WO2022142963A1 - Système vecteur pour le criblage de séquences régulatrices et application - Google Patents

Système vecteur pour le criblage de séquences régulatrices et application Download PDF

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WO2022142963A1
WO2022142963A1 PCT/CN2021/134329 CN2021134329W WO2022142963A1 WO 2022142963 A1 WO2022142963 A1 WO 2022142963A1 CN 2021134329 W CN2021134329 W CN 2021134329W WO 2022142963 A1 WO2022142963 A1 WO 2022142963A1
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library
random
sequence
fragment
tag
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Chinese (zh)
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施金秀
罗燕
肖晓丹
叶知晟
蓝田
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云舟生物科技(广州)股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the invention belongs to the field of bioengineering, and more particularly relates to a vector system and application for screening regulatory sequences.
  • the promoter or enhancer is a DNA sequence located in the upstream and downstream regions of the 5' of the structural gene, which can accurately interact with specific RNA polymerases and related transcription factors. Binding, thereby initiating the transcription initiation of downstream genes, is the most important cis-acting element for regulating gene expression.
  • Eukaryotic promoters contain three conserved sequences with important biological functions, namely the TATA box (TATA box) located in the -35--25 region, the CAAT box (CAAT box) located in the -80--70 region, and the - GC box in the 110--80 zone.
  • the TATA box is involved in regulating the precise transcription initiation of downstream genes, and the CAAT box and GC box are involved in regulating the frequency of transcription initiation.
  • the above three functional regions are important manifestations of the functional activity of promoters, not every promoter contains these three functional regions, and the change of any base or relative position of these three functional regions will often cause promoters Dramatic changes in activity and specificity.
  • the upstream promoter or enhancer activity and the regulatory sequences near the gene, such as 5'UTR and 3'UTR, are the key factors to determine whether the downstream gene can be expressed smoothly and whether the expression level is moderate. Therefore, in order to obtain better expression of the target gene. It is particularly important to transform and screen promoter regulatory sequences using molecular directed evolution technology in vitro.
  • Natural evolution is a long process of survival of the fittest and the accumulation of favorable mutations.
  • researchers simulate the natural evolutionary mechanisms of mutation, recombination and selection in vitro, so that evolution develops in the expected direction.
  • Early researchers mainly used physical methods, chemical methods, mutagenic strains or error-prone PCR to introduce random mutations into protein-coding genes, and then perform functional screening at the cellular or animal level to obtain new functions that can meet people's needs or Excellent performance protein. Although these methods can improve some properties of proteins to a certain extent, their diversity is far from meeting people's needs.
  • DNA shuffling involves dividing multiple related gene families from different sources into random fragments by DNaseI digestion or sonication, and then using the homology between the fragments as templates and primers for each other, these fragments are reassembled by primerless PCR (primerless PCR). To generate full-length genes, the process generates template switching or crossover events that increase the diversity of the mutant library.
  • the protein mutants were then amplified using specific 5' and 3' primers for different protein-coding frames, and cloned into relevant cloning vectors to form mutant libraries.
  • the library diversity ( ⁇ 106 or more) was verified by NGS sequencing. ), and finally perform functional screening at the cellular level or animal level to obtain a protein with improved properties.
  • This method is mainly aimed at the directed evolution of protein molecules. Different genes used as starting templates need to have a certain degree of homology, so as to generate in vitro homologous recombination between small fragments and introduce mutations to form a mutant library for screening.
  • promoter or enhancer DNA shuffling is to enhance the activity of the promoter or specifically change the expression characteristics of the gene.
  • promoter or enhancer shuffling are often extremely low in homology, so the above-mentioned DNA shuffling technology for protein molecules obviously cannot be used for the directed evolution of promoter regulatory sequences.
  • promoter shuffling generally adopts the following technical routes: (1) performing two rounds of error-prone PCR on a single promoter to recover PCR products (forming a large number of mutants with homologous sequences); (2) digesting with DNaseI or sonicating into Random fragments and recover; (3) use the recovered product as a template to carry out primer-free PCR; (4) add specific primers containing specific restriction sites to the primer-free PCR system to amplify the full-length promoter, and recover the specific size (5) The cloning vector and the full-length promoter mutant were digested and ligated with the corresponding restriction enzymes; (6) NGS sequencing was used to verify the diversity of the promoter library.
  • the technical problem to be solved by the present invention is to provide a vector system and application for screening regulatory sequences.
  • the purpose of the present invention is to overcome the limitations of the promoter shuffling method, solve the problem of insufficient library diversity due to the low homology of promoters from different sources and the inability to carry out efficient in vitro recombination, and provide a method for constructing a functional element library
  • a first aspect of the present invention provides a plasmid vector comprising: an index tag, a reporter gene and a barcode tag;
  • the barcode label is a random fragment with a length of 5-200 bp;
  • the number of the index tags is at least 1, and it is independently selected from random fragments with a length of 5-100 bp;
  • the expression product of the reporter gene is capable of self-emitting light or producing color change by catalyzing the substrate reaction, producing light or producing color change by catalyzing the substrate reaction, or producing emitted light or producing color change by irradiating excitation light, Or resistant to corresponding drug screening.
  • the barcode tag is a random fragment with a length of 40bp; the number of the index tags is 2, wherein index1 is a random fragment with a length of 30bp, index2 is a random fragment with a length of 30bp, and the reporter gene At least one selected from the group consisting of fluorescent protein, luciferase, LacZ gene or a resistance gene that can play a screening role, and the resistance gene includes a puromycin resistance gene.
  • Some provided herein include a vector backbone with a first terminator, a recombination site, a reporter gene, a multiple cloning site (MCS), a post-transcriptional regulatory sequence (WPRE), and a second terminator sequentially attached to the vector backbone.
  • MCS multiple cloning site
  • WPRE post-transcriptional regulatory sequence
  • the vector further comprises at least one enzyme cleavage site.
  • the expression product of the reporter gene is capable of self-emitting light or producing color change by catalyzing the reaction of the substrate, producing light or producing color change by catalyzing the reaction of the substrate, or by irradiating excitation light to produce emission light or producing Color changes, or resistance to corresponding drug screening.
  • the reporter gene is selected from at least one of fluorescent protein, luciferase, LacZ gene or a resistance gene that can play a screening role, such as a puromycin resistance gene.
  • TurboGFP is selected as the reporter gene.
  • the first terminator and the second terminator are elements capable of terminating transcription.
  • the terminator SV40 terminator hGH terminator, BGH terminator or rbGlob terminator.
  • both the first terminator and the second terminator are selected as BGH terminators, denoted as BGH-pA.
  • the plasmid vector sequentially comprises the following elements: pUC ori, 5' ITR, BGH pA, index1, index2, reporter gene, barcode tag, WPRE, BGH pA, 3' ITR, and a resistance selection marker.
  • an enzyme cleavage site and a random recombination regulatory sequence are also included between the index1 and index2.
  • the enzyme cleavage site is AsiSI; the number of the enzyme cleavage site is 2, located at both ends of the random recombination control sequence;
  • the random recombination regulatory sequence is a fragmented promoter fragment or a fragmented enhancer fragment.
  • the random recombination regulatory sequence is an enzymatically digested promoter fragment or an enzymatically digested enhancer fragment.
  • the fragmentation method in the step of preparing the random recombination regulatory sequence, includes enzymatic digestion, ultrasonication or artificial synthesis.
  • the enzyme digested by the enzyme is DnaseI.
  • the enzyme digested by the enzyme is DnaseI;
  • the promoter is selected from hRO, hRK, mCAR, ProA1, CMV, EF1A, EFS, CAG, CBh, SFFV, MSCV, SV40, mPGK, hPGK, UBC, Nanog, Nes, Tuba1a, Camk2a, SYN1, Hb9, Th, NSE, GFAP, Iba1, hRHO, hBEST1, Prnp, Cnp, K14, BK5, mTyr, cTnT, ⁇ MHC, Myog, ACTA1, MHCK7, SM22a, EnSM22a, Runx2, OC, Col1a1, Col2a1, aP2, Adipoq, Tie1, Cd144, CD68, CD11b, Afp, Alb, TBG, MMTV,
  • the plasmid vector includes the following elements in sequence: pUC ori, 5'ITR, BGH pA, index1, AsiSI restriction site, index2, Kozak, TurboGFP gene, barcode tag, WPRE, BGH pA, 3'ITR and Amp resistance screening marker; wherein, the barcode label is a random fragment with a length of 40bp, the index1 is a random fragment with a length of 30bp, and the index2 is a random fragment with a length of 30bp;
  • the plasmid vector sequentially includes the following elements: pUC ori, 5'ITR, BGH pA, index1, AsiSI restriction site, random recombination control sequence, AsiSI restriction site, index2, Kozak, TurboGFP gene , barcode tags, WPRE, BGH pA, 3'ITR and Amp resistance screening markers; wherein, the length of the random recombination control sequence is 50-2000bp, which is the promoter fragment after DnaseI digestion or the enhancer fragment after enzyme digestion , the barcode label is a random fragment with a length of 40bp, the index1 is a random fragment with a length of 30bp, and the index2 is a random fragment with a length of 30bp.
  • the second aspect of the present invention provides a method for constructing the plasmid vector, wherein barcode tags, index tags and random recombination control sequences are inserted into a backbone vector containing a reporter gene.
  • the present invention does not limit the insertion order of barcode tags, index tags or random recombination control sequences, and also does not limit the insertion order thereof.
  • Any method of connecting a vector and a nucleic acid fragment that can be used in the art can be used in the present invention.
  • the insert fragment and the vector are ligated after enzyme digestion, or the fragment and the vector are ligated by Gibson cloning reaction.
  • the insertion of the barcode tag is as follows: preparing a barcode tag carrying the homology arm of the backbone vector, making it react with the linearized backbone vector through Gibson cloning, and constructing a tag library.
  • the insertion of the index tag and the random recombination regulatory sequence includes:
  • the tag library is linearized; the fragments are then ligated to the linearized tag library to obtain a library of regulatory sequences.
  • the method for preparing the random recombination regulatory sequence comprises enzymatically digesting the promoter or enhancer.
  • the enzyme is the DnaseI enzyme.
  • the preparation of the homology arm 1-index tag 1-enzyme cleavage site 1-random recombination regulatory sequence-enzyme cleavage site 2-index tag 2-homology arm 2 fragment specifically includes:
  • the primer F and primer R are annealed to form a Y-shaped adapter; the structure of the primer F is homology arm 1-index tag 1-restriction site 1-protection sequence 1; the structure of the primer R is protection sequence 2- Restriction site 2-index tag 2-homology arm 2; the protection sequence 1 and protection sequence 2 are complementary;
  • a linear fragment is obtained by PCR on the random long fragment of the functional element containing the Y-shaped linker
  • the linear fragment is ligated with the linearized tag library to construct a library of regulatory sequences.
  • the method for constructing the plasmid vector of the present invention further includes removing the random recombination regulatory sequence from the regulatory sequence library by enzyme digestion to obtain an index tag library.
  • the third aspect of the present invention provides the application of the vector described in the first aspect of the present invention in library construction or functional element screening.
  • the fourth aspect of the present invention provides a method for library construction, using a Y-shaped linker to integrate randomly interrupted sequences into the vector.
  • the integration site is the recombination site of the vector.
  • the Y-shaped linker is structurally divided into a complementary region and a non-complementary region.
  • non-complementary sequences at the 5' end of the Y-shaped linker respectively comprise the first homology arm, the second homology arm, the first index sequence and the second index sequence from the front and rear ends of the backbone vector cloning site,
  • the complementary sequence at the 3' end contains the restriction enzyme cleavage site.
  • the structure of the Y-shaped linker is a first homology arm, a first index sequence, an enzyme cutting site, a random sequence embedding site, an enzyme cutting site, a second index sequence and a second homology arm.
  • the homologous sequence facilitates subsequent Gibson cloning reactions with the backbone vector.
  • the enzyme cleavage site is different from the enzyme cleavage site on the vector described in the first aspect of the present invention, and the enzyme cleavage site can be used for sequencing verification of functional elements after functional screening.
  • the enzyme cleavage site is selected as an AsiSI enzyme cleavage site.
  • the Y-shaped linker is prepared by annealing PCR primers.
  • downstream primer of the PCR also has an enzyme cleavage site.
  • the Y-shaped linker is prepared by mixing primer A: GGGCTCACCTCAGGCTACGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCGATCGCTTCATTC (SEQ ID NO.3) and primer B Phos-GAATGAAGCGATCGCNNNNNNNNNNNNNNNNNNNNCCCTGACGTAGGCTGACGGC (SEQ ID NO.4) after mixing.
  • the method according to the third aspect of the present invention comprises the following steps:
  • the label in step S01 is a random sequence with about 40 bases
  • the random tag sequence is located downstream of the fluorescent tracer screening gene TurboGFP,
  • the random tag sequence is located between the fluorescent tracer screening gene TurboGFP and polyA, and the Barcode sequence can be determined at the mRNA level, thereby indirectly determining its corresponding functional element sequence.
  • step S01 More specifically, the specific operations of step S01 are:
  • the vector is linearized by single enzyme cleavage in step a.
  • the upstream primer of the primer in step b contains the random tag sequence.
  • both the upstream and downstream primers of the primers in step b contain restriction enzyme cleavage sites.
  • the PCR fragment thus amplified can be ligated with the digested vector backbone after digestion.
  • the ligation product is transformed into E. coli for storage.
  • XbaI is used to cut the backbone vector
  • the primers used are: F-terminal primer: CACCAAGGAAGCCCTCGAGGACGCGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGATCCCGACCTACCGACCCAGCTTTC (SEQ ID NO.1) and R-terminal primer AGGCGAAGACGCGGAAGAGG (SEQ ID NO.2).
  • the specific technical route of step S01 is as follows: using a specific restriction enzyme to excise the backbone vector MCS and part of the element sequence, recovering the backbone large fragment, using the 5' end to carry the random tag Barcode sequence and The primers of the homology arm PCR amplify the backbone vector to obtain a PCR product with Barcode; the PCR product is subjected to Gibson cloning reaction with the backbone vector recovered by enzyme digestion to construct a tag library, or recorded as a Barcode library.
  • the diversity of the tagged Barcode library can also be verified by high-throughput NGS sequencing.
  • step S02 utilizes the Y-shaped linker to integrate the randomly interrupted sequence of the functional element into the vector.
  • step S02 is:
  • step d the functional element fragments are randomly broken into fragments smaller than 100 bp.
  • the functional element fragments are randomly broken into fragments of about 50 bp in step d.
  • the nucleic acid fragments are randomly interrupted and then blunted to form blunt-ended short fragments of different sizes.
  • the nucleic acid fragments of the functional elements in step d are nucleic acid fragments of multiple functional elements of a specific function.
  • the yield of random long fragments of functional elements containing Y-shaped linkers can be increased by PCR, and the random long fragments of functional elements containing Y-shaped linkers can be reconfigured into double-stranded DNA fragments.
  • the PCR product was purified and then ligated with the tagged Barcode library.
  • the primers used in this step are: F2:CGGTGGGCTCTATGGTGAGACGCCAGCCGTGGGCTCACCTCAGGCTACGG (SEQ ID NO.5);
  • R2 GTCTAGACCTCGAGGAGACGCCACGGCTGCCGTCAGCCTACGTCAGGG (SEQ ID NO. 6).
  • the Y-shaped adapter in step e is prepared by annealing PCR primers.
  • downstream primer of the PCR also has an enzyme cleavage site.
  • the Y-shaped linker is prepared by mixing primer A: GGGCTCACCTCAGGCTACGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCGATCGCTTCATTC (SEQ ID NO.3) and primer BPhos-GAATGAAGCGATCGCNNNNNNNNNNNNNNNNNNNNCCCTGACGTAGGCTGACGGC (SEQ ID NO.4) after mixing.
  • the specific technical route of step S02 is as follows: PCR amplification and recovery of several functional elements with the same tissue specificity or specific or unknown functions respectively; Random fragments of 100bp and blunt ends are recovered, and the target size band is recovered, such as a small band of about 50bp; the Y-type adaptor that has undergone annealing reaction is added to the random blunt-end short fragment for ligation and PCR reaction, and the obtained structure is the first identical.
  • the first index sequence is denoted as index1
  • the second index sequence is denoted as index2.
  • the Barcode library obtained in step S01 was specifically digested with XcmI, and the 4845bp fragment was recovered as the library backbone; the functional element random fragment and the library backbone were ligated and transformed into Escherichia coli DH10B, Obtain the promoter library.
  • step S03 the specific technical route of step S03 is as follows: the functional element library constructed in step S02 is digested with enzymes, the random sequence embedding site is removed, the vector backbone is recovered and self-ligated, and an index tag library is constructed.
  • the fragments obtained by cleaving the functional element library are added to the ligation reaction by means of a small amount of multiple additions, so that the ligation reaction that occurs in the ligation reaction is an intramolecular ligation reaction as much as possible, even if a single linearized fragment is self-circularized Ligation; and transforming the ligation product into E. coli DH10B to obtain an indexed tag library.
  • the functional element library was digested with enzymes, random fragments of functional elements were cut out, the backbone was recovered and self-ligated, so that index1 and index2 could be simultaneously analyzed in a high-throughput sequencing reaction.
  • Sequencing with Barcode the maximum sequencing read length of high-throughput sequencing NGS is 1 kb
  • an index tag library is constructed, and the inventor named the library as the Marriage library.
  • the index1, index2 and Barcode sequences in the Marriage library were amplified by PCR for high-throughput sequencing and NGS sequencing. The corresponding relationship between the three can be determined through data analysis.
  • the fifth aspect of the present invention provides the application of the method described in the third aspect of the present invention in screening functional elements.
  • the sixth aspect of the present invention provides a method for screening functional elements, comprising the step of building a library, and the method for constructing the library is the method described in the third aspect of the present invention.
  • the method according to the sixth aspect of the present invention comprises the following steps:
  • the promoter library is firstly transfected into specific cells or microinjected into experimental animals. If it is a viral vector, it needs to be packaged into virus particles to infect cells or live animals, and then the fluorescence expression of TurboGFP is observed.
  • index1 and index2 specific sequences can be obtained through the corresponding relationship between index1, index2 and Barcode in the Marriage library, and finally Using index1 and index2 of known sequences as primers, and using the functional element library as a template, PCR amplifies specific functional elements, and finally selects functional elements with excellent performance (such as small fragments, high specificity, and strong priming ability). sequence.
  • the existing specific functional element shuffling technology is based on in vitro homologous recombination in protein shuffling, and often only a single functional element can be used as an initial shuffling template, resulting in insufficient library diversity.
  • the invention provides a library construction method with high library diversity, abandons the in vitro homologous recombination technology used in protein shuffling, and successfully solves the problem of similar The defect that functional elements with specific or unknown functions cannot be effectively recombined.
  • This method mainly involves the construction of three libraries, namely, the construction of Barcode library, functional element library and marriage library, which can quickly and high-throughput realize promoter or
  • the construction method of highly diverse enhancers can also be applied to the construction and screening of other functional elements, which lays a solid foundation for the final screening of functional elements with excellent performance.
  • Figure 1 is a schematic diagram of the Y-type connector
  • Fig. 3 constructs the vector map of label Barcode library
  • Fig. 4 constructs the vector map of promoter library
  • Fig. 5 constructs the vector map of index tag Marriage library
  • Figure 6 The ratio of random segment fragments and adapters; wherein, the ratio of random short fragments added to lane 1 and Y-type adapter is 1:3; the ratio of lane 2 is 1:1;
  • Figure 7 Fluorescence image of retinal slices after in situ subretinal injection in mice, the brighter red fluorescence in panel A represents the distribution of cone cells, panel B is the distribution and expression of the entire library in the retina, panel C is panel A The co-staining map of Figure B, the yellow-orange fluorescently labeled cells in Figure C can be used for subsequent identification of promoter sequences;
  • AAV vector is selected as an example, and the plasmid map is shown in FIG. 2 .
  • the purpose of the present invention can be achieved by adopting the vectors used for conventional library construction.
  • Such as pUC18, pBR322 vectors, etc. different vectors can be selected according to the subsequent screening methods and application scenarios.
  • Genomic functional elements refer to the elements involved in the regulation of gene expression, mainly including cis-acting elements and trans-acting elements. Common ones include: promoters, enhancers Enhancers, silencers, regulatory regions and sequences, inducible elements, activators and repressors, etc.
  • Label Barcode a label for high-throughput sequencing process, to distinguish different samples.
  • the index is an index for further distinguishing different samples containing the same label Barcode in the high-throughput sequencing process.
  • a method for constructing a functional element library comprising the construction of three kinds of libraries, respectively constructing a Barcode library, a functional element library and a Marriage library, and specifically comprising the following steps:
  • the 40 N bases in the F-terminal primer represent random sequencing tag Barcode sequences.
  • the underline of the F-terminal primer is the restriction site of Mlu I; the restriction site of Tfi I is located on the transcriptional regulatory element WPRE of the vector backbone, and the amplification products of primers F and R contain the restriction site of Tfi I.
  • DNase I to digest the nucleic acid fragments of the functional elements (the conditions and times for the digestion of fragments of different lengths are different), so that the promoter fragments are randomly cut into short fragments of different sizes;
  • End Repair Module blunts the ends of the short fragments to form blunt-ended short fragments of different sizes, which are purified and recovered to obtain random short fragments of functional elements;
  • Primer F2 GGGCTCACCTCAGGCTACGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCGATCGCTTCATTC (SEQ ID NO. 3) and primer R2Phos-GAATGAA GCGATCGC NNNNNNNNNNNNNNNNNNNNNNCCCTGACGTAGGCTGACGGC (SEQ ID NO. 4) were mixed and prepared by annealing. After mixing, a Y-type adapter (containing an AsiSI restriction site) was formed by annealing.
  • primer B is the restriction site of AsiSI.
  • the random short fragments of the functional element and the Y-type adapter are mixed in a certain proportion, and then the ligation reaction is performed to generate a long fragment of the functional element containing the Y-type adapter;
  • the long fragments of functional elements are screened by agarose gel electrophoresis, gel cutting and recovery, and the long fragments of functional elements within the expected range are recovered and purified;
  • the primer sequence of PCR is: F2: CGGTGGGCTCTATGGTGAGACGCCAGCCGTGGGCTCACCTCAGGCTACGG (SEQ ID NO.5);
  • R2 GTCTAGACCTCGAGGAGAGACGCCACGGCTGCCGTCAGC CTACGTCAGGG (SEQ ID NO. 6).
  • the index1, index2 and Barcode sequences in the index tag Marriage library were amplified by PCR for high-throughput sequencing, and the corresponding relationship between the three was determined by data analysis.
  • the inventors selected four photoreceptor cell-specific promoters hRO, hRK, mCAR and ProA1 as raw materials for DNA shuffling.
  • the difference in seed promoter strength was hRO ⁇ hRK>mCAR>ProA1.
  • the ProA1 promoter is a promoter that is specifically expressed only in cone cells, but its full length is about 2 kb, which is obviously not suitable for AAV vectors.
  • the full-length hRK promoter is only about 500 bp and can be expressed in both cone and rod cells, but its specificity does not meet the expected requirements.
  • the hRO and mCAR promoters were only expressed in rod cells, again not as expected. Therefore, the inventors used these four promoters to carry out random DNA recombination, selected random recombination fragments with a size of about 500 bp and cloned them into an AAV vector to form a promoter library, and then packaged the obtained highly diverse promoter library into type 8 At the same time, the control virus was used as the reference for the targeting of cone cells, and the subretinal in situ injection was performed at the animal level. By observing the fluorescence expression of TurboGFP (green reporter gene) and Tdtomato (red reporter gene), we screened out those with excellent characteristics. Random recombinant promoter, the specific experimental steps are as follows:
  • E6050S Use End Repair Module
  • primer F2 GGGCTCACCTCAGGCTACGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNCCCCTGACGTAGGCTGACGGC (SEQ ID NO. 4) and primer R2: Phos-GAATGAA GCGATCGC NNNNNNNNNNNNNNNNNNNNNNCCCTGACGTAGGCTGACGGC (SEQ ID NO. 4) to form Y-type adaptor (containing AsiSI restriction site) by annealing;
  • the primer sequence is: F2: CGGTGGGCTCTATGGTGAGACGCCAGCCGTGGGCTCACCTCAG GCTACGG (SEQ ID NO.5);
  • R2 GTCTAGACCTCGAGGAGACGCCACGGCTGCCGTCAGCCTACGTCAGGG (SEQ ID NO. 6).
  • the random recombination promoter fragment and the library backbone are ligated, and transformed into Escherichia coli DH10B to obtain a promoter library;
  • PCR amplify the index1, index2 and Barcode sequences in the index tag of the Marriage library for NGS sequencing, and determine the corresponding relationship between the three through data analysis.
  • the diversity of the library was determined to be 8.5 ⁇ 10 6 by sequence analysis.
  • the present embodiment also provides a method for screening functional elements, comprising the following steps:
  • index1 and index2 sequences as primers and the promoter library as a template, PCR amplifies the corresponding promoter fragments;
  • Figure 6 shows the fluorescence images obtained by subretinal orthotopic injection in mice after premixing the library virus with the control ProA1-Tdtomato virus. Since the ProA1 promoter only specifically targets cone cells, the brighter red fluorescence in Figure A represents the distribution of cone cells, Figure B shows the distribution and expression of the entire library in the retina, and Figure C is Figure A The co-staining map of Figure B, the yellow-orange fluorescently labeled cells in Figure C can be used for subsequent identification of promoter sequences.
  • the library construction method provided by the present invention can achieve the effect of high diversity of the library, successfully solve the defect that functional elements with similar characteristics or specific or unknown functions cannot be effectively recombined, and can quickly and high-throughput.
  • a construction method that realizes high diversity of promoters, enhancers or other functional elements, and can screen out functional element sequences with excellent performance (such as smaller fragments, high specificity, and strong activation ability).
  • CMV_en, HBB_en and SV40_en enhancers as raw materials for DNA shuffling, these three enhancers can play a role in gene regulation, the size of the CMV_en enhancer is 300bp, the size of the HBB_ enhancer is 3kb, and the size of the SV40_en enhancer is 237bp.
  • HBB enhancer In order to obtain a new, shorter and positively regulated HBB enhancer, we used these three enhancers for random recombination, and selected a random recombination fragment with a size of about 800bp-1k and cloned it into a mammalian enhancer containing the SCP1_mini promoter
  • the enhancer library was formed on the sub-test expression vector ( Figure 8), and then the obtained enhancer library was transiently transfected into K562 cells, and cells with different fluorescence intensities were screened by flow sorting, and the random recombination enhancer that met the purpose was further screened. son.
  • the ligation product is subjected to agarose electrophoresis, and the recombination fragment of the enhancer with a size of about 800bp-1kb is cut into gel for recovery and purification to obtain the final random recombination enhancer fragment.
  • the specific sequence of the candidate enhancer can be obtained through the Sanger sequence.

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Abstract

La présente invention concerne un procédé de construction de banques pour des banques très diverses, en abandonnant la technologie de recombinaison homologue in-vitro utilisée dans la réorganisation des protéines, et en introduisant un adaptateur de type Y pour se connecter à un fragment aléatoire de taille inférieure à 100 pb après avoir été digéré par la DNase I. La présente invention résout avec succès le problème lié à l'impossibilité de recombiner efficacement des éléments fonctionnels ayant des caractéristiques similaires ou des fonctions spécifiques ou inconnues. Une banque de marqueurs, une banque d'éléments fonctionnels et une banque de marqueurs indices sont construites, et la diversité des éléments fonctionnels peut être obtenue rapidement et à haut débit, posant ainsi les bases d'un criblage final afin d'obtenir des éléments fonctionnels ayant d'excellentes performances.
PCT/CN2021/134329 2020-12-31 2021-11-30 Système vecteur pour le criblage de séquences régulatrices et application WO2022142963A1 (fr)

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