阅读说明：本技术 基于aav病毒的基因编辑表达盒 (AAV virus-based gene editing expression cassette ) 是由 褚贝贝 杨国宇 王江 刘忠虎 汪新建 钟凯 鲁维飞 郭豫杰 于 2018-09-11 设计创作，主要内容包括：本发明涉及基因编辑领域。具体而言,本发明涉及基于AAV病毒的基因编辑表达盒。更具体而言,本发明涉及基于AAV病毒的基因编辑表达盒,以及包括所述表达盒的载体和利用所述表达盒或载体的基因编辑方法。(The present invention relates to the field of gene editing. In particular, the invention relates to AAV virus-based gene editing expression cassettes. More particularly, the present invention relates to AAV virus-based gene editing expression cassettes, as well as vectors comprising the expression cassettes and gene editing methods using the expression cassettes or vectors.)

1. An expression cassette comprising two Inverted Terminal Repeats (ITRs) at the 5 'end and the 3' end of the expression cassette, respectively, and a first promoter located between the two inverted terminal repeats, a first polynucleotide encoding a Cas9 polypeptide operably linked to the first promoter, a plurality of tandem promoter-sgRNA units, wherein a spacer sequence is present between the tandem promoter-sgRNA units, and wherein the expression cassette is no more than 5.0kb in size.

2. The expression cassette of claim 1, wherein the number of promoter-sgRNA units is 2,3, 4, or more.

3. The expression cassette of claim 1, wherein the 5 'inverted terminal repeat AAV2ITR 5' sequence located at the 5 'end of the expression cassette is set forth in SEQ ID NO. 7, and the 3' inverted terminal repeat AAV2ITR3 'sequence located at the 3' end of the expression cassette is set forth in SEQ ID NO. 8.

4. The expression cassette of claim 1, wherein the promoter in the promoter-sgRNA unit is a tRNA coding sequence.

5. The expression cassette of claim 1, wherein the first promoter is the EF1 α promoter of SEQ ID NO 10.

6. The expression cassette of claim 4, wherein said tRNA coding sequence is any mammalian tRNA, e.g., GlntRNA, Pro tRNA, Gly tRNA, Asn tRNA, Cys tRNA, Glu tRNA.

7. The expression cassette of claim 4, wherein said tRNA coding sequence is Gln tRNA shown in SEQ ID NO 9.

8. The expression cassette of claim 4, wherein the promoter is mouse gamma herpes virus-68 (MHV68) RNA.

9. The expression cassette of claim 1, wherein the spacer sequence has a length of no more than 40bp, such as 10bp, 20bp or 40bp, preferably 20bp or 40bp, most preferably 20 bp.

10. The expression cassette of claim 1, wherein the Cas9 polypeptide is staphylococcus aureus (staphyloccocusareureus) Cas9(SaCas9), optionally linked to a Nuclear Localization Sequence (NLS), and the sgRNA in the promoter-sgRNA unit is the sgRNA corresponding to SaCas 9.

11. The expression cassette of claim 1, comprising, in order from the 5 'to 3' direction, AAV2ITR 5', EF1 α promoter, SaCas9 expression sequence operably linked to EF1 α promoter, no more than 4 tandem tRNA coding sequences-sgRNA units corresponding to SaCas9, and AAV2ITR 3'.

12. The expression cassette of claim 1, comprising a nucleotide sequence as set forth in SEQ ID NO 1-3.

13. A recombinant vector comprising the expression cassette of any one of claims 1-12.

14. The recombinant vector of claim 13, wherein the vector is an adeno-associated viral vector.

15. A kit comprising the expression cassette of any one of claims 1 to 12 or the recombinant vector of any one of claims 13 to 15.

16. A method of gene editing comprising the step of delivering the expression cassette of any one of claims 1 to 12 or the recombinant vector of any one of claims 13 to 14 to a cell of a subject.

Technical Field

The present invention relates to the field of gene editing. In particular, the invention relates to AAV virus-based gene editing expression cassettes. More particularly, the present invention relates to AAV virus-based gene editing expression cassettes, as well as vectors comprising the expression cassettes and gene editing methods using the expression cassettes or vectors.

Background

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and related proteins 9(CRISPR-associated proteins 9, Cas9) have become a revolutionary tool in the fields of basic biological research, biochemistry, agriculture, medical industry and the like (Barrangou et al, CRISPR protocols acquired resistance in proof viruses [ J ] Science,2007,315(5819) 1709. quadrature. 1712; Doudna and Charpy, Genome analysis. high sensitivity of Genome engineering with respect to genetic engineering with CRISPR-9 [ J ] Science, 346, 1258096; growth, Development and design of genetic engineering with respect to biological samples [ 17. J ] Cas J ] 35. CRISPR, Cas J ] 26, and 5. CRISPR J ] 9. CRISPR J. 12. CRISPR, CRISPR-11. CRISPR, CRISPR-J. 157, CRISPR-associated proteins [ 10. J ] 1. J. 10. CRISPR, CRISPR-associated proteins J. 10. J. CRISPR. 10. J. CRISPR. 10. 12. CRISPR. 10. J. 12. CRISPR. 12, CRISPR-associated proteins J. 10. 12. CRISPR. 10. CRISPR. 10. branched, CRISPR. branched, 12. branched, branched, 2016,34(9):933-941.). The method has the advantages of simple design, convenient operation and low cost, can be used for cutting or combining specific DNA or RNA sequences, and gradually becomes a standard application program of technologies such as gene editing, gene regulation, gene therapy and the like. In 2012, Doudna et al ligated CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to construct a single-stranded guide RNA (sgRNA) vector, and confirmed that the DNA fragment could be cleaved in vitro together with Cas 9. It is only necessary to alter the sequence in the sgRNA which is complementary to the gene of interest to cause double-strand break (DSB) (Jinek et al, A programmable dual-RNA-bound DNA endonuclearase in adaptive bacterial linearity [ J ] Science,2012,337(6096): 816-821.). The broken DNA is generally repaired by two pathways: mainly non-homologous end joining (NHEJ), resulting in random Insertion or deletion (insert or deletion, Indel) of bases at The disconnection position (Lieber, The mechanism of double-strand DNA break repair by The non-homologous DNA end-joining pathway [ J ]. AnnuRev Biochem,2010,79: 181) to form a frameshift mutation, thereby resulting in gene knock-out; the other is homologous recombination repair pathway (HDR), which can specifically repair the cleavage site by using a template with homologous arms (San Filippo et al, Mechanism of eukaryotic homology recombination [ J ] Annu Rev Biochem,2008,77: 229-once 257) to perform the functions of gene insertion, deletion, mutation, etc. The use of the CRISPR/Cas9 system for genome editing in eukaryotic cells was also published by Zhang and Church in 2013, and has milestone significance in genetic studies (Cong et al, multiple genome engineering CRISPR/Cas systems [ J ] Science,2013,339(6121): 819. 823; Mali P et al, RNA-guided human genome engineering via Cas9[ J ] Science,2013,339(6121): 823. 826).

The sgRNA plays a role in guiding the Cas9 protein to target a target DNA as an important component of CRISPR/Cas9 technology. In order to simultaneously cleave multiple target sites, it is necessary to use multiple sgRNA vectors. However, co-transfection of multiple plasmids in cells tends to cause inefficient transfection (Wang et al, One-step generation of plasmid DNA by CRISPR/Cas-mediated gene engineering [ J ] Cell,2013,153(4): 910-. Cao et al constructed up to 6 tandem lentiviral vectors each initiating transcription of sgRNA with the U6 promoter in a one-step cloning approach and demonstrated that they could function in cells (Cao et al, An easy and efficient independent CRISPR/Cas9platform with improved specificity for multiple gene targeting [ J ]. Nucleic Acids Res,2016,44(19): e 149.). However, lentiviruses are carried in larger amounts than AAV, and can effectively exert the multi-gene editing function of the Cas9 system. Yin and the like construct AAV vectors which contain 4 sgRNAs connected In series and start transcription by U6 promoters by an In-Fusion method, and HIV-1 model mice are infected after the viruses are packaged, so that HIV Provirus (Yin et al, In Vivo infection of HIV-1Provirus by saCas9and multiple Single-Guide RNAs In Animal Models [ J ] Mol Ther,2017,25(5): 1168-1186) can be effectively eliminated, but the recombinant vector connected with 4 sgRNAs In series has larger length, the packaged virus activity is poorer, and larger virus amount is needed In use, so that the test cost is increased.

AAV is considered the most promising viral vector for development, and has the advantages of non-integration into the host genome, low immunogenicity, non-pathogenicity, and the like. However, its carrying capacity is small (about 4.7kb), limiting its range of use. In view of the broad prospects of AAV-mediated CRISPR/Cas9 system gene editing in the biomedical field, there is a need to optimize AAV vectors to overcome the above-mentioned deficiencies.

Summary of The Invention

In one aspect, the invention provides an expression cassette comprising two Inverted Terminal Repeats (ITRs) located at the 5 'end and the 3' end of the expression cassette, respectively, and a first promoter located between the two inverted terminal repeats, a first polynucleotide encoding a Cas9 polypeptide operably linked to the first promoter, a plurality of tandem promoter-sgRNA units, wherein a spacer sequence is present between the tandem promoter-sgRNA units, and wherein the expression cassette is no more than 5.0kb in size.

In some embodiments, the number of promoter-sgRNA units is 2,3, 4, or more.

In some embodiments, the inverted terminal repeat AAV2ITR5 'sequence located 5' to the expression cassette is shown in SEQ ID NO. 7 and the inverted terminal repeat AAV2ITR3 'sequence located 3' to the expression cassette is shown in SEQ ID NO. 8.

In some embodiments, the promoter in the promoter-sgRNA unit is a tRNA coding sequence.

In some embodiments, the first promoter is the EF1 α promoter shown in SEQ ID NO. 10.

In some embodiments, the tRNA coding sequence is any mammalian tRNA, e.g., Gln tRNA, Pro tRNA, Gly tRNA, Asn tRNA, Cys tRNA, Glu tRNA.

In some embodiments, the tRNA coding sequence is a Gln tRNA shown in SEQ ID NO 9.

In some embodiments, the promoter is mouse gamma herpes virus-68 (MHV68) RNA

In some embodiments, the spacer sequence is no more than 40bp in length, for example 10bp, 20bp or 40bp, preferably 20bp or 40bp, most preferably 20 bp.

In some embodiments, the Cas9 polypeptide is Staphylococcus aureus (Staphylococcus aureus) Cas9(SaCas9), optionally linked to a Nuclear Localization Sequence (NLS), can be followed by a transcription termination signal PolyA, and the sgRNA in the promoter-sgRNA unit is the sgRNA corresponding to SaCas 9.

In some embodiments, an expression cassette of the invention comprises, in order from the 5'-3' direction, AAV2ITR 5', EF1 α promoter, SaCas9 expression sequence operably linked to EF1 α promoter, no more than 4 tandem tRNA coding sequences-sgRNA units corresponding to SaCas9, and AAV2ITR 3'.

In some embodiments, the nucleic acid sequence comprises the nucleotide sequence set forth as SEQ ID NOS: 1-3.

In another aspect, the present invention provides a recombinant vector comprising the expression cassette of the invention.

In some embodiments, the vector is an adeno-associated viral vector.

In another aspect, the invention provides a kit comprising an expression cassette of the invention or a recombinant vector of the invention.

In another aspect, the present invention provides a method of gene editing comprising the step of delivering the expression cassette of the present invention or the recombinant vector of the present invention to a cell.

Drawings

FIG. 1 shows a schematic diagram of a pX601(EF1 α -tRNA) recombinant vector.

FIG. 2 shows a schematic representation of the scaffold-U6/tRNA recombinant vector. FIG. 2a is a scaffold-U6 recombinant vector, and FIG. 2b is a scaffold-tRNA recombinant vector; the underlined parts indicate the insertion of spacer sequences of 0, 10, 20, 40bp at this site.

FIG. 3 shows a schematic structural diagram of tandem sgRNA recombinant vectors, ITR is an inverted terminal repeat sequence, NLS is a nuclear localization signal sequence, HA is an HA tag, Scaf is a scaffold sequence, EF1 α, CMV, tRNA and U6 are corresponding promoters, t4, 1t4, 2t4 and 4t4 are recombinant vectors which respectively contain tandem 4 sgRNAs with interval sequence lengths of 0, 10, 20 and 40bp and take EF1 α and tRNA as promoters, U4, 1U4, 2U4 and 4U4 are recombinant vectors which respectively contain tandem 4 sgRNAs with interval sequence lengths of 0, 10, 20 and 40bp and take CMV and U6 as promoters.

Fig. 4 shows the results of T7 endonuclease I detection of gene editing of tandem sgRNA recombinant vectors in NIH3T3 cells. FIG. 4a shows the detection results of different tandem groups of T7 endonuclease I methods at the mMSTN-sgRNA1 site; FIG. 4b shows the detection results of different tandem groups of T7 endonuclease I methods at the mMSTN-sgRNA2 site; FIG. 4c shows the result of detection by T7 endonuclease I method for different tandem groups of mTyr-sgRNA3 sites; FIG. 4d shows the detection results of the T7 endonuclease I method for different tandem groups of mRosa26-sgRNA2 sites. SU is an sgRNA group which takes U6 as a promoter; st is an sgRNA group using tRNA as a promoter; m is 50bp DNA Ladder; c^-Is a negative control. The arrow indicates the cut fragment of interest.

Fig. 5 shows T7 endonuclease I detection of AAV-DJ mediated effect of tandem sgRNA recombinant vectors on gene editing in NIH3T3 cells. FIG. 5a shows the detection results of different tandem groups of T7 endonuclease I methods at the mMSTN-sgRNA1 site; FIG. 5b shows the detection results of different tandem groups of T7 endonuclease I methods at the mMSTN-sgRNA2 site; FIG. 5c shows the result of detection by T7 endonuclease I method for different tandem groups of mTyr-sgRNA3 sites; FIG. 5d shows the detection results of the T7 endonuclease I method for different tandem groups of mRosa26-sgRNA2 sites. M is 50bp DNA Ladder; c^-Is a negative control. The arrow indicates the cut fragment of interest.

Detailed Description

Unless otherwise indicated or defined, all terms used have the ordinary meaning in the art that will be understood by those skilled in the art. Reference is made, for example, to standard manuals, such as Sambrook et al, "Molecular Cloning: Laboratory Manual" (2 nd edition), Vol.1-3, Cold Spring Harbor Laboratory Press (1989); lewis, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al, "Immunology" (2 nd edition), Gower Medical Publishing, London, New York (1989), and the general prior art cited herein; moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically recited may be and have been performed in a manner known per se to those of skill in the art. Reference is also made, for example, to standard manuals, the general prior art mentioned above and to other references cited therein.

As used in the specification and in the claims, ordinal indicators, such as first, second and third, for different structures or method steps should not be construed to indicate any particular structure or step, or any particular order or configuration of such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

Recombinant expression cassette

In one aspect, the invention provides an expression cassette comprising two Inverted Terminal Repeats (ITRs) located at the 5 'end and the 3' end of the expression cassette, respectively, and a first promoter located between the two inverted terminal repeats, a first polynucleotide encoding a Cas9 polypeptide operably linked to the first promoter, a plurality of tandem promoter-sgRNA units, wherein a spacer sequence is present between the tandem promoter-sgRNA units, and wherein the expression cassette is no more than 5.0kb in size.

In some embodiments, the number of promoter-sgRNA units is 2,3, 4, or more.

As used herein, the term "CRISPR" refers to regularly clustered interspaced short palindromic repeats, which constitute a family of loci typically consisting of short and highly conserved DNA repeats, e.g., repeats 1-40 times and at least part of 24-50 base pairs of a palindrome. The repeated sequences are typically species specific and are separated by variable sequences of constant length, e.g., 20-58 base pairs. The CRISPR locus may also encode one or more proteins and one or more RNAs that are not translated into a protein. Thus, a "CRISPR-Cas" system is a system that is identical to or derived from a bacterium or archaea and contains at least one Cas protein encoded by or derived from a CRISPR locus.

The abbreviation "Cas" as used herein refers to a CRISPR-associated moiety, e.g. a protein from a type II system such as Cas9 or a derivative thereof.

As used herein, the terms "Cas 9 polypeptide," "Cas 9 nuclease," or "Cas 9 enzyme" are used interchangeably and generally refer to nucleases found in naturally occurring CRISPR systems. Cas9 polypeptides can recognize and/or cleave a target nucleic acid structure by interacting with a guide RNA, such as an artificial gRNA (e.g., sgRNA). Examples of "Cas 9 polypeptides" include Cas9 nuclease or variants thereof. The Cas9 nuclease may be a Cas9 nuclease from a different species, for example from Staphylococcus (Staphylococcus).

In embodiments of the invention, SacAS9 (shown in SEQ ID NO:7) and variants thereof derived from Staphylococcus aureus (Staphylococcus aureus) and the CRISPR system derived from Staphylococcus aureus can be used. Each Cas9 polypeptide depends on a different recognition site or PAM, PAM of SaCas9 is 5 '-NNGRRT-3', where N is any nucleotide and R is a purine. Each having a different sgRNA scaffold sequence, forming the 3' portion of the single guide RNA. The length of the target sequence-specific 5' portion of the sgRNA also varies among Cas9 enzymes, with Sa using 18 to 24 nucleotide target sequences.

Examples of such Cas9 nuclease variants include, but are not limited to, highly specific variants of Cas9 nuclease, such as the SaCas9 nuclease variants described in PCT/US2016/049147, PCT/US2016/020756, and the like.

In CRISPR systems, the Cas9 enzyme is directed to cleave DNA target sequences by sgrnas. The sgRNA includes at least two portions having two functions. The first portion is a targeting portion of the sgRNA, which is at the 5' end of the sgRNA relative to the second portion. The first portion of the sgRNA is complementary to the strand of the target sequence. The target sequence is immediately 5' to the PAM sequence of Cas9 on the target DNA. The length of the portion of the sgRNA that is complementary to the target sequence can be between 10 nucleotides, 13 nucleotides, 15 nucleotides, 18 nucleotides, 20 nucleotides, 22 nucleotides, or 24 nucleotides, or any number of nucleotides between 10 and 30. The portion of the sgRNA that is complementary to the target sequence should be able to hybridize to the sequence in the target strand and optimally be completely complementary to the target sequence. The exact length and location of the complementary portion of the sgRNA depends on the Cas9 enzyme with which it is paired. The Cas9 enzyme selected required the sgRNA to be designed specifically for the enzyme and controlled the design of the sgRNA.

Some other "Cas 9 polypeptides" useful in the invention can be found, for example, in http:// www.addgene.org/criprpr/guide/.

As used herein, the term "promoter-sgRNA unit" refers to a construct or fragment in which a promoter and a sgRNA are operably linked, wherein the sgRNA comprises sequences specific for a target sequence and scaffold sequences required to make up the sgRNA. Sequences specific for the target sequence are typically about 20bp to about 30bp in length, about 20bp, about 21bp, about 22bp, about 23bp, about 24bp, about 25bp, about 26bp, about 27bp, about 28bp, about 29bp, or about 30 bp. The scaffold sequence is typically no more than 80bp, no more than 79bp, no more than 78bp, no more than 77bp, no more than 76bp, no more than 75bp, no more than 74bp, no more than 73bp, no more than 72bp, no more than 71bp, or no more than 70 bp. The promoter-sgRNA unit can be operably linked to other elements required for expression vectors, such as inverted terminal repeats, PolyA, etc., to construct a vector capable of expressing a desired protein, and can also be linked to additional promoters and nucleotide sequences operably linked to the additional promoters.

As used herein, two or more promoter-sgRNA units can be connected in series on an expression cassette to construct a recombinant vector containing multiple sgrnas for multigene editing while cutting multiple target sites, thereby saving experimental cost and time. As used herein, the incorporation of spacer sequences between tandem promoter-sgRNA units can increase the efficiency of multiple gene editing.

As used herein, the term "spacer sequence" refers to a nonsense nucleotide fragment of any length that does not encode any product nor has any regulatory function, but merely separates the tandem promoter-sgRNA units. The spacer sequence may be a fragment of any bp length, preferably no more than 40bp, such as 40bp, 30bp, 20bp, 10bp, more preferably 40bp or 20bp, even more preferably 20 bp.

As used herein, "gRNA" and "guide RNA", "sgRNA" and "single guide RNA" are used interchangeably to refer to an RNA molecule capable of forming a complex with a Cas9 polypeptide and, due to some complementarity to a target sequence, capable of targeting the complex to the target sequence. For example, in Cas 9-based gene editing systems, grnas typically consist of crRNA and tracrRNA molecules that are partially complementary to form a complex, where the crRNA comprises a sequence that is sufficiently complementary to a target sequence to hybridize to the target sequence and direct the CRISPR complex (Cas9+ crRNA + tracrRNA) to specifically bind to the target sequence. However, it is known in the art to design single guide rnas (sgrnas) that contain both the characteristics of crRNA and tracrRNA. It is within the ability of the person skilled in the art to design suitable gRNA sequences based on the Cas9 polypeptide used and the target sequence to be edited.

The term "recombinant" expression cassette or vector, as used herein, refers to the presence of two or more nucleic acid regions that are not naturally associated with each other. In the present invention, the recombinant expression cassette or recombinant vector may be used interchangeably with the expression cassette or expression vector, respectively.

As used herein, the term "operably linked" describes a linkage between a regulatory element and a gene or coding region thereof. That is, gene expression is typically under the control of certain regulatory elements, such as, but not limited to, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. By "operably linked" to a regulatory element is meant that the gene or coding region is under the control or influence of the regulatory element. In the present invention, the regulatory elements include promoters, enhancers, transactivators, and the like.

In some embodiments, the inverted terminal repeat AAV2ITR5 'sequence located 5' to the expression cassette is shown in SEQ ID NO. 7 and the inverted terminal repeat AAV2ITR3 'sequence located 3' to the expression cassette is shown in SEQ ID NO. 8.

In some embodiments, the inverted terminal repeats of the expression cassette flank the expression cassette for packaging in an adeno-associated virus (AAV) vector. One skilled in the art can design the sequence between two ITRs according to the spirit and needs of the present invention.

As used herein, the term "inverted terminal repeat" or "ITR" refers to AAV viral cis-elements so named for their symmetry. These elements are important for efficient amplification of the AAV genome. The indispensable minimal limiting elements of ITR function are assumed to be the Rep-binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' for AAV 2) and the terminal resolution site (TRS; 5 '-AGTTGG-3' for AAV 2) plus variable palindromic sequences that allow hairpin formation. According to the invention, the ITR contains at least these 3 elements (RBS, TRS and sequences allowing hairpin formation). Furthermore, in the present invention, the term "ITRs" refers to known ITRs of native AAV serotypes (e.g., ITRs of serotypes 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or 11 AAV), chimeric ITRs formed by fusion of ITR elements derived from different serotypes, and functional variants thereof.

In some embodiments, the first promoter is the EF1 α promoter shown in SEQ ID NO. 10.

In some embodiments, the promoter in the promoter-sgRNA unit is a tRNA coding sequence.

In some embodiments, the tRNA coding sequence is any mammalian tRNA, e.g., Gln tRNA, Pro tRNA, Gly tRNA, Asn tRNA, Cys tRNA, Glu tRNA.

In some embodiments, the tRNA coding sequence is a Gln tRNA shown in SEQ ID NO 9.

In some embodiments, the promoter is mouse gamma herpes virus-68 (MHV68) RNA.

As used herein, the term "promoter" includes those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells, as well as those sequences that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences), tissue-specific promoters may primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, particular organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes), in some embodiments, a vector includes one or more polymerase III promoters (e.g., 1, 2,3, 4, 5, or more polymerase III promoters), one or more polymerase II promoters (e.g., 1, 2,3, 4, 5, or more polymerase II promoters), one or more polymerase I promoters (e.g., 1, 2,3, 4, 5, or more polymerase I promoters), or a combination thereof.

In embodiments of the invention, polymerase III promoters including, but not limited to, tRNA coding sequences, preferably fragments smaller than the U6 promoter, polymerase II promoters including, but not limited to, the EF1 α promoter, preferably fragments smaller than the CMV promoter, optionally, other shorter elements, such as a polyA tail, etc., may be utilized in the invention.

As used herein, the terms "tRNA" and "tRNA coding sequence" are used interchangeably to refer to a very short about 70bp long RNA polymerase III dependent promoter present in a wild-type tRNA coding gene that is capable of expressing high levels of functional sgRNA. As is known to those skilled in the art, the promoter required for tRNA transcription is located in the transcription region downstream of the transcription initiation site, and is therefore also referred to as the downstream promoter (downtream promoter) or the internal promoter (Internalpromoter) or as the Internal Control Region (ICR), which is dependent on RNA polymerase III. the tRNA internal promoter contains two separate box A and box B, and the distance between box A and box B is wider, where box A acts as a promoter and box B acts as an enhancer. TF IIIC binds to box A and box B so that TF IIIB binds immediately upstream of the initiation site, TF IIIB binds to the initiation site and is linked to TF IIIC, and TF IIIB is responsible for the correct positioning of RNA polymerase III binding to initiate transcription. The tRNA coding sequence used herein can thus function as a promoter and can express at least 2 full-length sgrnas, leaving >800bp of available space for additional sites required for Cas9 transcription and function, e.g., RNA polymerase II dependent promoters, NLS, and poly (a), etc., or, as described in embodiments of the invention, space for a reporter molecule. The sgrnas are specific for a range of DNA targets, and are also specific for Cas9 polypeptides smaller than SpCas9, such as SaCas 9.

Previous work focused on using the U6 promoter to drive sgRNA transcription. Although very efficient, the U6 promoter is about 254bp long, and thus two U6 promoters would need to exceed 10% of the overall packaging capacity of the AAV vector. It is therefore desirable to identify RNA polymerase III promoters that are smaller than U6 and are equally effective. As used herein, a tRNA of mammalian or viral origin is capable of driving expression of a sgRNA. The invention utilizes human tRNA coding sequences for expressing high levels of sgrnas. In other embodiments, tRNA coding sequences of viral origin can also be used.

An RNA polymerase III promoter is operably linked to a single guide RNA (sgrna). In one embodiment, the sgRNA comprises a 5 'portion complementary to the sense strand of the target DNA sequence and a conserved, structured 3' end capable of binding Cas 9. The target DNA may comprise any DNA sequence encoding a gene for which mutation and/or deletion is desired. The potential target sequence must be located just 5' of the PAM sequence recognized by the Cas9 polypeptide in the target DNA sequence. The expression cassette may comprise only one RNA polymerase III promoter operably linked to the sgRNA, or two or more RNA polymerase III promoter-sgRNA combinations may be included in the expression cassette. In a single gene or target sequence, the use of two or more sgrnas targeting two target sequences is sufficient to modify one or more target sequences.

Examples of tRNA coding sequences include, but are not limited to, Gln tRNA, Pro tRNA, Gly tRNA, Asn tRNA, CystRNA, Glu tRNA, murine gamma herpes virus-68 (MHV68) RNA or any mammalian tRNA (see, e.g., MefferdaL, et al.

As used herein, the "EF 1 α promoter" is a strong mammalian expression promoter of about 212bp derived from the pEF-BOS plasmid (Mizushima and Nagata, 1990) and is about half the size of the CMV promoter (584 bp). the EF1 α promoter is a constitutive promoter and is very stable in expression levels in cells, regardless of cell type.

In some embodiments, the Cas9 polypeptide is Staphylococcus aureus (Staphylococcus aureus) Cas9(SaCas9), optionally linked to a Nuclear Localization Sequence (NLS), and the sgRNA in the promoter-sgRNA unit is the sgRNA corresponding to SaCas 9.

In a preferred embodiment, the Cas9 polypeptide is staphylococcus aureus Cas9(SaCas9), preferably the Cas9 polypeptide is followed by a transcription termination signal PolyA.

In some embodiments, an expression cassette of the invention comprises, in order from the 5'-3' direction, AAV2ITR 5', EF1 α promoter, SaCas9 expression sequence operably linked to EF1 α promoter, no more than 4 tandem tRNA coding sequences-sgRNA units corresponding to SaCas9, and AAV2ITR 3'.

In some embodiments, the expression cassettes of the invention comprise a nucleotide sequence as set forth in SEQ ID NOS 1-3.

In another aspect of the invention, the invention provides an expression cassette comprising a nucleotide sequence as shown in SEQ ID NOs 1-6, preferably comprising a nucleotide sequence as shown in SEQ ID NOs 1-3, wherein N is a sequence specific for a target sequence, which specific sequence can be designed as desired by one skilled in the art according to known techniques and means, followed by a sgRNA scaffold sequence corresponding to SaCas9, as shown in SEQ ID No. 11.

Recombinant vector

In another aspect, the invention provides a recombinant vector comprising, consisting of, or consisting essentially of an expression cassette of the invention.

In some embodiments, the vector is an adeno-associated viral vector.

As used herein, the term "vector" is meant to include any element, e.g., plasmid, phage, transposon, cosmid, chromosome, artificial chromosome (YAC or BAC), virus, etc., that is capable of transferring and/or transporting a nucleic acid composition to, into, and/or to a specific location in a host cell. The term thus includes cloning and expression tools, as well as viral and non-viral vectors, and possibly naked or combined DNA. However, the term does not include cells that produce gene transfer vectors, such as retroviral packaging cell lines.

For the purposes of the present invention, "recombinant virus", "recombinant vector", or "recombinant viral vector" refers to a virus that has been genetically altered, for example by the addition or insertion of heterologous nucleic acid compositions to particles. In some embodiments, the recombinant virus comprises AAV. Thus, for example, "recombinant AAV virus" and "recombinant AAV vector" also express the same meaning. The recombinant AAV vector comprises at least one AAV capsid ("capsid"), and a recombinant AAV (vector) genome contained within the capsid.

For the purposes of the present invention, a "recombinant AAV genome" or "recombinant AAV vector genome" refers to an AAV genome comprising heterologous sequences. Typically, recombinant AAV genomes are designed in such a way that all viral genes are replaced with heterologous sequences (e.g., expression cassettes), leaving only the cis elements necessary for the complete genome, i.e., Inverted Terminal Repeats (ITRs), DNA packaging signals, and origins of replication. Alternatively, the genome-essential cis-elements may be those as described in the prior art (Musatov et al, ACIS-acting element which direct cellular uptake-associated virus replication and packaging, J Virol. Decumber 2002; 76(24): 12792-. The recombinant AAV genome is part of a recombinant AAV vector.

The expression cassette of the invention can be introduced directly into the cell to be edited by methods known to those skilled in the art, for example by linking the expression cassette of the invention to a plasmid, or by transfecting the cell directly via liposomes. Alternatively, the expression cassette of the present invention may be packaged into a vector and the cells transfected.

AAV vectors have the advantage that they can be concentrated to typically ≧ 10 per ml¹⁴Titer of viral particles, which is the level of vector that has the potential to transduce all virus-infected cells. Furthermore, AAV-based vectors have established safety records, do not integrate into the target cell genome at significant levels, thus avoiding the potential for insertional activation of deleterious genes.

Techniques and means for incorporating the expression cassettes of the invention into AAV viral vectors are well known to those skilled in the art. As used herein, a plasmid containing an expression cassette of the invention is packaged into an AAV virus by co-transfection with a packaging plasmid and a helper plasmid to obtain a recombinant AAV virus.

Reagent kit

In another aspect, the invention provides a kit comprising an expression cassette of the invention and a recombinant vector.

The kit generally includes a label indicating the intended use and/or method of use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

Method for gene editing

In another aspect, the present invention provides a method of gene editing comprising the step of delivering the expression cassette of the present invention or the recombinant vector of the present invention to a cell of a subject.

The recombinant vector is introduced into the cell using standard transfection techniques. Introduction of molecules (e.g., plasmids or viruses) into cells can also be accomplished using other techniques known to those skilled in the art, such as calcium phosphate transfection or electroporation.

As used herein, the term "subject" refers to both humans and non-human animals. The term "non-human animal" of the present disclosure includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, mice, chickens, amphibians, reptiles, and the like.

As used herein, the term "introducing" or "delivery" refers to the delivery of a plasmid or vector of the present invention for recombinant protein or nucleotide expression to a cell or to a cell and/or tissue and/or organ of a subject. Such introduction or delivery may be performed in vivo, in vitro or ex vivo. Plasmids for recombinant protein or polypeptide expression can be introduced into cells by: transfection, which typically means the insertion of heterologous DNA into a cell by chemical means (e.g., calcium phosphate transfection, Polyethyleneimine (PEI) or lipofection); physical methods (electroporation or microinjection); infection, which generally refers to the introduction of a substance by an infectious agent, i.e., a virus; or transduction, which in microbiology refers to the stable infection of cells with viruses, or the transfer of genetic material from one microorganism to another by viral material (e.g., bacteriophage). The vectors of the invention for recombinant polypeptide, protein or oligonucleotide expression may be delivered by physical means (e.g., calcium phosphate transfection, electroporation, microinjection or lipofection), or by preparing the vectors of the invention with a pharmaceutically acceptable carrier (carrier) for in vitro, ex vivo or in vivo delivery to a cell, tissue, organ or subject.

Examples

The embodiments described herein are for illustrative purposes and are not intended to limit the scope of the invention, which may be modified by those skilled in the art in light of the spirit and teachings of the present invention. Any feature described in relation to some embodiments may be used in combination with any other embodiment, unless stated otherwise or apparent from the context.