CN118345117A - Construction production and use of recombinant adeno-associated virus large gene packaging expression vector - Google Patents
Construction production and use of recombinant adeno-associated virus large gene packaging expression vector Download PDFInfo
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- CN118345117A CN118345117A CN202310055399.2A CN202310055399A CN118345117A CN 118345117 A CN118345117 A CN 118345117A CN 202310055399 A CN202310055399 A CN 202310055399A CN 118345117 A CN118345117 A CN 118345117A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The application discloses construction, production and application of a recombinant adeno-associated virus large gene packaging expression vector. A construction system for recombinant adeno-associated viral vector packaging expression large genes comprises a first packaging expression vector and a second packaging expression vector; from the 5 'to the 3' end, the first packaging expression vector comprises a first ITR element or a similar element and a first packaging expression cassette comprising a promoter of a positive strand sequence, a gene of interest and a polyadenylation signal; from the 5 'to the 3' end, the second packaging expression vector comprises a second ITR element or similar element and a second packaging expression cassette comprising a polyadenylation signal of negative strand sequence, a gene of interest and a promoter; the base of the positive strand and the base of the negative strand are complementary paired. The positive and negative chain DNA molecules of the adenovirus-associated large gene packaging expression vector are paired through the 3' -end part of the DNA molecules after the cells are transduced, and are copied to form a complete large gene expression frame, so that the transcription expression protein has high efficiency, accuracy and high stability, and can be effectively applied to the protein expression of large genes and AAV gene therapy.
Description
Technical Field
The invention relates to the fields of gene vectors, gene therapy and gene editing, in particular to construction, production and application of a large gene packaging expression vector (LARGE GENE expression ado-associated virus, lgAAV) for recombinant adeno-associated virus.
Background
Gene therapy is directed to patients suffering from genetic mutations caused by abnormal gene expression patterns, including treatment or prevention of genetic diseases caused by gene deficiency, abnormal regulation or expression, such as diseases caused by under-expression or over-expression, malignant tumors, etc., and can be treated, prevented or alleviated by providing the patients with corrective genetic material. Currently, gene delivery vectors mainly include non-viral vectors and viral gene delivery vectors. Among the many available viral-derived gene vectors, for example, recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc., recombinant adeno-associated virus (rAAV) gene vectors are becoming increasingly popular.
Adeno-associated virus (AAV) belongs to the parvoviridae (Parvoviridae), and has become the most promising gene vector for current gene therapy due to its advantages of non-pathogenicity, low immunogenicity, broad spectrum infectivity, etc. AAV is a single-stranded DNA virus without envelope, and carries a linear single-stranded DNA genome of 4.6-4.7 kb, two ends of the DNA genome of AAV are respectively provided with an inverted terminal repeat sequence of 145bp, ITR, wherein 125 bases near the terminal are of a longer palindromic structure, and the AAV can fold by base complementation pairing, so that the ITR presents a T-shaped hairpin structure. Positive and negative strand DNA genomes carrying wild type inverted terminal repeats are packaged into AAV viral capsids with the same probability, thereby producing the same number of positive strand, or negative strand AAV virions. When AAV infects cells, enters cell nucleus, after capsid removal, the repetitive sequence at the tail end of genome is folded to form a T-shaped palindromic structure, a second strand is synthesized by taking the 3' -tail end as a primer to form a double-stranded molecule, and then gene expression is started. The AAV genomic DNA can also be complementarily paired with the negative strand to form a double-stranded molecule, thereby promoting gene expression. In addition, the ITRs between molecules spontaneously bind to form dimers and multimers, and such molecules are capable of sustained expression of exogenous genes in cells over a long period of time, even throughout the life.
The rAAV vector packaging gene size is 4.7kb, thus limiting the application of AAV gene therapy in large genetic diseases. Major strategies currently aimed at AAV delivery of large gene expression include: the first approach is cis-splicing, taking advantage of the property of AAV vectors that can join AAV genomes together by homologous recombination of ITR sequences to form concatamers. One of the AAV vectors carries a promoter, a 5' portion coding region, and a splice donor signal; another AAV vector carries a splice acceptor signal, the remaining coding region and a polyA signal. When two AAV are joined end-to-end to form a concatemer, the recombinant ITR sequence is removed by splicing to form the complete gene expression cassette.
In the second strategy, two AAV vectors with homologous coding gene sequences form a large gene expression cassette in the cell by homologous recombination.
The third method is a hybrid two-vector strategy, combining the two methods. Independent AAV vector genomes are brought together by recombination (in this case, the starting vector is deliberately split into left and right halves with 5 'and 3' splice elements, respectively) by virtue of tandem activity of the AAV genome, and splicing is performed to ensure that the correct transgenic protein is produced after infection. One study showed that insertion of a 872bp highly recombinant alkaline phosphatase sequence into an AAV vector with an intron splice site resulted in high levels of transgene independent homologous recombination of the alkaline phosphatase sequence after introduction of the split vector in an animal. This strategy may increase the expression of the intact functional protein.
A fourth approach is to construct the full-length gene into the same packaging plasmid, such that AAV virions of gene segments truncated at different locations will be generated during viral packaging, i.e. a mixed population of AAV vectors contains truncated genes of different lengths. After transduction of these AAV vectors, the different AAV vector genomes are annealed and DNA synthesized at complementary regions by homologous recombination of overlapping regions of the two different AAV vector genomes or by single stranded templates to produce complete gene expression cassettes.
A fifth approach is that split AAV vectors can be designed to package larger AAV genomes using protein intron (intein) -mediated protein trans-splicing techniques. Similar to intron-mediated RNA splicing, intein catalyzes protein splicing and results in precise ligation of two separate polypeptides by trans-splicing. When larger AAV cassettes are delivered using this technology, multiple AAV vectors (each encoding one of the target protein fragments flanked by short split inteins) are delivered to the same cell. Protein trans-splicing then occurs and full-length protein is formed. This method has been successfully used to deliver dystrophin, FVIII, CFTR, CRISPR-Cas9, ABCA4 and 290kDa centrosome protein (CEP 290) in animals.
In a word, the existing AAV large gene expression method has low large gene expression efficiency, unstable performance and even mechanism, and cannot be effectively applied to AAV gene therapy of large gene genetic diseases.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a construction, production and use of a recombinant adeno-associated virus large gene packaging expression vector, in order to solve the problems in the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
A first aspect of the present invention provides a construction system for packaging expression of large genes from recombinant adeno-associated viral vectors, said packaging expression DNA having a length of greater than 4.7kb, said construction system comprising a first packaging expression vector and a second packaging expression vector,
From the 5 'to the 3' end, the first packaging expression vector comprises a first ITR element or similar element and a first gene expression cassette comprising a promoter of a positive strand sequence, a gene of interest and a polyadenylation signal;
From the 5 'to the 3' end, the second packaging expression vector comprises a second ITR element or similar element and a second expression cassette comprising a polyadenylation signal of negative strand sequence, a gene of interest and a promoter;
the base of the positive strand and the base of the negative strand are complementary paired.
In certain embodiments, portions of the positive and negative strands are base complementary paired.
In certain embodiments, the first ITR element or like element and the second ITR element or like element are selected from AAV viral ITR elements or like elements.
Preferably, the AVV viral ITR element comprises a Rep Binding Element (RBE), a D region sequence, a Rep protein cleavage sequence (trs), an a sequence, a B sequence, and a C sequence.
Preferably, the similar element is selected from one or more of DttRR element and DtrsA 'B' element.
More preferably, the DttRR element consists of a Rep protein binding element (RBE) and a Rep protein cleavage sequence (trs).
More preferably, the DtrsA 'B' element consists of the D element, trs sequence, a 'sequence and B' sequence in the AAVITR element.
In certain embodiments, the ITR element has a coding sequence set forth in SEQ ID NO. 32.
In certain embodiments, the coding sequence of the DttRR element is shown as SEQ ID NO. 33.
In certain embodiments, the coding sequence of the DtrsA 'B' element is shown as SEQ ID NO 34.
In certain embodiments, the promoter is selected from one of CMV, CAG, ef-a and hU 6. The promoter may also be selected from other gene expression promoters.
In certain embodiments, the large gene is selected from one or more of the group consisting of engineered expanded EGFP, luciferase, CRISPR/CAS 9; preferably, the CRISPR/CAS9 is selected from spCas9.
In certain embodiments, the positive strand of the first packaging expression vector and the negative strand of the second packaging expression vector are complementarily paired at the 3' end portion to synthesize the second strand to form the complete gene expression cassette of interest.
In a second aspect the invention provides a recombinant adeno-associated viral vector comprising a first packaged expression vector as described above and a second recombinant adeno-associated viral vector comprising a second packaged expression vector as described above.
In certain embodiments, after simultaneous transduction of the host cell with the first recombinant adeno-associated viral vector and the second recombinant adeno-associated viral vector, the DNA molecules of the two recombinant adeno-associated viral vectors are complementarily paired at the 3' end portion, initiating synthesis of DNA, forming a complete large gene expression cassette.
A third aspect of the invention provides a host cell transduced with a recombinant adeno-associated viral vector as described above.
In certain embodiments, the host cell is selected from the group consisting of a human cell, other mammalian cell.
A fourth aspect of the invention provides the use of a construction system as described above or a recombinant adeno-associated viral vector as described above in the preparation of a gene therapy drug and/or vaccine or for gene editing.
In a fifth aspect the invention provides a gene therapy agent comprising a construction system as described above or a recombinant adeno-associated viral vector as described above.
In a sixth aspect the invention provides a method of gene editing comprising a construction system as described above or a recombinant adeno-associated viral vector as described above.
A seventh aspect of the invention provides a genetic recombination method comprising the use of a recombinant adeno-associated viral vector as described above or by a construction system as described above.
Compared with the prior art, the invention has the following beneficial effects:
The recombinant adeno-associated virus large gene packaging expression vector can package left and right fragments of a large gene expression frame efficiently, the left and right fragment DNA is in a positive strand and negative strand complementary pairing relationship, the left and right fragment DNA is paired with a 3' -end part complementary sequence to form a partial double strand, and the complementary strand is synthesized to form a complete large gene expression frame, so that the large gene is expressed efficiently.
Drawings
FIG. 1 shows a schematic diagram of the DNA sequence structure of AAV2 Flip ITR (RBE, trs sites are shown).
FIG. 2 shows a schematic diagram of the construction and packaging of recombinant adeno-associated virus vectors of the invention.
FIG. 3 shows a schematic diagram of the construction and packaging of lgAAV-CMV-Luci-7.6kb recombinant adeno-associated virus vector of the invention.
FIG. 4 shows a schematic diagram of the construction and packaging of lgAAV-CMV-EGFP-8.3kb recombinant adeno-associated viral vector of the invention.
FIG. 5 shows a schematic diagram of the construction and packaging of lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated viral vector of the invention.
FIG. 6 shows SDS-PAGE patterns of capsid proteins of rAAV-EGFP recombinant adeno-associated viral vector, lgAAV-CMV-Luci-7.6kB recombinant adeno-associated viral vector, lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated viral vector and lgAAV-spCAS9-gRNA-NF kB-7.0kB recombinant adeno-associated viral vector of the present invention. Wherein MW is protein molecular weight standard, 1 is rAAV-EGFP,2 is lgAAV-CMV-Luci-7.6kB recombinant adeno-associated virus vector, 3 is lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated virus vector, 4 is lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector. .
FIG. 7 shows DNA neutral agarose electrophoresis patterns of rAAV-EGFP recombinant adeno-associated viral vector, lgAAV-CMV-Luci-7.6kB recombinant adeno-associated viral vector, lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated viral vector and lgAAV-spCAS9-gRNA-NF kB-7.0kB recombinant adeno-associated viral vector in the invention. Wherein MW is protein molecular weight standard, 1 is rAAV-EGFP,2 is lgAAV-CMV-Luci-7.6kB recombinant adeno-associated virus vector, 3 is lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated virus vector, 4 is lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector. .
FIG. 8 shows DNA basic agarose electrophoresis patterns of rAAV-EGFP recombinant adeno-associated viral vector, lgAAV-CMV-Luci-7.6kB recombinant adeno-associated viral vector, lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated viral vector and lgAAV-spCAS9-gRNA-NF kB-7.0kB recombinant adeno-associated viral vector in the invention. Wherein MW is protein molecular weight standard, 1 is rAAV-EGFP,2 is lgAAV-CMV-Luci-7.6kB recombinant adeno-associated virus vector, 3 is lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated virus vector, 4 is lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector. .
FIG. 9 shows a fluorescence detection of lgAAV-CMV-Luci-7.6kb recombinant adeno-associated viral vector of the invention for expression of Luciferase in mouse muscle.
FIG. 10 shows a fluorescence detection of the recombinant adeno-associated viral vector lgAAV-CMV-EGFP-8.3kb in the present invention for EGFP expression in the retina of the mouse eye.
FIG. 11 shows functional verification of expression of large genes in HEK293 cells transduced with lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated viral vectors of the invention.
Detailed Description
The present application provides a viral vector for producing recombinant adeno-associated virus (AAV) capable of packaging and expressing large genes.
The invention develops a design and packaging technology of a recombinant adeno-associated virus large gene (lgAAV) packaging expression vector. The packing production method of lgAAV carrier mainly adopts the principle that two complementary single-chain monopole AAV DNA molecules are utilized, and through homologous sequence complementary pairing, 3' -end extends to synthesize DNA in cells, so that complete DNA double-chain molecule is formed, and protein is expressed. In packaging single stranded monopolar DNA AAV vectors, ITR sequences, i.e., dtrsA ' B ' BC ' CA, or engineered ITR sequences (similar elements), i.e., comprising only the Rep protein binding element (Rep binding element, RBE) in the ITR structure, and the Rep protein cleavage site (terminal resolution site, trs) or DtrsA ' B ', are utilized. ITR, RBE-trs, dtrsA ' B ' packaging elements are respectively arranged on the positive strand of a large gene to be packaged and the 5' end of the negative strand, and the positive strand DNA vector of a recombinant adeno-associated virus large gene (lgAAV) packaging expression vector is packaged and produced, and when the negative strand DNA vector is applied, the positive strand vector and the negative strand vector containing the same DNA molecular number are mixed, and cells are transduced, so that the large gene can be expressed. The positive strand rAAV DNA molecule and the negative strand rAAV DNA molecule produced by packaging are partially complementary at the 3 '-end, complementary pairing is carried out in cells, meanwhile, the 3' -end starts to synthesize DNA, a DNA double-stranded molecule with the target length is formed, and the target protein is transcribed and synthesized. The packaging method of lgAAV vectors related by the invention is different from the existing rAAV large gene expression method in that the traditional rAAV large gene expression method mainly depends on homologous recombination between double-stranded DNA and RNA gene editing, and has low efficiency, poor performance and poor application effect. lgAAV vector large gene packaging expression is based on complementary pairing between two single-stranded DNA molecules of two single-stranded monopole AAV vectors packaged respectively, 3' DNA synthesis is carried out to form double chains, and the protein is transcribed and expressed, so that the efficiency is high, the accuracy is high, the stability is high, and the vector large gene packaging expression can be effectively applied to AAV gene therapy of large gene genetic diseases. The large gene expression recombinant adeno-associated viral vectors (lgAAV) of the invention comprise conventional, or variant, AAV protein capsids, as well as variant AAV genome components.
Adeno-associated virus (AAV) is a single stranded DNA virus that is used as a vector to transfer foreign genes into patients during gene therapy. AAV genome is a linear, single-stranded DNA (ssDNA) of about 4.7kb in size, with 145bp Inverted Terminal Repeats (ITRs) on the left and right sides and 2 Open Reading Frames (ORFs) in the middle, and 125 nucleotides of the ITRs form palindromic sequences as initiation elements for replication initiation, capable of folding back on itself to form hairpin structures, which are the only cis-regulatory elements in AAV life cycle, with the left ORF encoding replication protein (Rep) and the right ORF encoding structural protein (Cap). AAV is a natural replication-defective virus that requires the aid of helper viruses to complete the replication cycle, such as Adenovirus (AV), herpes simplex virus (HSV-1), and the like. In the absence of helper virus, AAV genes integrate into host genes and remain latent in proviral form.
ITR plays a key role in viral replication and packaging processes and is involved in the integration and escape of viral genomes on the host genome. The CG content in the sequence is up to above 80%, the former 125bp (1-125) sequence can be divided into A, B, B ', C', C, A 'and other different sections in turn, wherein A, A', B ', C and C' are reversely complementary to form a T-shaped hairpin structure which is used as the origin of DNA self replication of AAV, and the subsequent 20bp forms a special D sequence.
The A-A' region
The tandem repeat 5'-GAGCGAGC GAGC GCGC-3' in the A-A 'region is the core sequence in the A-A' region and even throughout the ITR, the most well-defined function of which is to bind to the Rep protein and is therefore referred to as the Rep binding site (Rep Binding Element, RBE). RBE involves replication of the AAV genome, transcription, and proviral integration, and almost all of the important functions of ITRs are initiated by Rep binding to RBE. The A-A' region is also involved in the formation of specific trs sequences (terminal resolution site), and the trs sequences in the ITR are involved in the rescue and replication pathways of AAV.
The B-B 'and C-C' regions, both palindromic messages also have a role in binding to Rep, and the sequence (5 '-CTTTG-3') called RBE 'located in the B-B' region has a strong affinity for Rep.
The A-A ', B-B ' and C-C ' regions of the ITR all form palindromic double-stranded structures, with only the D region being single-stranded.
In the application, the first packaging expression vector, the positive chain vector and the positive vector can be exchanged, and the second packaging expression vector, the negative chain vector and the load body can be exchanged.
A first aspect of the present invention provides a construction system for packaging expression of large genes from recombinant adeno-associated viral vectors, said packaging expression DNA being greater than 4.7kb in length, said construction system comprising a first packaging expression vector and a second packaging expression vector;
from the 5 'to the 3' end, the first packaging expression vector comprises a first ITR element or a similar element and a first packaging expression cassette comprising a promoter of a positive strand sequence, a gene of interest and a polyadenylation signal;
From the 5 'to the 3' end, the second packaging expression vector comprises a second ITR element or similar element and a second packaging expression cassette comprising a polyadenylation signal of negative strand sequence, a gene of interest and a promoter;
the base of the positive strand and the base of the negative strand are complementary paired.
Specifically, the first packaging expression vector is single-stranded DNA, and comprises ITR elements or similar elements, promoters, target genes and polyadenylation signals from the 5 'end to the 3' end, and the second packaging expression vector is single-stranded DNA, and comprises complementary strands of the ITR elements or similar elements and the target genes from the 5 'end to the 3' end. The base complementary pairing of the positive strand and the negative strand is partial base complementary pairing, and the length of the complementary pairing region is not less than 1KB, and may be 1.1KB, 1.8KB, and 2.4KB, for example. The principle of the invention is that the AAV packaged single-stranded monopole DNA synthesizes a complete large gene DNA expression frame after partial base complementation pairing in a host cell, and expresses the protein encoded by the corresponding large gene.
In the construction system of the invention, the ITR element or the like is selected from one or more of an AAV viral ITR element, dttRR element, and DtrsA 'B' element. In certain embodiments, the AVV viral ITR element comprises a Rep Binding Element (RBE), a D region sequence, a Rep protein cleavage sequence (trs), an a sequence, a B sequence, and a C sequence. Preferably, the ITR element has a coding sequence as set forth in SEQ ID NO. 32. In other embodiments, the DttRR element consists of a Rep protein binding element (RBE) and a Rep protein cleavage sequence (trs). Preferably, the coding sequence of DttRR elements is shown in SEQ ID NO. 33. In other embodiments, the DtrsA 'B' element consists of the D element, trs sequence, a 'sequence, and B' sequence in the AAVITR element. Preferably, the coding sequence of the DtrsA 'B' element is shown in SEQ ID NO. 34.
ITR sequence:
Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa(SEQ ID NO:32)
DttRR sequence:
aggaacccctagtgatggagttggagttggctgcgcgctcgctcgctcactgctgcgcgctcgctcgctcactg(SEQ ID NO:33)
DtrsA 'B' sequence:
aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaa(SEQ ID NO:34)
in the construction system of the present invention, the 3 '-end of the positive-strand expression vector does not contain ITR elements or the like, and the 3' -end of the negative-strand expression vector does not contain the complementary strand of ITR elements or the like.
In the construction system of the present invention, the large gene is a gene having a length of more than 4.7 kb.
In the construction system of the invention, a connecting sequence is connected between the ITR element or the like of the forward expression vector and the promoter. A connecting sequence is connected between the complementary strand of the ITR element or the similar element of the negative chain expression vector and the second fragment of the target gene. In certain embodiments, such as stuff, stuff2.
In the construction system, the length of the complementary region between the first segment of the target gene and the second segment of the target gene is not less than 1KB.
In the construction system of the invention, the promoter is one selected from CMV, CAG, ef-a and hU 6. The promoter may also be selected from other gene expression promoters.
In the construction system, the positive strand of the first packaging expression vector and the negative strand of the second packaging expression vector are in partial complementary pairing at the 3' -end, and the second strand is synthesized to form the complete target gene expression frame.
In the construction system, the large gene is selected from one or more of EGFP, luciferase, CRISPR/CAS9 modified and enlarged; preferably, the CRISPR/CAS9 is selected from spCas9. In a specific embodiment, taking the modification of an expanded EGFP as an example, the sequence of the construction system DttRR-3.1kbstuff1-CEP-2.8kbstuff2-R ' R't ' t ' D ' is SEQ ID NO 29; taking the transformation of enlarged luciferases as an example, a system DttRR-2.4kb stuff1-CLP-1.8kb stuff2-R ' R't ' t ' D ' with the sequence of SEQ ID NO:30 is constructed; taking the example of engineering an expanded CRISPR/CAS9, the sequence of construction system ITRL-spCas9-gRNA12 (NFkB) is SEQ ID NO:31.
DttRR-3.1kbstuff1-CEP-2.8kbstuff2-R'R't't'D':
aggaacccctagtgatggagttggagttggctgcgcgctcgctcgctcactgctgcgcgctcgctcgctcactgacgcgtgaagaaagacggcgaagtgcggggcagcatcaacagattcaagaccagcgactacgtgaaagaagccaaacagctgctgaaggtgcagaaggcctaccaccagctggaccagagcttcatcgacacctacatcgacctgctggaaacccggcggacctactatgagggacctggcgagggcagccccttcggctggaaggacatcaaagaatggtacgagatgctgatgggccactgcacctacttccccgaggaactgcggagcgtgaagtacgcctacaacgccgacctgtacaacgccctgaacgacctgaacaatctcgtgatcaccagggacgagaacgagaagctggaatattacgagaagttccagatcatcgagaacgtgttcaagcagaagaagaagcccaccctgaagcagatcgccaaagaaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccggcaagcccgagttcaccaacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagattattgagaacgccgagctgctggatcagattgccaagatcctgaccatctaccagagcagcgaggacatccaggaagaactgaccaatctgaactccgagctgacccaggaagagatcgagcagatctctaatctgaagggctataccggcacccacaacctgagcctgaaggccatcaacctgatcctggacgagctgtggcacaccaacgacaaccagatcgctatcttcaaccggctgaagctggtgcccaagaaggtggacctgtcccagcagaaagagatccccaccaccctggtggacgacttcatcctgagccccgtcgtgaagagaagcttcatccagagcatcaaagtgatcaacgccatcatcaagaagtacggcctgcccaacgacatcattatcgagctggcccgcgagaagaactccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagaccaacgagcggatcgaggaaatcatccggaccaccggcaaagagaacgccaagtacctgatcgagaagatcaagctgcacgacatgcaggaaggcaagtgcctgtacagcctggaagccatccctctggaagatctgctgaacaaccccttcaactatgaggtggaccacatcatccccagaagcgtgtccttcgacaacagcttcaacaacaaggtgctcgtgaagcaggaagaaaacagcaagaagggcaaccggaccccattccagtacctgagcagcagcgacagcaagatcagctacgaaaccttcaagaagcacatcctgaatctggccaagggcaagggcagaatcagcaagaccaagaaagagtatctgctggaagaacgggacatcaacaggttctccgtgcagaaagacttcatcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacctgctgcggagctacttcagagtgaacaacctggacgtgaaagtgaagtccatcaatggcggcttcaccagctttctgcggcggaagtggaagtttaagaaagagcggaacaaggggtacaagcactgacagccctgctgctgcctctggctctgctgctgcatgccgctagacccgattacaaggatgacgacgataagggtggtggtggttctatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattttttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctacgcgacatcaagattacattctggagtcccatcaaggttcagtggcagtgggtctggaacagatttttctctcaccattagcaaactggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccacttacgttcggcgctgggacaaagttggaacttaaaggtggtggtggttctggcggcggcggctccggaggaggaggatcgctgcaacagtctggacctgagttggtgaagcctggggcttcagtgaagatttcctgcaaggcttctggatacacattcactgactactacatgaattgggtgaagcttagccatggaaagagccttgagtggattggagatattgttcctaacaatggtgatactacttacaaccagaatttcagaggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggactctgcagtctattactgtgcaagattcagtaattacgtttacccttttgactactggggccaaggcaccactatcacagtctccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaaggatacgcgtctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgtcaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggattcgaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcgaacatcgattgaattccccggggatccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaactctcgagagatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccaaccgtaatccggacctcacccagctccctggtggctttcatcaactctcgctgtacatctccgggcggttcctacggccatctctccattggtaccatgagcccttctttaggattcccacctcagatgagtcatcaaaaaggaacttcacctccctatggagtccagccctgtgtaccacatgactctactcggggttcaatgatgcttcacccccagtcccggggaccacgtgcaacctgccagctgaagtcagagctggatatgatggttggcaagtgcccggaggaccctttggaaggggacatgtctagccccaactccacaggcacacaggatcacctgttggggatgctggatgggcgggaggacctggagagagaggagaagcctgagcctgagtctgtgtatgagacagactgccgctgggatggttgcagccaggagttcgattcccaggagcagctggtgcaccacatcaacagtgagcatatccacggggagcggaaggaattcgtgtgccattggggaggttgctccagggagctgaggcccttcaaggcccaatacatgctggtggtgcacatgcgcagacacacgggcgagaagccacacaagtgcacgtttgaaggctgtcggaagtcctattcacgccttgaaaacctcaagacgcaccttcggtcgcacacgggtgagaagccttacatgtgtgagcaagaaggttgcagcaaggcctttagcaatgccagtgaccgcgccaagcaccagaatcggacccactccaatgagaagccatacgtgtgcaagctccccggctgcaccaagcgctacacagatcccagctcgctccgcaaacacgtgaagacagtgcatggtccggatgcccacgtgaccaagcggcatcgaggggatggccccttgccacgggctcagcccctctccacagtggagcccaagcgggaaagggaaggaggatccggcagggaagagagcagactgactgtgcccgagagtgccatgccgcagcagagccccggagcgcagtcctcttgcagcagcgaccactccccagcaggcagtgcggccaacacggacagcggcgtggagatggccggcaacgccgggggcagcactgaggacttgtccagcttggatgaaggaccttgtgtctcggccaccggactctccacgcttcgccgcctggagaaccttaggctggatcagctgcatcagctccggcccatagggtctcggggtctcaaactgcccagcttaacccacgctggcgcacctgtgtctcgccgtctgggccccccagtctccctggaccgccgcagcagcagctccagcagcatgagctctgcttacacagtcagccgcaggtcctccctggcatcccctttcccgccgggaaccccaccagagaatggggcatcgtcactacctggcctcacacctgctcagcactacatgctccgtgccagatatgcttcagccagggggagtggcaccccgcccactgcagctcacagcctggatcggatgggaggtctttctgttcctccttggagaagccgaaccgagtacccgggatacaacccaaatgcaggggtcactcggagggccagtgacccagcccgggctgctgaccacccagctccagccagagtccagcggttcaagagcctgggatgtgtccacacgccccctagtgtggcaacgggacggaacttcgatccccaccaccctacctctgtctattcgccacagccccccagcatcaccgaaaatgttgccatggatactagggggctacaggaggagccagaggttggaacttctgtgatgggcaatggtctgaacccatacatggatttttcctccactgatactctgggatatgggggacccgaggggacggcagctgagccttatgaagctaggggtccaggttccctgcctcttgggcctggtccaccaaccaactatggccctggccactgtgcccagcaggtctcctatcctgatcccaccccagaaaactggggtgagttcccttctcatgctggggtgtaccctagcaataaggctccgggtgctgcctatagccagtgtcctcgacttgagcattatggacaagtgcaggtaaaaccagaacaagggtgcccagtggggtctgactccaccggattggcaccctgcctcaatgcccaccccagtgaagggtccccaggcccgcagcctctgttttcacatcatccccagctccctcagccccagtatccccagtcgggtccctatcctcagcctccccatggttatctctcaacagaacccaggcttggcctcaatttcaacccctcctcctctcattccacaggacagctcaaagctcagctggtgtgtaattacgttcagtcgcagcaggaattgttgtgggagggaagaaaccggggagggctccccaaccaggaactcccataccagagccccaagtttctggggggttcccaagttagtcagagccctgccaagaccccagcagcagcggcggcagcatatggatctggctttgcacctgcttcggccaatcacaaatcaggctcctatcctgccccttcaccctgccatgaaactttcaccgtgggagtaaacaggccttcccacaggccagcagcaccaccccgacttctgcccccgctgtccccttgctatgggcccctcaaggtgggggataccaaccccagctgtggccatcctgaggtgggcaggttaggagcaggccctgccttgtaccctggtaaccacgtg(SEQ ID NO:29)
DttRR-2.4kb stuff1-CLP-1.8kb stuff2-R'R't't'D'：
aggaacccctagtgatggagttggagttggctgcgcgctcgctcgctcactgctgcgcgctcgctcgctcactgacgcgtggctgttcaaagaggccaacgtggaaaacaacgagggcaggcggagcaagagaggcgccagaaggctgaagcggcggaggcggcatagaatccagagagtgaagaagctgctgttcgactacaacctgctgaccgaccacagcgagctgagcggcatcaacccctacgaggccagagtgaagggcctgagccagaagctgagcgaggaagagttctctgccgccctgctgcacctggccaagagaagaggcgtgcacaacgtgaacgaggtggaagaggacaccggcaacgagctgtccaccaaagagcagatcagccggaacagcaaggccctggaagagaaatacgtggccgaactgcagctggaacggctgaagaaagacggcgaagtgcggggcagcatcaacagattcaagaccagcgactacgtgaaagaagccaaacagctgctgaaggtgcagaaggcctaccaccagctggaccagagcttcatcgacacctacatcgacctgctggaaacccggcggacctactatgagggacctggcgagggcagccccttcggctggaaggacatcaaagaatggtacgagatgctgatgggccactgcacctacttccccgaggaactgcggagcgtgaagtacgcctacaacgccgacctgtacaacgccctgaacgacctgaacaatctcgtgatcaccagggacgagaacgagaagctggaatattacgagaagttccagatcatcgagaacgtgttcaagcagaagaagaagcccaccctgaagcagatcgccaaagaaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccggcaagcccgagttcaccaacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagattattgagaacgccgagctgctggatcagattgccaagatcctgaccatctaccagagcagcgaggacatccaggaagaactgaccaatctgaactccgagctgacccaggaagagatcgagcagatctctaatctgaagggctataccggcacccacaacctgagcctgaaggccatcaacctgatcctggacgagctgtggcacaccaacgacaaccagatcgctatcttcaaccggctgaagctggtgcccaagaaggtggacctgtcccagcagaaagagatccccaccaccctggtggacgacttcatcctgagccccgtcgtgaagagaagcttcatccagagcatcaaagtgatcaacgccatcatcaagaagtacggcctgcccaacgacatcattatcgagctggcccgcgagaagaactccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagaccaacgagcggatcgaggaaatcatccggaccaccggcaaagagaacgccaagtacctgatcgagaagatcaagctgcacgacatgcaggaaggcaagtgcctgtacagcctggaagccatccctctggaagatctgctgaacaaccccttcaactatgaggtggaccacatcatccccagaagcgtgtccttcgacaacagcttcaacaacaaggtgctcgtgaagcaggaagaaaacagcaagaagggcaaccggaccccattccagtacctgagcagcagcgacagcaagatcagctacgaaaccttcaagaagcacatcctgaatctggccaagggcaagggcagaatcagcaagaccaagaaagagtatctgctggaagaacgggacatcaacaggttctccgtgcagaaagacttcatcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacctgctgcggagctacttcagagtgaacaacctggacgtgaaagtgaagtccatcaatggcggcttcaccagctttctgcggcggaagtggaagtttaagaaagagcggaacaaggggtacaagcactgacagccctgctgctgcctctggctctgctgctgcatgccgctagacccgattacaaggatgacgacgataagggtggtggtggttctatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattttttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctacgcgacatcaagattacattctggagtcccatcaaggttcagtggcagtgggtctggaacagatttttctctcaccattagcaaactggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccacttacgttcggcgctgggacaaagttggaacgcgtctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgtcaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggattcgaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcgaacatcgatatggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaactcgagagatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaacctgccagctgaagtcagagctggatatgatggttggcaagtgcccggaggaccctttggaaggggacatgtctagccccaactccacaggcacacaggatcacctgttggggatgctggatgggcgggaggacctggagagagaggagaagcctgagcctgagtctgtgtatgagacagactgccgctgggatggttgcagccaggagttcgattcccaggagcagctggtgcaccacatcaacagtgagcatatccacggggagcggaaggaattcgtgtgccattggggaggttgctccagggagctgaggcccttcaaggcccaatacatgctggtggtgcacatgcgcagacacacgggcgagaagccacacaagtgcacgtttgaaggctgtcggaagtcctattcacgccttgaaaacctcaagacgcaccttcggtcgcacacgggtgagaagccttacatgtgtgagcaagaaggttgcagcaaggcctttagcaatgccagtgaccgcgccaagcaccagaatcggacccactccaatgagaagccatacgtgtgcaagctccccggctgcaccaagcgctacacagatcccagctcgctccgcaaacacgtgaagacagtgcatggtccggatgcccacgtgaccaagcggcatcgaggggatggccccttgccacgggctcagcccctctccacagtggagcccaagcgggaaagggaaggaggatccggcagggaagagagcagactgactgtgcccgagagtgccatgccgcagcagagccccggagcgcagtcctcttgcagcagcgaccactccccagcaggcagtgcggccaacacggacagcggcgtggagatggccggcaacgccgggggcagcactgaggacttgtccagcttggatgaaggaccttgtgtctcggccaccggactctccacgcttcgccgcctggagaaccttaggctggatcagctgcatcagctccggcccatagggtctcggggtctcaaactgcccagcttaacccacgctggcgcacctgtgtctcgccgtctgggccccccagtctccctggaccgccgcagcagcagctccagcagcatgagctctgcttacacagtcagccgcaggtcctccctggcatcccctttcccgccgggaaccccaccagagaatggggcatcgtcactacctggcctcacacctgctcagcactacatgctccgtgccagatatgcttcagccagggggagtggcaccccgcccactgcagctcacagcctggatcggatgggaggtctttctgttcctccttggagaagccgaaccgagtacccgggatacaacccaaatgcaggggtcactcggagggccagtgacccagcccgggctgctgaccacccagctccagccagagtccagcggttcaagagcctgggatgtgtccacacgccccctagtgtggcaacgggacggaacttcgatccccaccaccctacctctgtctattcgccacagccccccagcatcaccgaaaatgttgccatggatactagggggctacaggaggagccagaggttggaacttctgtgatgggcaatggtctgaacccatacatggatttttcctccactgatactctgggatatgggggacccgaggggacggcagctgagccttatgaagctaggggtccaggttccctgcctcttgggcctggtccaccaaccaactatggccctggccactgtgcccagcaggtctcctatcctgatcccaccccagaaggtaaccacgtg(SEQ ID NO:30)
ITRL-spCas9-gRNA12(NFkB)：
ctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgtcaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggattcgaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcgaacatcgattgaattcatggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtgactcgagagatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccagtttggttaattaacgtaccgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgcgagggaccagccaagatcggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttacggatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgggagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaactatagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacattttacatgataggcttggatttctataagagatacaaatactaaattattattttaaaaaacagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactataaatatcccttggagaaaagccttgtttgagactgtgggcatgagcacggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgaattacgttacataacttacggtaaccacgtg(SEQ ID NO:31)
Another aspect of the invention provides a recombinant adeno-associated viral vector comprising a first packaged expression vector as described above and a second recombinant adeno-associated viral vector comprising a second packaged expression vector as described above.
Another aspect of the invention provides a host cell transduced with a vector system as described above.
In the host cell of the present invention, the host cell is selected from the group consisting of human cells and other mammalian cells.
Another aspect of the invention provides the use of a vector system as described above in the preparation of a gene therapy drug and/or vaccine or for gene editing.
Another aspect of the invention provides a gene therapy agent comprising a vector system as described above.
Another aspect of the invention provides a method of constructing a large gene expression vector comprising constructing a positive and a negative expression vector in a vector system as described above.
Another aspect of the invention provides a method of gene editing comprising a construction system as described above or a recombinant adeno-associated viral vector as described above. Specifically, it comprises constructing a vector system as described above, corresponding to a complete gene expression cassette of interest comprising CRISPR/CAS9 and gRNA, and introducing the vector system into a host containing the gene to be edited.
Another aspect of the invention provides a genetic recombination method comprising the use of a recombinant adeno-associated viral vector as described above or by the construction system as described above. Specifically, it comprises constructing a vector system as described above, wherein the vector system corresponds to a complete gene expression cassette of interest comprising EGFP and luciferase, and introducing the vector system into a host.
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Example 1
As shown in FIG. 1, is an ITR gene element (DtrsA 'B' BC 'CA) consisting of two palindromic arms (B-B' and C-C '), one palindromic long stem (A-A') and the D region.
ITR145bp sequence:
Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcc cgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa(SEQ ID NO:32)
The application modifies ITR, and the modified ITR sequence (DttRR) only comprises a Rep protein binding element (RBE) and a Rep protein cleavage element (trs).
DttRR sequence:
aggaacccctagtgatggagttggagttggctgcgcgctcgctcgctcactgctgcgcgctcgctcgctcactg(SEQ ID NO:33)
D sequence:
Aggaacccctagtgatggag(SEQ ID NO:35)
Trs sequence:
agttgg
RBE sequence:
ctgcgcgctcgctcgctcactg(SEQ ID NO:36)
The application modifies ITR, and the modified ITR sequence (DtrsA 'B') is composed of a D element, a trs sequence, an A 'sequence and a B' sequence in an AAVITR element.
DtrsA 'B' sequence:
aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaa(SEQ ID NO:34)
EXAMPLE 2 construction of pFBD-DttRR-3.1kb Stuff1-CEP-2.8kb tuff2-R ' R't ' t ' D ' recombinant plasmid
In this example, a pFBD-DttRR-3.1kb Stuff1-CEP-2.8kb Stuff2-R ' R't ' t ' D ' recombinant plasmid was constructed, wherein CEP is an abbreviation for CMV-EGFP-PolyA, comprising the steps of:
2.1 construction of pFBD-DttRR-3.1kb Stuff1-CEP-ITR recombinant plasmid
Primer 1 and primer 2 are used, and the primer becomes renatured to obtain an oligomeric double chain DNA (OligodsDNA) with sticky ends.
Primer 1:
5’-CAGGAACCCCTAGTGATGGAGTTGGAGTTGGCTGCGCGCTCGCTCGCTCACTGCTGCGCGCTCGCTCGCTCACTGA-3’(SEQ ID NO:1)
primer 2:
5'-CGCGTCAGTGAGCGAGCGAGCGCGCAGCAGTGAGCGAGCGAGCGCGCAGCCAACTCCAACTCCATCACTAGGGGTTCCTGGTAC-3'(SEQ ID NO:2)
the variational reaction system is shown in Table 1.
TABLE 1
| Reagent(s) | Volume of |
| 10xPCR Buffer | 2μL |
| F (10 uM) primer 3 | 2μL |
| R (10 uM) primer 4 | 2μL |
| ddH2O | 14μL |
The variational reaction procedure is as follows: and (3) renaturating and cooling to room temperature at 95 ℃ for 30 min.
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃3.5min,25 cycles.
The resulting cohesive-end oligo duplex DNA (OligodsDNA) was ligated with the KpnI/MluI-inserted double-digested pFastBacdual-ITR-EGFP plasmid to give the pFBD-DttRR-CEP-ITR recombinant plasmid.
Construction methods and maps of pFastBacdual-ITR-EGFP plasmid are described in reference 1 (Li Taiming et al, section 1.2.1 of insect cell preparation AAV-ITR gene expression microcarrier, bioengineering journal, 2015,31 (8), pages 1232).
2.2 Construction of pFBD-DttRR-CEP-R ' R't ' t ' D ' recombinant plasmid
By using the primer 3 and the primer 4, the primer becomes renatured to obtain an oligomeric double strand DNA (OligodsDNA) with a sticky end.
Primer 3:
5’-GACCGCAGTGAGCGAGCGAGCGCGCAGCAGTGAGCGAGCGAGCGCGCAGCCAACTCCAACTCCATCACTAGGGGTTCCTA-3’(SEQ ID NO:3)
Primer 4:
5'-AGCTTAGGAACCCCTAGTGATGGAGTTGGAGTTGGCTGCGCGCTCGCTCGCTCACTGCTGCGCGCTCGCTCGCTCACTGCG-3'(SEQ ID NO:4)
The variational reaction system is shown in Table 2.
TABLE 2
| Reagent(s) | Volume of |
| 10xPCR Buffer | 2μL |
| F (10 uM) primer 3 | 2μL |
| R (10 uM) primer 4 | 2μL |
| ddH2O | 14μL |
The variational reaction procedure is as follows: and (3) renaturating and cooling to room temperature at 95 ℃ for 30 min.
The obtained oligo duplex DNA (OligodsDNA) with sticky end is connected with the pFBD-DttRR-CEP-ITR recombinant plasmid obtained in the step 2.1 of inserting RsrII/HindIII double enzyme cutting, and the pFBD-DttRR-CEP-R ' R't ' t ' D ' recombinant plasmid is obtained.
2.3 Construction of pFBD-DttRR-3.1kb Stuff1-CEP-R ' R't ' t ' D ' recombinant plasmid
PCR amplification was performed using SaCas9-gene as template and primer 5 and primer 6 to give a sticky-ended 3.1kb stuff1 fragment.
Primer 5:5'-TCACTGACGCGTCCTGAACGACCTGAACAATCT-3' (SEQ ID NO: 5)
Primer 6:5'-AACTAGACGCGTATCCTTAGCGAGGGGGCAGGG-3' (SEQ ID NO: 6)
The PCR reaction system is shown in Table 3.
TABLE 3 Table 3
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃3min,25 cycles.
The resulting cohesive-terminated 3.1kb stuff1 fragment was ligated with the pFBD-DttRR-CEP-R't ' t ' D ' recombinant plasmid of step 2.2 treated with MluI single enzyme cleavage alkaline phosphatase to give pFBD-DttRR-3.1kb stuff1-CEP-R't ' D ' recombinant plasmid.
2.4 Construction of pFBD-DttRR-3.1kb Stuff1-CEP-2.8kb Stuff2-R ' R't ' t ' D ' recombinant plasmid
PCR was performed using Gli-1 as a template and primer 7 and primer 8 to obtain a sticky-end 2.8kb stuff2 fragment.
Primer 7:5'-TTTGTAGGTAACCAACCGTAATCCGGACCTCACC-3' (SEQ ID NO: 7)
Primer 8:5'-GCACGTGGTTACCAGGGTACAAGGCAGGGCCTGC-3' (SEQ ID NO: 8)
The PCR reaction system is shown in Table 4.
TABLE 4 Table 4
| Reagent(s) | Volume of |
| KOD-Plus-Neo | 2.5μL |
| 10xPCR Buffer | 5μL |
| 25mM MgSO4 | 5μL |
| 2mM dNTPs | 5μL |
| F (10 uM) primer 7 | 0.6μL |
| R (10 uM) primer 8 | 0.6μL |
| SaCas9-gene templates | 1-50ng |
| Water and its preparation method | To 50 mu L |
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃3min,25 cycles.
The resulting cohesive end 2.8kb Stuff2 fragment was ligated with the BstEII single cleavage alkaline phosphatase treated pFBD-DttRR-3.1kb Stuff1-CEP-R't ' t ' D ' recombinant plasmid of step 2.3 to give pFBD-DttRR-3.1kb Stuff1-CEP-2.8kb Stuff2-R't ' D ' recombinant plasmid, and the sequence of DttRR-3.1kb Stuff1-CEP-2.8kb Stuff2-R't ' t ' D ' was shown in SEQ ID NO:29.
EXAMPLE 3 pFBD-DttRR-2.4kb Stuff1-CLP-1.8kb Stuff2-R ' R't ' t ' D ' construction of recombinant plasmid
In this example 3, a pFBD-DttRR-2.4kb stuff1-CLP-1.8kb stuff2-R ' R't ' t ' D ' recombinant plasmid was constructed, comprising the steps of:
pFBD-DttRR-2.4kb Stuff1-CLP-1.8kb Stuff2-R ' R't ' t ' D ', CLP is an abbreviation for CMV-Luciferase-PolyA.
3.1 Construction of pFBD-DttRR-CLP-R ' R't ' t ' D ' recombinant plasmid
PCR amplification was performed using the luciferase gene as a template and primer 9 and primer 10 to obtain a sticky-end luciferase fragment.
Primer 9:5'-TCGAACATCGATATGGAAGATGCCAAAAACATT-3' (SEQ ID NO: 9)
Primer 10:5'-AGATCTCTCGAGTTACACGGCGATCTTGCCGCC-3' (SEQ ID NO: 10)
The PCR reaction system is shown in Table 5.
TABLE 5
| Reagent(s) | Volume of |
| KOD-Plus-Neo | 2.5μL |
| 10x PCR Buffer | 5μL |
| 25mM MgSO4 | 5μL |
| 2mM dNTPs | 5μL |
| F (10 uM) primer 9 | 0.6μL |
| R (10 uM) primer 10 | 0.6μL |
| Luciferase gene template | 1-50ng |
| Water and its preparation method | To 50 mu L |
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃3min,25 cycles.
The resulting cohesive-end luciferase fragment was ligated with the pFBD-DttRR-CEP-R't ' t ' D ' recombinant plasmid of step 2.2 of example 2, which was digested with ClaI/XhoI, to construct a pFBD-DttRR-CLP-R ' R't ' t ' D ' recombinant plasmid.
3.2 Construction of pFBD-DttRR-2.4kb Stuff1-CLP-R ' R't ' t ' D ' recombinant plasmid
PCR amplification was performed using the SaCas9 gene as template and primer 11 and primer 12 to give a sticky-ended 2.4kb stuff1.
Primer 11:5'-TCACTGACGCGTGGCTGTTCAAAGAGGCCAACGT-3' (SEQ ID NO: 11)
Primer 12:5'-AACTAGACGCGTTCCAACTTTGTCCCAGCGCCGA-3' (SEQ ID NO: 12)
The PCR reaction system is shown in Table 6.
TABLE 6
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃3min,25 cycles.
The resulting sticky-ended 2.4kb stuff1 fragment was ligated to the pFBD-DttRR-CLP-R't' t 'D' recombinant plasmid of step 3.1 in this example treated with MluI single enzyme cleavage alkaline phosphatase, to construct a pFBD-DttRR-2.4kb stuff1-CLP-R 'R't 'D' recombinant plasmid.
3.3 Construction of pFBD-DttRR-2.4kb Stuff1-CLP-1.8kb Stuff2-R ' R't ' t ' D ' recombinant plasmid
PCR amplification was performed using the Gli-1 gene as a template and using primer 13 and primer 14, to obtain a 1.8kb stuff2 fragment with a cohesive end.
Primer 13:5'-TTTGTAGGTAACCTGCCAGCTGAAGTCAGAGCTG-3' (SEQ ID NO: 13)
Primer 14:5'-GCACGTGGTTACCTTCTGGGGTGGGATCAGGATA-3' (SEQ ID NO: 14)
The PCR reaction system is shown in Table 7.
TABLE 7
| Reagent(s) | Volume of |
| KOD-Plus-Neo | 2.5μL |
| 10xPCR Buffer | 5μL |
| 25mM MgSO4 | 5μL |
| 2mM dNTPs | 5μL |
| F (10 uM) primer 13 | 0.6μL |
| R (10 uM) primer 14 | 0.6μL |
| Gli-1 gene as template | 0.6μL |
| Water and its preparation method | To 50 mu L |
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃2min,25 cycles.
The resulting 1.8kb stuff2 fragment with sticky ends was ligated to the pFBD-DttRR-2.4kb stuff1-CLP-R't' t 'D' recombinant plasmid of step 3.2 in this example treated with BstEII single enzyme, and the sequence of DttRR-2.4kb stuff1-CLP-1.8kb stuff2-R't' t 'D' was constructed as shown in SEQ ID NO:30.
EXAMPLE 4 pFBD-expression cassette 1.0 kb-ITRL-CMV-spCas9-PolyA-U6-gRNA1/2 (NFkB) -RXH recombinant plasmid
In this example, a pFBD-expression cassette of 15.0KB-ITRL-CMV-spCas9-PolyA-U6-gRNA1/2 (NF-KB) -RXH was constructed. The method comprises the following steps:
4.1 construction of pFBL-CEP-RXH recombinant plasmid
The primer 15 and the primer 16 are used for renaturation to obtain an oligomeric double strand DNA (OligodsDNA) with sticky ends.
Primer 15:5'-GACCGTAATCTCTAGATTGAGGAACAA-3' (SEQ ID NO: 15)
Primer 16:5'-AGCTTTGTTCCTCAATCTAGAGATTACG-3' (SEQ ID NO: 16)
The variational reaction system is shown in Table 8.
TABLE 8
| Reagent(s) | Volume of |
| 10xPCR Buffer | 2μL |
| F (10 uM) primer 15 | 2μL |
| R (10 uM) primer 16 | 2μL |
| ddH2O | 14μL |
The variational reaction procedure is as follows: and (3) renaturating and cooling to room temperature at 95 ℃ for 30 min.
The obtained oligo duplex DNA (OligodsDNA) with sticky end is connected with pFastBacdual-ITR-EGFP plasmid which is cut by RsrII/HindIII double enzyme, and pFBD-ITRL-CEP-RXH recombinant plasmid is constructed.
4.2 Construction of pFBD-ITRL-CMV-spCas9-polyA-RXH recombinant plasmid
And (3) carrying out PCR amplification by using the spCas9 as a template and adopting a primer 17 and a primer 18 to obtain the spCas9 fragment with the sticky end.
Primer 17:5'-TCGAACGAATTCATGGACTATAAGGACCACGAC-3' (SEQ ID NO: 17)
Primer 18:5'-AGATCTCTCGAGTCACTTTTTCTTTTTTGCCTG-3' (SEQ ID NO: 18)
The PCR reaction system is shown in Table 9.
TABLE 9
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃4min,25 cycles.
The resulting cohesive end spCas9 fragment was ligated to the pFBD-ITRL-CEP-RXH recombinant plasmid of step 4.1 of this example, which was digested with EcoRI/XhoI, to construct a pFBD-ITRL-CMV-spCas9-polyA-RXH recombinant plasmid.
4.3 Construction of pFBD-ITRL-CMV-spCas9-polyA-U6-gRNA1/2 (NFkB) -RXH recombinant plasmid
PCR amplification is carried out by using U6-gRNA1/2 (NF-KB) as a template and adopting a primer 19 and a primer 20 to obtain the U6-gRNA1/2 (NF-KB) expression frame fragment with the sticky end.
Primer 19:5'-TTTGTAGGTAACCAGTTTGGTTAATTAACGTACCGA-3' (SEQ ID NO: 19)
Primer 20:5'-GCACGTGGTTACCGTAAGTTATGTAACGTAATTCAA-3' (SEQ ID NO: 20)
The PCR reaction system is shown in Table 10:
Table 10
| Reagent(s) | Volume of |
| KOD-Plus-Neo | 2.5μL |
| 10xPCR Buffer | 5μL |
| 25mM MgSO4 | 5μL |
| 2mM dNTPs | 5μL |
| F (10 uM) primer 19 | 0.6μL |
| R (10 uM) primer 20 | 0.6μL |
| U6-gRNA1/2 (NF-KB) gene template | 1-50ng |
| Water and its preparation method | To 50 mu L |
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃1min,25 cycles.
The resulting cohesive end U6-gRNA1/2 (NF-KB) expression cassette was ligated to the pFBD-ITRL-CMV-spCas9-polyA-RXH recombinant plasmid of step 4.2 in this example treated with BstEII single enzyme cleavage alkaline phosphatase, and a pFBD-ITRL-CMV-spCas9-polyA-U6-gRNA1/2 (NF-KB) -RXH recombinant plasmid was constructed.
4.4 Construction of pFBD-expression cassette 15.0kb-ITRL-CMV-spCas9-PolyA-U6-gRNA1/2 (NFkB) -RXH recombinant plasmid
PCR amplification was performed using CMV-spCas9-PolyA-U6-gRNA1/2 (NF-KB) as a template and primer 21 and primer 22 to obtain a cohesive end expression cassette 15.0KB fragment.
Primer 21:5'-TCTCCCGGTACCATGATCAAGTTCCGGGGCCAC-3' (SEQ ID NO: 21)
Primer 22:5'-CATGGTGCTAGCGTAAGTTATGTAACGTAATTC-3' (SEQ ID NO: 22)
The PCR reaction system is shown in Table 11.
TABLE 11
| Reagent(s) | Volume of |
| KOD-Plus-Neo | 2.5μL |
| 10×PCR Buffer | 5μL |
| 25mM MgSO4 | 5μL |
| 2mM dNTPs | 5μL |
| F (10 uM) primer 21 | 0.6μL |
| R (10 uM) primer 22 | 0.6μL |
| CMV-spCas9-PolyA-U6-gRNA1/2 (NF-KB) template | 1-50ng |
| Water and its preparation method | To 50 mu L |
The PCR reaction procedure was: 95 ℃ for 4min;95℃10s,56℃30s,72℃5min,25 cycles.
The resulting cohesive end expression cassette 15.0KB fragment was ligated with the pFBD-ITRL-CMV-spCas9-polyA-U6-gRNA1/2 (NF-KB) -RXH recombinant plasmid of step 4.3 in this example, which was digested with KpnI/NheI, to construct the pFBD-expression cassette 15.0KB-ITRL-CMV-spCas9-PolyA-U6-gRNA1/2 (NF-KB) -RXH plasmid, the ITRL-spCas9-gRNA12 (NFkB) sequence was shown in SEQ ID NO:31.
EXAMPLE 5 viral packaging
The recombinant adeno-associated virus large gene packaging expression vector packaging system can be an insect cell baculovirus packaging system or a mammalian cell packaging system. The insect cells were Sf9 cells.
First, one or two recombinant plasmids expressing the AAV replication protein Rep, expressing the AAV capsid protein Cap (Rep and Cap genes on one or two different plasmids, respectively), and the other plasmid containing lgAAV DNA genome, will be expressed. E.coli DH10Bac competent cells were transformed respectively, colonies containing recombinant bacmid were white, colonies without recombination were blue, white colonies were picked up for amplification, and recombinant bacmid was extracted.
Then, two or three recombinant bacmid are respectively transfected into insect cell Sf9 cells by using insect cell transfection reagent, and after 4-5 days, cell supernatant is collected to obtain the P1 generation insect cell recombinant baculovirus. And (3) amplifying the P1 generation recombinant baculovirus by twice infecting Sf9 insect cells to obtain the P3 generation recombinant baculovirus. The titer of the P3-generation baculoviruses was determined using the plaque assay, viral titer (pfu/mL) =1/dilution x plaque number x 1/volume inoculated per well.
Finally, two or three recombinant baculoviruses of the P3 generation are co-infected with Sf9 insect cells, and lgAAV vector virus particles are obtained by packaging. Specific procedures can be referred to as 2(Urabe M,Ding C,Kotin RM.Insect cells as a factory to produce adeno-associated virus type 2 vectors.Hum Gene Ther.2002, 11, 1; 13 (16) 1935-43.Doi:10.1089/10430340260355347.Pmid: 12427305). The method comprises the following steps:
5.1 preparation of recombinant Bacmid
1) Mu.L DH10Bac competent cells were thawed slowly on ice for 1-3min.
2) 500Ng of the recombinant plasmid DNA obtained in example 2, example 3 and example 4 was added, respectively, and gently mixed.
3) Placed on ice for 30 minutes, heat-shocked at 42 ℃ for 90 seconds, immediately transferred to ice and placed on ice for 3 minutes.
4) 890. Mu.L of LB medium was added thereto, and the mixture was shaken at 37℃for 2-3 hours at 225 rpm.
5) To the center of a prefabricated 90 mmKTG-resistant agarose LB solid culture dish containing 50. Mu.g/mL kanamycin (kan), 77. Mu.g/mL gentamycin (Gen), 10. Mu.g/mL tetracycline (Tet) was added dropwise 40. Mu.L 2% (20 mg/mL) of X-gal and 20. Mu.L 20% (200 mg/mL) of IPTG. The plate was spread evenly on the surface using a sterile applicator and incubated in an oven until all liquid had disappeared.
6) KTG-resistant solid agarose plates were plated with a gradient of 100. Mu.L and 300. Mu.L of bacterial solution.
7) After 48h at 37℃2 white clones were picked and streaked onto new KTG-resistant solid agarose plates, overnight at 37 ℃.
8) Selecting two monoclonal bacterial plaques for PCR identification; the primers used for PCR identification of Bacmid are the target genes F/R and PUC M13F/R respectively.
9) Taking out and identifying the correct Bacmid bacterial plaque, inoculating to 10mL LB (Kan+, gen+, tet+) shaking bacteria for 16-18h, extracting and separating recombinant Bacmid DNA by using an OMEGA kit, measuring Bacmid concentration by using an experimental method according to a kit instruction, sub-packaging, and freezing at-20 ℃.
5.2 Transfection of Sf9 cells with recombinant baculovirus Bacmid
5.2.1 Preparation of transfection reagents
200ML of 1 XHBS solution: 0.954g Hepes, 1.754g NaCl, 150mL sterilized ddH2O, adjusting pH to 7.4 with 1M NaOH, fixing volume to 200mL, aseptically filtering in a safe cabinet, and preserving at 4deg.C.
PEI 20 mL: 0.043g PEI and 1mL absolute ethyl alcohol, after fully dissolving, the volume is fixed to 20mL by using 1 XHBS solution, and the freezing and thawing are repeated three times (-20 ℃ for freezing and thawing at room temperature), and the storage is carried out at 20 ℃.
5.2.2 Cell plating
And (3) paving: taking a 6-hole plate, sucking Sf9 suspension culture cells for counting, so that the density of the plated cells is 2X 10 6 cells/mL, 2mL of each hole is used, and the cell survival rate is more than 95%.
5.2.3 Transfection (amount per well)
And (3) solution A: PEI: 6. Mu.L PEI and 94. Mu.L 1 XHBS were added and mixed, and the mixture was allowed to stand for 4 minutes.
And (2) liquid B: mu.g of DNA was taken out of the pellet (the pellet was inactivated at 65℃for 30 minutes in advance), and 1 XHBS was used to make up the pellet so that the final volume was 100. Mu.L, and gently mixed.
100. Mu.L of solution A was added to solution B, mixed well and incubated for 30 minutes at room temperature. Add to Sf9 cell-plated wells and incubate for 96 hours.
5.2.4 Amplified Virus
1) Isolation of P1
After confirming that Sf9 cells are in the late stage of infection (96 h), 2mL of virus-containing medium is collected per well into a sterile 15mL centrifuge tube, and centrifuged at 500g for 5 min to remove cell debris.
The supernatant was taken into sterile EP tubes and stored at 4℃in the absence of light. If long-term storage is desired, packaging and freezing at-80deg.C.
2) Amplification of virus to obtain P2
Cells were suspended at MOI of 0.1,8mL, at a density of 2X 10 6 cells/mL, and the required P1 volume was calculated to be 1.6mL. The cells were incubated at 27℃for 72h, and the suspension-cultured cells were collected in a sterile 15mL centrifuge tube and centrifuged at 500g for 5 minutes. The supernatant was dispensed into sterile EP tubes, the viral supernatant was P2, stored at 4℃in the dark, and if long-term storage was desired, the aliquots were frozen at-80 ℃.
3) Amplified virus acquisition P3
P3 was obtained by amplification in the above manner, and the cells were collected by culturing in suspension at a MOI of 0.1 and a density of 2X 10 6 cells/mL, 10mL of the suspension in 200. Mu.L of the P2 stock solution, and culturing for 72 hours.
The commonly available P1 virus titers are between 1X 10 6-1×107 and P2 titers are between 1X 10 7-1×108.
4) Virus package
Cells were collected by adding 5mL of each of P3-generation Rep, cap and baculovirus of the target gene to cells cultured in suspension at a MOI of 1, 100mL and at a density of 5X 10 6 cells/mL, and culturing for 72 hours. Respectively obtaining corresponding recombinant adeno-associated virus vectors, which are respectively: lgAAV-CMV-Luci-7.6kB, lgAAV-CMV-CEP-8.3kB and lgAAV-spCAS-gRNA-NF kB-7.0 kB.
EXAMPLE 6 Virus purification, titre detection and identification
In this example 6, the lgAAV-CMV-Luci-7.6kB, lgAAV-CMV-CEP-8.3kB and lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated viral vectors obtained in example 5 were purified and identified. The method comprises the following steps:
6.1 purification of viral suspensions
The virus suspension obtained in example 5 was purified by iodixanol density gradient ultracentrifugation. The method comprises the following steps:
6.1.1 obtaining cell lysates
1) After 72h of co-infection, 1000g, 5min were centrifuged to collect the cells.
2) The supernatant was discarded and the cells obtained in step 1) were resuspended in 10mL of Lysis Buffer to give a cell suspension. The preparation method of the 1L Lysis Buffer solution comprises the following steps: 8.766g NaCl,6.055g Tris,950mL DDH 2 O,5M HCl to adjust pH to 8.5, constant volume to 1L, sterile filtration in a safe cabinet, and preservation at 4deg.C.
3) Freezing the cell suspension obtained in the step 2) by liquid nitrogen, melting at 37 ℃, and repeating for 3-4 times until the cell suspension is clear.
4) The supernatant was collected by centrifugation at 4℃and 5000g for 30 min; the pellet was resuspended in 4-5mL PBS and the supernatant collected by centrifugation again; and combining the supernatants of the secondary centrifugation to obtain a combined supernatant.
5) DNase (10. Mu.l/mL) and RNase (1. Mu.l/mL) were added to the combined supernatant obtained in step 4), and the mixture was digested in a water bath at 37℃for 1 hour to obtain a cell lysate.
6.1.2 Iodixanol density gradient ultracentrifugation to purify viruses
1) 60% Iodixanol was diluted with PBS-MK (1 XPBS, 1mM MgCl 2, 2.5 mMKCl) to 15%, 25%, 40% and 58% phases, 15% added solid NaCl to a final concentration of 1M.
2) In 15% phase, 25% phase was added with 0.5% phenol red solution to 1.5. Mu.l/mL and 58% phase was added with 0.5% phenol red solution to 0.5. Mu.l/mL.
3) 8ML of 15%,6mL of 25%,8mL of 40% and 5mL of 58% iodixanol diluent were added sequentially from the bottom of a 39mL rapid seal tube with a flat-mouth spinal needle of 1.27 x 120mm, avoiding air bubbles.
4) The cell lysate from step 6.1.1 was carefully overlaid on iodixanol density gradient solution, filled into the tube (flush with the nozzle line), filled with Lysis Buffer make-up volume if necessary, trimmed, and centrifuged at 69000rpm at 18 ℃ for 1.5h (BECKMAN COULTER centrifuge 70Ti rotor).
5) The quick seal tube was pierced from the bottom and the first 4mL (corresponding to 58% phase) was discarded and the 6mL of solution in the tube was collected.
6) Ultrafiltration tube (Amicon Uitra-4 Centrifugai Filters,Ultracel-100 k), 3000g, 5min centrifugation. PBS (1 XPBS, soy pal, 5M NaCl was added to a final concentration of 350 mM) was repeatedly washed until iodixanol was present at a residual concentration <
0.1%, And finally the pathogenicity of the packaging volume per 100mL is compressed to about 200 mu L.
7) Adding glycerol to final concentration of 5%, sterile filtering, and packaging at-80deg.C to obtain purified virus.
6.2 Detection of viral titres
The titer of the recombinant adeno-associated virus vector after purification is detected by adopting fluorescent quantitative PCR (RT-PCR), and the RT-PCR method comprises the following steps:
1) The plasmid ITR-CMV-EGFP-PolyA-ITR was diluted 10-fold, then subjected to gradient dilution, and 5 gradients were diluted as a standard curve.
2) The purified recombinant adeno-associated viral vector was diluted 10-fold, then subjected to gradient dilution, 4 gradients.
3) Buffer was formulated (protected from light) and the formulation is given below.
2×SuperpreMix Plus(SYBR Green)10μL
Primer 23:5'-TCCGCGTTACATAACTTACGG-3' (SEQ ID NO: 23) 0.3. Mu.L
Primer 24:5'-GGGCGTACTTGGCATATGAT-3' (SEQ ID NO: 24) 0.3. Mu.L
dd H2O 4.4μL
4) Sample addition, 20 μl system: 5. Mu.L of template+15. Mu.L of Buffer from step 3), two duplicate wells were made per gradient.
5) RT-PCR (Roche LightCycler) procedure:
Stage 1 Pre-denaturation Reps at 95℃for 600s
Stage 2 cyclic reaction Reps at 95℃for 10s
55℃ 30s
72℃ 30s
Stage 3 dissolution Curve Reps at 95℃for 15s
60℃ 60s
95℃ 15s
6.3 Identification
6.3.1SDS-PAGE analysis of recombinant adeno-associated viral vector capsid protein after 6.2 purification
1) And (3) glue preparation:
① Separator gum (10%): in a separate gel formulation beaker was added (9.093 mL) as follows: 3.69mL of double distilled water, 2.97mL of 30% acrylamide glue solution (acrylamide: methylene bisacrylamide=29:1), 2M Tris,pH8.8 2.25mL, 90 mu L of 10% ammonium persulfate and 90 mu L, TEMED mu L of 10% SDS, and dripping the separation glue between two glass plates by using a dropper until the liquid level reaches the position of 1cm at the lower edge of the comb. Slowly adding 75% ethanol with a dropper, standing, and pouring out 75% ethanol layer after polymerization of the separation gel.
② Concentrated gum (5%): the following charges (2.357 mL) were added in a formulation beaker of concentrated gum: 1.83mL of double distilled water, 0.39mL of 30% acrylamide glue solution (acrylamide: methylene bisacrylamide=29:1), 0.5M Tris,pH6.8 0.75mL, 30 mu L of 10% ammonium persulfate and 30 mu L, TEMED mu L of 10% SDS, and after uniform mixing, a dropper is used for adding concentrated glue to cover the separation glue between two glass plates until the separation glue is full, and a comb is gently inserted. Standing for coagulation to obtain gel plate.
2) Sample treatment:
The virus samples were prepared using the same procedure as described above for the construction, packaging, and purification production of the lgAAV-CMV-Luci-7.6kB, lgAAV-CMV-CEP-8.3kB, and lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated virus vectors.
Viral samples were mixed at a ratio of 5 x loading buffer=2:1, boiled for 10 minutes, centrifuged instantaneously after bathing, and all samples were taken for electrophoresis.
3) Electrophoresis
① The gel plate made of two glass plates is vertically arranged on a power supply frame in the electrophoresis tank, so that the concave edge surface of the gel plate is abutted against the power supply frame.
② The gel plate and the power supply frame are fixed in a power supply groove according to the requirement, electrophoresis buffer solution is added according to the requirement, and the comb in the gel plate is gently pulled out.
③ The treated sample solution was collected, and 15. Mu.L of the sample solution was aspirated by a micropipette and slowly added to the notched portion (sample spot entrance) in the gel plate.
④ During electrophoresis, the voltage is controlled, the concentration gel is 90V, and the separation gel voltage is 120V. And stopping electrophoresis when the color bar of bromophenol blue in the observation gel is taken away to the position close to the bottom end by 1 cm.
⑤ Dyeing. The two glass plates outside the gel plate are gently pried by a blade or a thin plate, and the separation gel is cut off by the blade along the junction of the separation gel and the concentrated gel on the gel. The separation gel was then carefully transferred to a staining vessel, 100mL staining solution was added, capped and stained for 1-3 hours.
⑥ And (5) decoloring. The staining solution was poured off. Rinsing the dyed gel with water for several times, putting the gel into clear water, putting the gel into a microwave oven, heating the gel with high fire for 2 minutes, taking out the gel, slowly shaking the gel, and repeating the operation until the protein strips can be clearly displayed.
FIG. 6 is a SDS-PAGE electrophoresis of capsid proteins of rAAV-EGFP recombinant adeno-associated viral vector, lgAAV-CMV-Luci-7.6kB recombinant adeno-associated viral vector, lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated viral vector and lgAAV-spCAS9-gRNA-NF kB-7.0kB recombinant adeno-associated viral vector in this example. Wherein MW is protein molecular weight standard, 1 is rAAV-EGFP,2 is lgAAV-CMV-Luci-7.6kB recombinant adeno-associated virus vector, 3 is lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated virus vector, 4 is lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector.
As can be seen from FIG. 6, the capsid proteins of lgAAV-CMV-Luci-7.6kB recombinant adeno-associated virus vector, lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated virus vector and lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector were not different from the capsid proteins of the conventional AAV-EGFP recombinant adeno-associated virus vector, and were composed of three proteins VP1, VP2, and VP3, and the composition of the three proteins was substantially 1:1:10.
6.3.2 Identification of recombinant adeno-associated viral vector DNA genome
1) Neutral agarose electrophoresis analysis
① Agarose solution was prepared by adding an accurate amount of agarose powder and a quantitative amount of 1××tae running buffer to an Erlenmeyer flask or glass flask.
② Kimwapes paper was gently stoppered over the neck of the flask. If a glass bottle is used, the bottle stopper needs to be unscrewed. The suspension is heated in a boiling water bath or microwave oven until agarose is dissolved.
③ Cooling the clear solution to 40-50deg.C, adding 4S red staining solution, and rapidly pouring into gel. After the gel has completely solidified, the gel is placed in an electrophoresis tank, and a newly prepared 1 xTAE electrophoresis buffer solution is added until the gel is just covered.
④ Recombinant adeno-associated viral vectors were obtained using rAAV-CMV-EGFP-PolyA vectors prepared as described above and example 5. 10. Mu.L of recombinant adeno-associated viral vector stock was prepared, 1.1. Mu.L of 10 XPCR Buffer solution was added, and the mixture was put into a PCR machine for PCR reaction.
Wherein, the PCR reaction procedure is as follows: 95℃for 5 minutes, 72℃for 5 minutes, 55℃for 5 minutes, 37℃for 5 minutes, 80℃for 5 minutes, 72℃for 5 minutes, 55℃for 5 minutes, 37℃for 15 minutes.
⑤ The sample after renaturation was added with 0.2 times of 6 Xgel loading buffer.
⑥ All samples dissolved in 6 Xloading buffer were added to the loading wells. Electrophoresis was started for 50 minutes at a voltage of 100V.
⑦ Imaging: the gel was placed in a gel imager and recorded by photographing.
FIG. 7 is a gel electrophoresis chart of rAAV-EGFP vector, lgAAV-CMV-Luci-7.6kB recombinant adeno-associated virus vector, lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated virus vector, and lgAAV-spCAS9-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector in this example. Wherein MW is the molecular weight standard of DNA, 1 is rAAV-EGFP vector, 2 is lgAAV-CMV-Luci-7.6kB recombinant adeno-associated virus vector, 3 is lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated virus vector, 4 is lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector.
As can be seen from FIG. 7, lane 1 is a rAAV-CMV-EGFP vector whose DNA is mainly a 4.7kb double-stranded DNA molecule formed by complementary pairing of positive and negative strands, and which is diffuse due to mismatch in the complementary pairing.
Lane 2 is the lgAAV-CMV-Luci-7.6kb vector obtained in example 5, which was prepared by mixing designed positive and negative strand monopolar DNA molecules (both of which were approximately 4.7kb in length). Positive and negative strand DNA have partial complementarity and, when analyzed by gel electrophoresis on neutral agarose, will exhibit double-stranded DNA molecules of less than 4.7kb in length. A single-stranded DNA molecule that is partially non-complementary, monopolar, will, due to its smaller molecular weight, exhibit much smaller DNA bands, usually in a diffuse form.
Lane 3 is the lgAAV-CMV-EGFP-8.3kb vector obtained in example 3, mixed from designed positive and negative strand monopolar DNA molecules (both approximately 4.7kb in length). Positive and negative strand DNA have partial complementarity and, when analyzed by gel electrophoresis on neutral agarose, will exhibit double-stranded DNA molecules of less than 4.7kb in length. A single-stranded DNA molecule that is partially non-complementary, monopolar, will, due to its smaller molecular weight, exhibit much smaller DNA bands, usually in a diffuse form.
Lane 4 is lgAAV-spCAS9-gRNA-NF kB-6.7kB vector obtained in example 4, mixed from designed positive and negative strand monopolar DNA molecules (both approximately 4.7kB in length). Positive and negative strand DNA have partial complementarity and, when analyzed by gel electrophoresis on neutral agarose, will exhibit double-stranded DNA molecules of less than 4.7kb in length. A single-stranded DNA molecule that is partially non-complementary, monopolar, will, due to its smaller molecular weight, exhibit much smaller DNA bands, usually in a diffuse form.
2) Alkaline agarose gel electrophoresis analysis
① Preparation of alkaline agarose gel: an agarose solution was prepared by adding 0.36g of agarose powder and 27mL of distilled water to an Erlenmeyer flask or a glass flask. Kimwapes paper was gently stoppered over the neck of the Erlenmeyer flask. If a glass bottle is used, the bottle stopper needs to be unscrewed. The suspension was heated to agarose dissolution in a microwave oven with a medium fire. The clear solution was equilibrated in a water bath at 56℃for 5 minutes. 3mL of 10 Xalkaline gel electrophoresis buffer (equilibrated in a water bath at 56 ℃ for 2 minutes) was added and mixed well, and the gel was rapidly poured. After the gel has completely solidified, it is placed in an electrophoresis tank, and a newly prepared 1 Xelectrophoresis buffer (4 ℃) is added until the gel is just covered.
② Sample preparation: the rAAV-CMV-EGFP vector prepared as described above and the 3 recombinant adeno-associated viral vectors obtained in example 5 were used. 30. Mu.L of virus sample was taken, 3. Mu.L of proteinase K (20 mg/mL) was added, digested at 65℃for 15 minutes, centrifuged at 12000g for 5 minutes, 30. Mu.L of supernatant was taken, and 6. Mu.L of 6 Xalkaline gel loading buffer was added.
③ Electrophoresis: the DNA samples were all added to the wells and electrophoresis was started at a voltage < 3.5V/cm. (horizontal electrophoresis tank JY-SPAT 27V electrophoresis 3 h.)
④ Eluting: the gel was placed in 400mL of water for injection and eluted at 56rpm on a horizontal shaker for 1h, with water changed every 30 minutes.
⑤ Dyeing: the eluted gel was stained with 1 XTAE stain containing 0.5. Mu.g/mL EB (ethidium bromide).
⑥ Imaging: the gel was placed in a gel imager and recorded by photographing.
FIG. 8 shows alkaline agarose electrophoresis of rAAV-CMV-EGFP vector, lgAAV-CMV-Luci-7.6kB recombinant adeno-associated viral vector, lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated viral vector and lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated viral vector. Wherein MW is the molecular weight standard of DNA, 1 is rAAV-CMV-EGFP vector, 2 is lgAAV-CMV-Luci-7.6kB recombinant adeno-associated virus vector, 3 is lgAAV-CMV-EGFP-8.3kB recombinant adeno-associated virus vector, 4 is lgAAV-spCAS-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector.
As can be seen from FIG. 8, the DNA molecules packaged with the rAAV viral vector were all 4.7kb single-stranded DNA.
EXAMPLE 7 identification of Gene expression from the order
In this example, 3 recombinant adeno-associated viral vectors of lgAAV-CMV-Luci-7.6kB, lgAAV-CMV-EGFP-8.3kB, lgAAV-spCAS-gRNA-NF kB-7.0kB containing different genes of interest were obtained in example 5, and the genes of interest were identified, including the following:
7.1lgAAV-CMV-EGFP-8.3kb recombinant adeno-associated virus vector injection mouse eye gene expression identification
1) Mouse eye vitreous injection: after 4% chloral hydrate is used for anesthetizing a mouse with 0.01mL/g, 0.1mg/mL of procaine hydrochloride eye drops and 5mg/mL of compound topiramate eye drops are added into eyes of the mouse after the mouse is anesthetized, and the eyes of the mouse are anesthetized and mydriatic. The head position of the mouse is adjusted to enable the eyeball to keep the limbal level. A31G needle was used to puncture 1mm after limbus, and a Hamilton Hamilton G syringe was used to inject lgAAV-CMV-EGFP-8.3kb recombinant adeno-associated viral vector 1. Mu.L, 1E10 vg/eye at the puncture. The needle tip enters vertically, then is inclined, is slowly pushed and is left for 30 seconds after the needle is pushed, and the needle is rapidly withdrawn. 1mg/mL sodium hyaluronate eye drops are added into eyes of mice for moisturizing, and 4.88mg/mL levofloxacin eye drops are added into eyes of mice for anti-inflammatory.
Meanwhile, a control group is established, wherein the control group 1 is singly injected with a negative vector, the control group 2 is singly injected with a positive vector, and the control group 3 is rAAV6-CMV-EGFP vector.
2) Mouse fundus imaging:
4% chloral hydrate 0.01mL/g anesthetized mice, 5mg/mL compound topiramate eye drops are added dropwise into eyes of the mice, mydriasis is carried out on eyes of the mice, and then 1mg/mL sodium hyaluronate eye drops are added dropwise for moisturizing. After mydriasis of the mouse's eye, line scanning confocal fundus imaging system is used to image the mouse's fundus. Green fluorescence was observed on days 10 and 17 after injection of lgAAV-CMV-EGFP-8.3kb recombinant adeno-associated viral vector using 488nm solid state laser excitation.
FIG. 9 is a graph showing the results of a test of the expression of EGFP in the eyes of mice by lgAAV-CMV-Luci-7.6kb recombinant adeno-associated viral vector.
FIG. 9 shows that lgAAV-CMV-EGFP-8.3kb vector, positive and negative vectors were injected independently, and the positive and negative vectors were injected simultaneously, which showed strong fluorescence, indicating that after pairing of partial complementary sequences in the positive and negative DNA strands, the 3' -end DNA was synthesized with the paired DNA as template to form a complete DNA double strand, thus the complete EGFP expression cassette of the target gene was obtained, the EGFP of the target gene was transcribed, and the green fluorescence was generated.
In summary, the original AAV packages the single-stranded monopole DNA of 4.7kb of the positive and negative vectors, and after pairing by the DNA complementary sequence of 1.1kb in the cell, the EGFP expression cassette of full length 8.3kb was synthesized.
7.2LgAAV-CMV-Luci-7.6kb recombinant adeno-associated viral vector injection mouse muscle Gene expression identification
1) Intramuscular injection of mice:
4% chloral hydrate 0.01mL/g anesthetized mice, the anesthetized mice were placed ventrally up on a test bench, the syringe was rapidly inserted 1/4 at 90℃angle to the semitendinous muscle, 100 μ L lgAAV-CMV-Luci-7.6kb recombinant adeno-associated viral vector was injected, 5E10 vg/right leg.
Meanwhile, a control group is established, wherein rAAV6-CMV-Luci is injected into the left leg of the same mouse, and the injection dosage and the injection method are the same as lgAAV-CMV-Luci-7.6kb recombinant adeno-associated virus vector.
2) Chemiluminescent imaging:
4% chloral hydrate 0.01mL/g anesthetized mice were intraperitoneally injected with 100 μ L D-sodium fluorescein salt solution (15 mg/mL), and after 5 minutes, the anesthetized mice were placed ventrally up in a chemiluminescent imager for 5 minutes of exposure time, and then fluorescence was observed on days 10, 14, 21 and 28. FIG. 10 is a diagram showing the results of verification of the expression of Luciferase in mouse muscle by lgAAV-CMV-Luci-7.6kb recombinant adeno-associated viral vector.
As can be seen from FIG. 10, lgAAV-CMV-Luci-7.6kb vector, the single injection of positive and negative carriers has no fluorescence, the mixture of positive and negative carriers has strong fluorescence, which means that after partial complementary sequences in the positive and negative DNA chains are paired, the 3' -end DNA takes the paired DNA as a template to synthesize the complementary chain, and a complete DNA double strand is formed, so that a complete expression frame of the target gene Luci is provided, the target gene Luci is transcribed, luciferase is synthesized, and the substrate is decomposed, and fluorescence is generated.
The genome of the positive and negative vectors is 4.7kb single-stranded monopole DNA and there is no 3' ITR, so that a single positive vector, or the vector, cannot synthesize the second complementary DNA strand in the cell, and thus cannot express the gene, so that, in the early stage, the ordinary rAAV-CMV-Luci has gene expression, whereas lgAAV-CMV-Luci-7.6kb has no gene expression.
From the combination, it was found that the 4.7kb single-stranded monopolar DNA packaged by the AAV was paired by a 1.8kb DNA complementary sequence in the cell, and a full-length 7.6kb DNA expression cassette was synthesized, and luciferase protein was expressed.
Gene expression identification of HEK 293T cells transfected with 7.3lgAAV-spCAS9-gRNA-NFkB-7.0kb recombinant adeno-associated viral vector
1) Cell infection
HEK 293T cells were seeded in 24-well plates in an amount of 2E 5. The next day, the cell culture medium was aspirated, and after mixing the MOI5E6 virus suspension with 500. Mu.L of complete medium, added to the cells and culture continued for 6 days with fresh medium changed daily. Non-transviral cells served as controls.
2) Cell genome extraction
Cell genome extraction (Tiangen Biochemical technology Beijing Co., ltd.) was performed using a blood/cell/tissue genome DNA extraction kit. The cells in the adherence culture are firstly treated into cell suspension, then centrifuged for 1min at 10,000rpm (11,200 Xg), the supernatant is poured out, 200 mu L buffer GA is added, and the suspension is oscillated until the suspension is complete; mu.L of protease K solution was added and mixed well. 200 mu L of buffer GB is added, the mixture is fully and reversely mixed, the mixture is placed at 70 ℃ for 10min, the solution is clear in strain, and the mixture is centrifuged briefly to remove water drops on the inner wall of the tube cover. Adding 200 mu L of absolute ethyl alcohol, fully shaking and uniformly mixing for 15sec, wherein flocculent precipitation possibly occurs, and centrifuging briefly to remove water drops on the inner wall of the tube cover. The solution obtained in the previous step and the flocculent precipitate were both added to an adsorption column CB3 (the adsorption column was placed in a collection tube), centrifuged at 12,000rpm (13,400Xg) for 30sec, the waste liquid was poured off, and the adsorption column CB3 was placed back in the collection tube. To the adsorption column CB3, 500. Mu.L of the buffer solution GD was added, and the mixture was centrifuged at 12,000rpm (13,400Xg) for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was placed in a collection tube. 600. Mu.L of the rinse PW was added to the adsorption column CB3, centrifuged at 12,000rpm (13,400 Xg) for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was placed in a collection tube. Repeating once. The adsorption column CB3 was put back into the collection tube and centrifuged at 12,000rpm (13,400Xg) for 2min, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material. Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 mu L of elution buffer TE into the middle part of the adsorption film, standing for 2-5min at room temperature, centrifuging at 12,000rpm (about 13,400 Xg) for 2min, and collecting the solution into the centrifuge tube.
3) Amplifying genomic fragments containing the target site
Nested PCR amplification: 10. Mu.L of KOD one PCR premix, 200ng of genomic DNA template, 0.5. Mu.L of each of the upstream and downstream primers (10 uM), and water was added to make up to 20. Mu.L. The second round of PCR was performed using 0.2. Mu.L of the first round of PCR product as a template, 25. Mu.L of 2 XKOD one PCR premix, and 1.5. Mu.L of each of the upstream and downstream primers (10. Mu.M) were prepared to prepare a 50. Mu.L system. Amplification conditions: pre-denaturation at 98 ℃ for 20 seconds; denaturation at 98℃for 5 sec, annealing at 60℃for 10 sec, elongation at 68℃for 10 sec, 30 cycles. And cutting the gel to recover the target band of the second round of PCR.
Primer was used:
NFkB2-F1：5’-CCTGGGTGGTCTTCCCTGATC-3’(SEQ ID NO:25)
NFkB2-R1：5’-GCACCTTGTCACAAAGCAGATAAAC-3’(SEQ ID NO:26)
NFkB2-F2：5’-TGATCACAATGCTACTATGCCCTTGG-3’(SEQ ID NO:27)
NFkB2-R2：5’-CTGCTGTCTTGTCCATTCGAGAAATC-3’(SEQ ID NO:28)
synthesized by the biotechnology company of Jin Weizhi, su.
4) Annealing and enzyme cutting
Preparing a reaction solution: 300ng of PCR purified product, 2. Mu.L of 10 XNEBuffer 2, and sterile water was added to 19.7. Mu.L. The reaction solution was placed in a 95 ℃ water bath for 5 minutes, and then naturally cooled to room temperature. 0.3 mu L T Endonuclease I was added and the mixture was digested at 37℃for 1 hour. 1.5% agarose gel electrophoresis detection.
FIG. 11 shows the results of functional verification of lgAAV-spCAS9-gRNA-NF kB-7.0kB recombinant adeno-associated viral vector transduced HEK293 cells, expressed the target gene spCAS, and gRNA editing NFkB gene. Wherein MW is the molecular weight standard of DNA, 1 is rAAV-CMV-EGFP vector, 2 is lgAAV-spCAS9-gRNA-NF kB-7.0kB recombinant adeno-associated virus vector obtained in example 5.
As can be seen from FIG. 11, lgAAV-spCAS9-gRNA-NF kB-7.0 kB recombinant adeno-associated viral vector produced indels of NF kB gene after infection of cells, indicating spCAS functions.
The lgAAV-spCAS-gRNA-NF kB-7.0 kb DNA genome is 4.7kb single-stranded monopole DNA, and has no 3'ITR, and can not synthesize a second complementary DNA strand in cells, and the positive and negative DNA strands are only partially complementary paired, so that after the positive and negative DNA strands are partially paired, the 3' end DNA takes the paired DNA as a template to synthesize the complementary strand, and form a complete DNA double strand, thereby having a complete large gene expression frame, enabling a large gene to be transcribed, synthesizing protein and having a gene editing function. The result shows that the original lgAAV packaged 4.7kb single-stranded monopole DNA is paired by a 2.4kb DNA complementary sequence in the cell, and a full-length 7.0kb DNA expression frame is synthesized, and spCAS protein and gRNA are expressed.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (13)
1. A construction system for packaging expression of a large gene from a recombinant adeno-associated viral vector, wherein the packaged expression DNA is greater than 4.7kb in length, the construction system comprising a first packaged expression vector and a second packaged expression vector;
From 5 , to 3 ,, the first packaging expression vector comprises a first ITR element or similar element and a first packaging expression cassette comprising a promoter of a positive strand sequence, a gene of interest, and a polyadenylation signal; from 5 , to 3 ,, the second packaging expression vector comprises a second ITR element or similar element and a second packaging expression cassette comprising a polyadenylation signal of negative strand sequence, a gene of interest, and a promoter;
the base of the positive strand and the base of the negative strand are complementary paired.
2. The construction system of claim 1, wherein the first ITR element or like element and the second ITR element or like element are selected from one or more of an AAV viral ITR element, dttRR element, and DtrsA ,B, element, wherein the DttRR element consists of a Rep protein binding element (RBE) and a Rep protein cleavage sequence (trs) in an AAVITR element, and wherein the DtrsA ,B, element consists of a D element, trs sequence, a , sequence, and B , sequence in an AAVITR element.
3. The construction system of claim 1, wherein the ITR element has a coding sequence set forth in SEQ ID NO. 32;
And/or the coding sequence of DttRR element is shown as SEQ ID NO. 33;
and/or the coding sequence of DtrsA ,B, element is shown as SEQ ID NO. 34.
4. The construction system of claim 1, wherein the promoter is selected from one of CMV, CAG, ef a and hU6, or other gene expression promoters.
5. The construction system of claim 1, wherein the large gene is selected from one or more of the group consisting of engineered expanded EGFP, luciferase, CRISPR/CAS 9; preferably, the CRISPR/CAS9 is selected from spCas9.
6. The construction system of claim 1, wherein the positive strand of the first packaging expression vector and the negative strand of the second packaging expression vector are complementarily paired at the 3' end portion.
7. A recombinant adeno-associated viral vector comprising a first recombinant adeno-associated viral vector comprising the first packaged expression vector of any one of claims 1-6 and a second recombinant adeno-associated viral vector comprising the second packaged expression vector of any one of claims 1-6.
8. A host cell transduced with the recombinant adeno-associated viral vector of claim 7.
9. The host cell of claim 8, wherein the host cell is selected from the group consisting of a human cell, and other mammalian cells.
10. Use of the construction system according to any one of claims 1-6 or the recombinant adeno-associated viral vector according to claim 7 for the preparation of a gene therapy drug and/or vaccine or for gene editing.
11. A gene therapy agent comprising the construction system of any one of claims 1-6 or the recombinant adeno-associated viral vector of claim 7.
12. A method of gene editing comprising the construction system of any one of claims 1-6 or the recombinant adeno-associated viral vector of claim 7.
13. A method of genetic recombination comprising the use of a recombinant adeno-associated viral vector as defined in any one of claims 1 to 6 or as defined in claim 7.
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