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 'and 3' ends of the expression cassette, respectively, and a first promoter located between the two inverted terminal repeats, and operably linked to the first promoter a first polynucleotide encoding a Cas9 polypeptide, and a second promoter, and operably linked to the second promoter a second polynucleotide encoding a single guide rna (sgrna), wherein the expression cassette is no more than 4.3kb in size.

2. The expression cassette of claim 1, wherein the Cas9 polypeptide is Staphylococcus aureus (Staphyloccocusareureus) Cas9(SaCas9), optionally linked to a Nuclear Localization Sequence (NLS) as set forth in SEQ ID NO:7, and the sgRNA is the sgRNA corresponding to SaCas 9.

3. The expression cassette of claim 1, wherein the size of the first promoter and the second promoter together does not exceed 300 bp.

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

5. The expression cassette of claim 1, wherein the second promoter is a tRNA coding sequence.

6. The expression cassette of claim 5, wherein the tRNA coding sequence is a mammalian tRNA, e.g., a Gln tRNA, ProtRNA, Gly tRNA, Asn tRNA, Cys tRNA, or Glu tRNA.

7. The expression cassette of claim 5, wherein said tRNA coding sequence is Gln tRNA shown in SEQ ID NO 4.

8. The expression cassette of claim 1, wherein the second promoter is mouse gamma herpes virus-68 (MHV68) RNA.

9. 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. 2, and the 3' inverted terminal repeat AAV2ITR 3 'sequence located at the 3' end of the expression cassette is set forth in SEQ ID NO. 3.

10. The expression cassette of claim 1, wherein said first polynucleotide is further linked in-frame to a reporter molecule, said reporter molecule being no more than 800bp in size.

11. The expression cassette of claim 10, wherein the reporter molecule is selected from the group consisting of EGFP, mCherry, and NanoLuc.

12. The expression cassette of claim 1, comprising, in order from the 5'-3' direction, AAV2ITR 5', EF1 α promoter, SaCas9 expression sequence operably linked to EF1 α promoter, tRNA coding sequence, sgRNA corresponding to SaCas9 operably linked to tRNA coding sequence, and AAV2ITR 3'.

13. The expression cassette of claim 1, comprising a nucleotide sequence as set forth in SEQ ID NO 1, SEQ ID NO 8 or SEQ ID NO 9.

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

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

16. A kit comprising the expression cassette of any one of claims 1 to 13 or the recombinant vector of any one of claims 14 to 15.

17. A method of gene editing comprising the step of delivering the expression cassette of any one of claims 1 to 13 or the recombinant vector of any one of claims 14 to 15 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).

Adeno-associated virus (AAV) vectors are considered as the most potential viral vectors for development, and have the advantages of non-integration into host genome, low immunogenicity, and no pathogenicity. However, its carrying capacity is small (about 4.7kb), limiting its range of use. Cas9(SaCas9) from Staphylococcus aureus (Staphylococcus aureus), a small fragment (about 3159bp) suitable for assembly into adeno-associated viral vectors for in vivo studies. The gene editing by using a pX601 vector fused with AAV and SaCas9 technologies has been studied, but the gene editing efficiency of the method is low. And due to the small bearing capacity of AAV, a reporter molecule (reporter gene) cannot be further added into the vector during virus packaging, so that the detection of the packaged virus infection condition is inconvenient.

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. The present invention overcomes the above problems by constructing a novel recombinant vector.

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 3' end of the expression cassette, respectively, and a first promoter located between the two inverted terminal repeats, and operably linked to the first promoter, a first polynucleotide encoding a Cas9 polypeptide, and a second promoter, and operably linked to the second promoter, a second polynucleotide encoding a single guide rna (sgrna), wherein the expression cassette is no more than 4.3kb in size.

In some embodiments, the Cas9 polypeptide is Staphylococcus aureus (Staphylococcus aureus) Cas9(SaCas9) as shown in SEQ ID NO:7, optionally linked to a Nuclear Localization Sequence (NLS).

In other embodiments, the sgRNA is a sgRNA corresponding to SaCas 9.

In other embodiments, the size of the first promoter and the second promoter together does not exceed 400bp, 390bp, 380bp, 370bp, 360bp, 350bp, 340bp, 330bp, 320bp, 310bp, 300bp, or 290 bp.

In other embodiments, the first promoter is a promoter of RNA polymerase II selected from the group consisting of the EF1 α promoter, the CMV promoter, the CBA promoter, the hSynapsin promoter, the HSV-TK promoter, the SV40 early promoter and the LSP promoter, preferably the EF1 α promoter, more preferably the EF1 α promoter of SEQ ID NO. 5.

In other embodiments, the second promoter is an RNA polymerase III promoter selected from the group consisting of: the U6 promoter or tRNA coding sequence, preferably tRNA coding sequence. The tRNA coding sequence is any mammalian tRNA, including but not limited to Gln tRNA, Pro tRNA, Gly tRNA, Asn tRNA, Cys tRNA, Glu tRNA.

In a preferred embodiment of the invention, the tRNA coding sequence is Gln tRNA shown in SEQ ID NO 4.

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

In other embodiments, the inverted terminal repeat AAV2ITR 5 'sequence located 5' to the expression cassette is shown as SEQ ID NO. 2, and the inverted terminal repeat AAV2ITR 3 'sequence located 3' to the expression cassette is shown as SEQ ID NO. 3.

In other embodiments, the first polynucleotide is further linked in-frame to a reporter (reporter) molecule that is no more than 800bp, no more than 790bp, no more than 780bp, no more than 770bp, no more than 760bp, no more than 750bp, no more than 740bp, no more than 730bp, or no more than 720bp in size.

In other embodiments, the reporter (reporter gene) is selected from the group consisting of EGFP, mCherry, and NanoLuc.

In other 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, tRNA coding sequence, sgRNA corresponding to SaCas9 operably linked to tRNA coding sequence, and AAV2ITR 3'.

In other embodiments, the expression cassettes of the invention comprise a nucleotide sequence as set forth in SEQ ID NO 1, SEQ ID NO 8 or SEQ ID NO 9.

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.

In another aspect, the present invention provides a method of increasing gene editing efficiency, 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.

In some embodiments, sgRNA expression is reduced relative to wild-type expression.

In other embodiments, two or more grnas are present in a recombinant vector or expression cassette, and more than one target sequence is modified.

Drawings

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

Figure 2 shows a schematic of a recombinant vector containing fluorescent reporter molecules (reporter genes) (EGFP and mCherry).

FIG. 3 showsThe T7 endonuclease I detected the results of editing of endogenous genes in HEK293T cells. In fig. 3a, U6 represents the promoter group with U6 as sgRNA; tRNA represents a promoter group using tRNA as sgRNA; c^-As a negative control, M was 50bp DNAladder, the arrow indicates the cut target fragment, in FIG. 3b, EGFP represents pX601(EF1 α -tRNA) EGFP recombinant vector group, mCherry represents pX601(EF1 α -tRNA) mCherry recombinant vector group, C^-Negative control; m is 50bp DNA Ladder, and the arrow indicates the cleaved target fragment.

FIG. 4 shows the results of TA clone sequencing to identify the editing efficiency of the genes of the U6 set and tRNA set. The situation of TA clone sequencing mutation sequences of the U6 group MSTN-sgRNA1 site is shown as a; the situation of a TA cloning sequencing mutation sequence of a tRNA set MSTN-sgRNA1 site is shown as b; the situation of TA clone sequencing mutation sequences of the U6 group MSTN-sgRNA2 site is shown as c; the situation of the TA clone sequencing mutation sequence of the tRNA set MSTN-sgRNA2 site is shown as d. The dashed lines indicate missing bases, underlined and bolded letters indicate PAM sequences.

FIG. 5 shows the results of the efficiency of TA clone sequencing to identify gene editing in pX601(EF1 α -tRNA) EGFP group and pX601(EF1 α -tRNA) mCherry group.a is the case of TA clone sequencing mutant sequence at position TA in pX601(EF1 α -tRNA) EGFP group MSTN-sgRNA1, b is the case of TA sequencing mutant sequence at position TA in pX601(EF1 α -tRNA) mChery group MSTN-sgRNA1, c is the case of TA sequencing mutant sequence at position TA in pX601(EF1 α -tRNA) EGFP group MSTN-sgRNA2, d is the case of TA sequencing mutant sequence at position TA in pX601(EF1 α -tRNA) mChery group MSTN-sgRNA 2. the short horizontal line indicates missing base, and the PAM sequence is underlined and bold letters indicate PAM.

FIG. 6 shows the results of TA clone sequencing alignment, where pX601 is the ratio of sequencing mutations of the TA clones in pX601 group, pX601(tRNA) is the ratio of sequencing mutations of the TA clones in pX601(tRNA), pX601(EF1 α -tRNA) EGFP is the ratio of sequencing mutations of the TA clones in pX601(EF1 α -tRNA), pX601(EF1 α -tRNA) mCherry is the ratio of sequencing mutations of the TA clones in pX601(EF1 α -tRNA) mCherry group, U6 uses U6 as a promoter group, and tRNA as a promoter group.

FIG. 7 shows AAV-DJ-mediated gene editing effect in NIH3T3 cells, FIGS. 7a and 7d are fluorescence and bright field observations (200X) 7 days after HBAAV-GFP challenge, FIGS. 7b and 7e are pX601(EF1 α -tRNA) -EGFP-mCSk 9Fluorescence and brightfield observations (200 ×) 7 days after sgRNA2 challenge; FIGS. 7c and 7f are fluorescence and brightfield observations (200X) 7 days after pX601-EGFP-mPCsk9-sgRNA2 challenge; FIG. 7g is a flow cytometer detecting percent green fluorescence, where C⁺Is HBAAV-GFP group, 1 is pX601(EF1 α -tRNA) -EGFP-mPCsk9-sgRNA2 group, 2 is pX601-EGFP-mPCsk9-sgRNA2 group, and 7h is T7 endonuclease I method detection result, wherein C is^-As a negative control group, 1 was a pX601(EF1 α -tRNA) -EGFP-mPCsk9-sgRNA2 group, 2 was a pX601-EGFP-mPCsk9-sgRNA2 group, and the arrows indicate the cut-off target fragments, and FIG. 7i is a graph illustrating the fold of each virus titer and the fold of percent green fluorescence.

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, or consisting essentially of: 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, and a first polynucleotide encoding a Cas9 polypeptide operably linked to the first promoter, and a second polynucleotide encoding a single guide rna (sgrna) operably linked to the second promoter, wherein the expression cassette is no more than about 3.8kb, 3.9kb, 4.0kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, preferably no more than about 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, more preferably no more than about 4.2kb, 4.3kb, 4.4kb, most preferably no more than about 4.3 kb.

In some embodiments, the Cas9 polypeptide is Staphylococcus aureus (Staphylococcus aureus) Cas9(SaCas9) as shown in SEQ ID NO:7, optionally linked to a Nuclear Localization Sequence (NLS).

In other embodiments, the sgRNA is a 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.

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, "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 other embodiments, the size of the first promoter and the second promoter together is no more than 400bp, 390bp, 380bp, 370bp, 360bp, 350bp, 340bp, 330bp, 320bp, 310bp, 300bp or 290bp, preferably no more than 350bp, 340bp, 330bp, 320bp, 310bp, 300bp or 290bp, more preferably no more than 320bp, 310bp, 300bp or 290bp, even more preferably no more than 300 bp.

In further embodiments, the first promoter is a promoter of RNA polymerase II selected from the group consisting of the EF1 α promoter, the CMV promoter, the CBA promoter, the hSynapsin promoter, the HSV-TK promoter, the SV40 early promoter and the LSP promoter, preferably a promoter of NO more than 580bp, more preferably the EF1 α promoter, even more preferably the EF1 α promoter as shown in SEQ ID NO. 5.

In other embodiments, the second promoter is an RNA polymerase III promoter selected from the group consisting of: the U6 promoter or tRNA coding sequence, preferably a promoter of no more than 240bp, more preferably a tRNA coding sequence. The tRNA coding sequence is any mammalian tRNA, including but not limited to Gln tRNA, Pro tRNA, Gly tRNA, Asn tRNA, Cys tRNA, Glu tRNA. In a preferred embodiment of the invention, the tRNA coding sequence is Gln tRNA shown in SEQ ID NO 4. In other embodiments, the second 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 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 a sequence between two ITRs according to the spirit and needs of the present invention, preferably no more than 3700bp, more preferably no more than 3650bp, even more preferably no more than 3600 bp.

In other embodiments, the Inverted Terminal Repeat (ITR) sequence of AAV2 located 5 'to the expression cassette is shown in SEQ ID NO:2, and the Inverted Terminal Repeat (ITR) sequence of AAV2 located 3' to the expression cassette is shown in SEQ ID NO: 3.

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 other embodiments, the first polynucleotide is further linked in-frame to a reporter (reporter) molecule that is no more than 800bp, no more than 790bp, no more than 780bp, no more than 770bp, no more than 760bp, no more than 750bp, no more than 740bp, no more than 730bp, or no more than 720bp in size.

In other embodiments, the reporter (reporter gene) is selected from the group consisting of EGFP, mCherry, and NanoLuc.

As used herein, incorporation of a fluorescent reporter (reporter gene) into a recombinant vector facilitates detection of viral transduction efficiency, and viral infection and distribution, and provides a clear prediction of Cas9 editing effect. In embodiments of the invention, shorter reporter molecules (reporter genes) are preferred due to AAV genome size limitations, including but not limited to EGFP, mCherry, NanoLuc, and the like, with EGFP and mCherry being more preferred.

In a preferred embodiment of the invention, the expression cassette of the invention comprises, in order from the 5 'to 3' direction, AAV2ITR 5', EF1 α promoter, SaCas9 expression sequence operably linked to EF1 α promoter, tRNA coding sequence, sgRNA corresponding to SaCas9 operably linked to tRNA coding sequence, and AAV2ITR 3'.

In other embodiments, the expression cassettes of the invention comprise the nucleotide sequence shown in SEQ ID NO. 1, and the invention also provides expression cassettes comprising EGFP and mCherry comprising the nucleotide sequences shown in SEQ ID NO. 7 and 8, respectively. Wherein, N is a sequence specific to a target sequence, and a person skilled in the art can design the specific sequence as required according to known techniques and means, and then the specific sequence is followed by a Sa sgRNA scaffold sequence as shown in SEQ ID NO. 6.

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 a recombinant AAV 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. In the examples of the present invention, gene editing efficiency can be improved 2-fold using the method of the present invention.

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.

Surprisingly, although the gene editing efficiency was increased 2-fold, in some embodiments, sgRNA expression was reduced relative to wild-type expression.

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.