, an influenza virus hemagglutinin tag, a Myc tag, a VSV-G tag, a thioredoxin tag, a streptavidin tag; optionally, the transcriptional activation domain comprises VP64 and/or VPR; Optionally, the transcription repression domain comprises KRAB and/or SID; optionally, the nuclease comprises fokl; Optionally, the cleavage active polypeptide comprises a polypeptide having single-stranded RNA cleavage activity, a polypeptide having double-stranded RNA cleavage activity, a polypeptide having single-stranded DNA cleavage activity, or a polypeptide having double-stranded DNA cleavage activity; Optionally, the ligase comprises a DNA ligase and/or an RNA ligase; optionally, the functional domain is linked to the N-terminus, and/or the C-terminus, of the Cas protein; Optionally, the functional domain is inserted between the N-terminus and the C-terminus of the Cas protein; optionally, the one or more functional domains are linked to the N-terminus and/or C-terminus of the Cas protein, optionally through a linker; Optionally, the functional domain is inserted between the N-terminus and the C-terminus of the Cas protein through a linker.
  7. The fusion protein of claim 5, wherein the fusion protein has the structure Z1-Z2 (I '), or Z2-Z1 (II '), or Z3-Z1-Z4 (III ') from N-terminus to C-terminus; Wherein Z1 is cytosine deaminase or adenosine deaminase; z2 is the Cas protein of claim 1; Z3 is the N-terminal fragment of the Cas protein of claim 1; z4 is the C-terminal fragment of the Cas protein of claim 1; And each "-" is independently a bond or a linker.
  8. An isolated polynucleotide encoding the Cas protein of claim 1 or the fusion protein of claim 5; Optionally, the isolated nucleotide comprises a sequence that is optimized by humanization; Optionally, the polynucleotide additionally comprises an auxiliary element selected from the group consisting of a signal peptide, a secretory peptide, a tag sequence (e.g., 6 His), or a combination thereof, flanking the ORF of the variant; optionally, the polynucleotide is selected from the group consisting of genomic sequences, cDNA sequences, RNA sequences, or combinations thereof; optionally, the polynucleotide further comprises a promoter operably linked to the ORF sequence of the variant; Optionally, the promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter; optionally, the polynucleotide has been codon optimized for expression in eukaryotic cells; optionally, the polynucleotide is a polynucleotide that is codon optimized according to the codon preference of the host cell; optionally, the host cell comprises a prokaryotic cell or a eukaryotic cell; Optionally, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell, or a mammalian cell (including human and non-human mammals); Optionally, the host cell is a prokaryotic cell, such as E.coli; Optionally, the yeast cell is selected from one or more sources of the group consisting of Pichia, kluyveromyces, or combinations thereof; Optionally, the yeast cells include Kluyveromyces marxianus, more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis; Optionally, the host cell is selected from the group consisting of E.coli, wheat germ cells, insect cells, SF9, hela, HEK293, CHO, yeast cells, or combinations thereof; Optionally, the polynucleotide comprises a sequence having at least 70%, at least 75%, at least 80%, or at least 90% sequence identity to any one of SEQ ID NOs 16, 40, or 46.
  9. A guide RNA (gRNA), the guide RNA comprising (I) A homeotropic repeat (DIRECT REPEAT, DR) sequence and a polypeptide capable of binding to the Cas protein of claim 1 (Ii) A spacer (spacer) sequence capable of targeting a target sequence of a target DNA, the guide RNA configured to form a complex with the Cas protein.
  10. A vector comprising the polynucleotide of claim 9.
  11. A composite of a metal and a silicon-containing material, characterized by comprising: (i) A protein component selected from the group consisting of the Cas protein of claim 1, the fusion protein of claim 5, or a combination thereof, and (Ii) A nucleic acid component selected from the group consisting of the guide RNA of claim 9, a nucleic acid encoding the guide RNA of claim 9, a precursor RNA of the guide RNA of claim 9, a precursor RNA nucleic acid encoding the guide RNA of claim 9, or a combination thereof, the protein component and the nucleic acid component binding to each other to form a complex; Wherein the guide RNA comprises: (iii) A homeotropic repeat (DIRECT REPEAT, DR) sequence and a polypeptide capable of binding to the Cas protein of claim 1 (Iv) Spacer (spacer) sequences capable of targeting a target sequence of a target DNA.
  12. A CRISPR-Cas composition comprising: (i) A first component selected from the group consisting of a Cas protein of claim 1, a fusion protein of claim 5, a nucleotide sequence encoding a Cas protein of claim 1 or a fusion protein of claim 5, and any combination thereof, and (Ii) A second component which is a guide RNA comprising one or more of the guide RNAs of claim 9 or encodes the nucleotide sequence comprising one or more of the guide RNAs of claim 9, the guide RNA comprising: (iii) A homeotropic repeat (DIRECT REPEAT, DR) sequence and a polypeptide capable of binding to the Cas protein of claim 1 (Iv) A spacer (spacer) sequence capable of targeting a target sequence of a target DNA, the guide RNA configured to form a complex with the Cas protein; Optionally, the composition comprises a pharmaceutical composition; optionally, the dosage form of the composition is selected from the group consisting of a lyophilized formulation, a liquid formulation, or a combination thereof; Optionally, the dosage form of the composition is a liquid formulation; optionally, the dosage form of the composition is an injectable dosage form; Optionally, the composition is a cellular preparation.
  13. A CRISPR-Cas system is characterized in that, comprising one or more vectors, the one or more vectors comprising: (i) A first nucleic acid that is a nucleotide sequence encoding the Cas protein of claim 1 or the fusion protein of claim 5, optionally operably linked to a first regulatory element, and (Ii) A second nucleic acid encoding the nucleotide sequence of the guide RNA of claim 9; Optionally, the second nucleic acid is operably linked to a second regulatory element; the guide RNA comprises: (iii) A homeotropic repeat (DIRECT REPEAT, DR) sequence and a polypeptide capable of binding to the Cas protein of claim 1 (Iv) A spacer (spacer) sequence capable of targeting a target sequence of a target DNA, the guide RNA configured to form a complex with the Cas protein; wherein the first nucleic acid and the second nucleic acid are present on the same or different vectors.
  14. The complex of claim 11, the CRISPR-Cas composition of claim 12, or the CRISPR-Cas system of claim 13, wherein, The spacer sequence is greater than 17 nucleotides in length, preferably 17 to 100 nucleotides, more preferably 16 to 50 nucleotides (e.g., ,17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50 nucleotides), more preferably 17 to 50 nucleotides, more preferably 17 to 40 nucleotides, more preferably 18 to 39 nucleotides, and most preferably 18 to 37 nucleotides; Optionally, the target DNA is selected from double-stranded DNA, single-stranded DNA, RNA, genomic DNA, and extrachromosomal DNA; Optionally, the spacer sequence is linked to the 3' end of the co-repeat (DIRECT REPEAT, DR) sequence; optionally, the spacer (spacer) sequence comprises a complement of the target sequence; Optionally, the target sequence is located adjacent to the 3 'end of the motif (PAM) of the protospacer sequence and the PAM is 5' -TN, wherein N is A, T, G or C; Optionally, the target sequence is DNA from a prokaryotic or eukaryotic cell or a DNA sequence formed based on RNA reverse transcription, or the target sequence is non-naturally occurring DNA or a DNA sequence formed based on RNA reverse transcription; optionally, the target DNA is located inside or outside the cell; optionally, the cell is a eukaryotic cell or a prokaryotic cell; Optionally, the cell is selected from animal cells, plant cells, fungal cells; Optionally, the eukaryotic cell is a plant cell, mammalian cell, insect cell, arthropod cell, fungal cell, bird cell, reptile cell, amphibian cell, invertebrate cell, mouse cell, rat cell, primate cell, non-human primate cell, or human cell; Optionally, further comprising a DNA donor template that can be inserted at the locus of interest by Homology Directed Repair (HDR); optionally, the donor template nucleic acid has a length of 8-1000 nucleotides; optionally, the donor template nucleic acid has a length of 25-500 nucleotides; optionally, the vector comprises a plasmid, a viral vector; Optionally, the guide RNA comprises unmodified and modified guide RNAs; Optionally, the modified guide RNA comprises a chemical modification of a base; optionally, the chemical modification comprises a methylation modification, a methoxy modification, a fluorination modification or a thio modification; Optionally, the first regulatory element and/or the second regulatory element is a promoter, such as an inducible promoter, a constitutive promoter, a ubiquitous (ubiquitous) promoter, a cell type specific promoter or a tissue specific promoter; optionally, at least one component of the composition is non-naturally occurring or modified; Optionally, the DR sequence comprises a structure represented by formula IV 5'-R1a-Ba-R2a-L-R2b-Bb-R1b-3' (IV), Wherein segments R1a and R1b are reverse complement sequences and form a first stem (R1), the first stem (R1) having a plurality (2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10) of nucleotide pairs in the Cas protein; Segments Ba and Bb do not base pair with each other and form a bulge (B); Segments R2a and R2b are reverse complement sequences and form a second stem (R2), the second stem (R2) having a loop formed at the second stem of a plurality (2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10) of nucleotides; Optionally, the DR sequence has a secondary structure substantially identical to the secondary structure of the DR sequence set forth in any one of SEQ ID NOs 2,4, 6; optionally, the DR sequence has nucleotide additions, insertions, deletions or substitutions that do not result in substantial differences in secondary structure compared to the DR sequence set forth in any one of SEQ ID NOs 2,4, 6; Optionally, the DR sequence comprises or consists of a sequence selected from the group consisting of seq id nos: (i) A sequence shown in any one of SEQ ID NO 2,4 and 6; (ii) A sequence having a substitution, deletion or addition of one or more bases (e.g., a substitution, deletion or addition of 1,2,3,4, 5,6,7, 8, 9 or 10 bases) as compared to the sequence set forth in any one of SEQ ID NOs 2,4, 6; (iii) A sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOs 2,4, 6; (iv) A sequence which hybridizes under stringent conditions to a sequence as set forth in any one of (i) to (iii), or (V) A complement of the sequence set forth in any one of (i) - (iii); And, the sequence of any one of (ii) - (v) substantially retains the biological function of the sequence from which it was derived.
  15. A kit comprising one or more components selected from the group consisting of the Cas protein of claim 1, the fusion protein of claim 5, the polynucleotide of claim 8, the vector of claim 10, the complex of claim 11, the CRISPR-Cas composition of claim 12, and the CRISPR-Cas system of claim 13; optionally, the kit further comprises a label or instructions; Optionally, the kit is used for one or more of gene or genome editing, disease treatment, targeting a target gene, cleaving a gene of interest or a non-gene of interest.
  16. A delivery composition comprising a delivery vector or delivery medium and one or more selected from the group consisting of the Cas protein of claim 1, the fusion protein of claim 5, the polynucleotide of claim 8, the vector of claim 10, the complex of claim 11, the CRISPR-Cas composition of claim 12, and the CRISPR-Cas system of claim 13; Optionally, the delivery vehicle is a particle; Optionally, the delivery vehicle is selected from a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microbubble, a gene gun, or a viral vector (e.g., replication defective retrovirus, lentivirus, adenovirus, or adeno-associated virus); Optionally, the delivery medium comprises a nanoparticle, a liposome, an exosome, a microvesicle, an electrotransport device, or a gene gun.
  17. A host cell comprising the Cas protein of claim 1, the fusion protein of claim 5, the polynucleotide of claim 8, the vector of claim 10, the complex of claim 11, the CRISPR-Cas composition of claim 12, the CRISPR-Cas system of claim 13, or the delivery composition of claim 16; Optionally, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell, or a mammalian cell (including human and non-human mammals); Optionally, the host cell is a prokaryotic cell, such as E.coli; Optionally, the yeast cell is selected from one or more sources of Pichia yeast, kluyveromyces yeast, or a combination thereof, preferably, the yeast cell comprises Kluyveromyces yeast, more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis; Optionally, the host cell is selected from the group consisting of E.coli, wheat germ cells, insect cells, SF9, hela, HEK293, CHO, yeast cells, or combinations thereof.
  18. An enzyme preparation comprising the Cas protein of claim 1, the fusion protein of claim 5, the complex of claim 11, the CRISPR-Cas composition of claim 12, the CRISPR-Cas system of claim 13, or the delivery composition of claim 16; optionally, the enzyme formulation comprises an injection, and/or a lyophilized formulation.
  19. A medicine box, which comprises a medicine box body, characterized by comprising the following steps: A first container, and the CRISPR-Cas composition of claim 12 or the CRISPR-Cas system of claim 13 or containing the CRISPR-Cas composition of claim 12 or the CRISPR-Cas system of claim 13 in the first container; Optionally, the CRISPR-Cas composition of claim 12 or the CRISPR-Cas system of claim 13 is contained in the first container; Optionally, the composition is a pharmaceutical composition; optionally, the pharmaceutical composition is in a dosage form selected from the group consisting of a lyophilized formulation, a liquid formulation, or a combination thereof; optionally, the dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form; Optionally, the kit further comprises instructions.
  20. A medicine box, which comprises a medicine box body, characterized by comprising the following steps: (a1) A first container, and the Cas protein of claim 1, or the fusion protein of claim 5, or the encoding gene thereof, or the expression vector thereof, or a medicament containing the Cas protein of claim 1, or the fusion protein of claim 5, or the encoding gene thereof, or the expression vector thereof, in the first container; (b1) An optional second container, and the guide RNA of claim 9 or an expression vector thereof, or a medicament containing the guide RNA of claim 9 or an expression vector thereof, located in the second container; Optionally, the first container and the second container are different containers; Optionally, the drug of the first container is a unilateral preparation containing the Cas protein of claim 1, or the fusion protein of claim 5, or the encoding gene thereof, or the expression vector thereof; Optionally, the drug in the second container is a unilateral formulation comprising the guide RNA or the expression vector thereof of claim 9; optionally, the pharmaceutical dosage form is selected from the group consisting of a lyophilized formulation, a liquid formulation, or a combination thereof; Optionally, the dosage form of the medicament is an oral dosage form or an injection dosage form; Optionally, the kit further comprises instructions.

Description

Cas protein, CRISPR-Cas system containing same and application of CRISPR-Cas system Cross Reference to Related Applications The present application claims the benefit and priority of patent application number CN202311664660.5, titled "a Cas protein, CRISPR-Cas system comprising it and its use", filed on 12/06/2023, the entire contents of which, including any sequence listings and figures, are incorporated herein by reference in their entirety. Reference to electronic sequence list The present disclosure contains an electronic sequence list ("P2024-3068 xlb.xml", created by "WIPOSequence" software according to WIPO standard st.26), which is incorporated herein by reference in its entirety. The symbol "T" is used to denote T in DNA and U in RNA according to WIPO Standard ST.26. Thus, in the list of sequences prepared according to st.26, whenever the sequence is RNA, T in the sequence should be considered U. Technical Field The present disclosure relates to the field of gene editing, in particular, to a Cas protein, a CRISPR-Cas system comprising the same, and applications thereof. Background Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR/Cas systems, are currently considered to be immunity to phage infection by bacteria and archaebacteria. The CRISPR-Cas system of prokaryotic adaptive immunity is an extremely diverse set of protein effectors (proteineffector), non-coding elements and locus structures that can be engineered and used for gene editing, target detection, and disease treatment applications. There are a variety of Cas proteins and corresponding editing techniques available, and there remains a need in the art for new Cas proteins and CRISPR-Cas systems to meet the diverse application needs. Disclosure of Invention The primary object of the present disclosure is to provide novel Cas proteins and CRISPR-Cas systems to meet diverse application needs. In one aspect, the present disclosure provides a Cas protein comprising an OBD domain, REC domain, ruvC domain, helical domain, nuc domain. In some embodiments, the RuvC domain comprises RuvC-I, ruvC-II and RuvC-III domains. In some embodiments, the Cas protein does not comprise a HNH domain and a PI domain. In some embodiments, the RuvC-III domain is located between the Nuc-I domain and the Nuc-II domain. In some embodiments, the OBD domain is a bi-split domain (bi-split domain), comprising an OBD-I and an OBD-II domain. In some embodiments, the OBD-I domain is located at the N-terminus and the Nuc-II domain is located at the C-terminus. In some embodiments, the Cas protein does not perform a nucleic acid cleavage function with a tracrRNA. In another aspect, the present disclosure provides a fusion protein comprising a Cas protein of the present disclosure, and one or more functional domains. In some embodiments, the functional domain is selected from the group consisting of a localization signal, a reporter protein, a Cas protein targeting moiety, a DNA binding domain, an epitope tag, a transcriptional activation domain, a transcriptional repression domain, a nuclease, a deamination domain, a methylase, a demethylase, a transcriptional release factor, an HDAC, a lytic active polypeptide, a ligase, an integrase, a transposase, a recombinase, a polymerase, and a base excision repair inhibitor (e.g., uracil-DNA glycosylase inhibitor (UGI)). In some embodiments, the functional domain includes one or more of enzymatic activity to a target sequence, methylase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, dimyristoylation activity, glycosylation activity (e.g., from an O-GlcNAc transferase) and deglycosylation activity. In some embodiments, the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain. In one aspect, the present disclosure provides an isolated polynucleotide encoding a Cas protein described herein or a fusion protein described herein. In one aspect, the present disclosure provides an isolated nucleic acid molecule comprising a structure according to formula IV: 5'-R1a-Ba-R2a-L-R2b-Bb-R1b-3'(IV), wherein segments R1a and R1b are reverse complement sequences and form a first stem (R1), the first stem (R1) having a plurality (2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10) of nucleotide pairs in the Cas protein; Segments Ba and Bb do not base pair with each other and form a bulge (B); Segments R2a and R2b are reverse complement sequences and form a second stem (R2), the second stem (R2) having loops formed at a plurality (2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10) of base pairs, and L is a pl