WO2017215517A1 - 测序文库构建中5'和3'接头连接副产物的去除方法 - Google Patents

测序文库构建中5'和3'接头连接副产物的去除方法 Download PDF

Info

Publication number
WO2017215517A1
WO2017215517A1 PCT/CN2017/087678 CN2017087678W WO2017215517A1 WO 2017215517 A1 WO2017215517 A1 WO 2017215517A1 CN 2017087678 W CN2017087678 W CN 2017087678W WO 2017215517 A1 WO2017215517 A1 WO 2017215517A1
Authority
WO
WIPO (PCT)
Prior art keywords
linker
rna
base
sgrna
sequence
Prior art date
Application number
PCT/CN2017/087678
Other languages
English (en)
French (fr)
Inventor
吴立刚
杨其元
Original Assignee
中国科学院上海生命科学研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海生命科学研究院 filed Critical 中国科学院上海生命科学研究院
Publication of WO2017215517A1 publication Critical patent/WO2017215517A1/zh

Links

Images

Classifications

    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • 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

Definitions

  • the present invention pertains to the field of nucleic acid sequencing, particularly RNA sequencing based on linker ligation, and in particular to the removal of 5' and 3' linker byproducts in sequencing library construction.
  • RNA sequencing methods for deep sequencing of RNA mainly include random primers or oligo-dT primer reverse transcription method and two-step linker reverse transcription method [Reuter, JA, DVSpacek and MPSnyder, High-Throughput Sequencing Technologies , Molecular Cell, 2015, 58(4): p. 586-597].
  • the former is commonly used for the construction of long transcript RNAs such as mRNA, and the latter is more widely used for the construction of any RNA deep sequencing library suitable for ligation reactions including interrupted long transcript RNA, such as small RNA.
  • Sequencing [Munafo, DB and GB Robb, Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA, RNA, 2010, 16(12): p.
  • CLIP sequencing [Licatalosi, DD, etc., HITS- CLIP yields genome-wide insights into brain alternative RNA processing, Nature, 2008, 456 (7221): p. 464-469]
  • RIP sequencing [Helwak, A. et al., Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding, Cell , 2013, 153(3): p.654-665]
  • GRO sequencing Core, LJ, JJ Waterfall and JTLis, Nascent RNA sequencing reveals significant pausing and divergent initiation at human promoters, Science, 2008, 322 (5909): P.1845-8] and so on.
  • RNAs small non-coding RNAs
  • ncRNAs non-coding RNAs
  • RNA sequencing is a library of existing small RNA or RNA fragments due to technical limitations.
  • the construction method is still difficult to detect small RNA in a small sample (less than 100 ng total RNA).
  • the procedure for library construction of small RNA or RNA fragments first requires ligation of the 3' adapter sequence at the 3' end of the small RNA, followed by inactivation of the reaction, and complementation with the 3' linker sequence using reverse transcription primers.
  • the first aspect of the invention provides a method of cleaving an RNA-DNA: cDNA hybrid duplex, the method comprising the steps of mixing a Cas enzyme, an sgRNA and the RNA-DNA: cDNA hybrid duplex; wherein the DNA comprises the Cas enzyme
  • the identified pro-spacer adjacent motif (PAM) the sgRNA specifically binds to a portion of the cDNA strand; and the Cas enzyme specifically recognizes the sgRNA and cleaves the hybrid duplex.
  • the sgRNA sequence consists of a target region and a Cas recognition region, wherein the base sequence of the target region is 15 to 25 bases long, preferably 18 to 18 inches long from the RNA-DNA sequence.
  • a 22-base fragment consisting of the first base of the PAM sequence, or the last base of the fragment and the first base of the PAM sequence are separated by 8 or less, preferably 5 or less. Base.
  • the Cas enzyme is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2 , Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2 , Csf3, Csf4, homologs thereof or modified forms thereof.
  • the Cas enzyme is a Cas9 enzyme, including but not limited to Cas9 (SpCas9) from S. pyogenes, Cas9 (SaCas9) from S. aureus, and Cas9 from S. thermophilus (St1Cas9).
  • the first base of the PAM is the DNA immediately adjacent to the RNA
  • the first base, the target region of the sgRNA consists of 15 to 25 bases of the RNA near the DNA side, or 8 bases apart from the first base of the PAM sequence.
  • the RNA consists of a fragment of 15 to 25 bases in length.
  • the first base of the PAM sequence is the m-th base of the DNA near the side of the RNA, m ⁇ 2, and the target region of the sgRNA is RNA and fragments of the DNA consist.
  • At least half of the base sequences of the fragments are sequences of the RNA.
  • the RNA is a 5' linker used in the construction of an RNA sequencing library that is a 3' linker used in the construction of an RNA sequencing library.
  • the mixing is carried out under conditions suitable for cleavage of the RNA-DNA: cDNA hybrid duplex by the Cas enzyme.
  • a second aspect of the invention provides a method of removing 5' and 3' linker ligation products produced during the construction of an RNA sequencing library, the method comprising:
  • the 3' linker comprises a anterior region sequence proximity motif (PAM) recognized by the Cas enzyme; the sgRNA is capable of specifically binding to a portion of a cDNA strand produced by reverse transcription; and the Cas enzyme is specific for The sgRNA is sexually recognized and the 5' and 3' linker ligation byproducts are cleaved.
  • PAM anterior region sequence proximity motif
  • the sgRNA sequence consists of a target region and a Cas recognition region, wherein the base sequence of the target region is 15 to 25 bases long by the 5' and 3' linker ligation byproducts
  • the fragment consists of a length of 18 to 22 bases, and the fragment is immediately adjacent to the first base of the PAM sequence, or the last base of the fragment is separated from the first base of the PAM sequence by 8 or less.
  • the bases are within 5 or less.
  • the Cas enzyme is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2 , Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2 , Csf3, Csf4, homologs thereof or modified forms thereof.
  • the Cas enzyme is a Cas9 enzyme, including but not limited to Cas9 (SpCas9) from S. pyogenes, Cas9 (SaCas9) from S. aureus, and Cas9 from S. thermophilus (St1Cas9).
  • the first base of the PAM sequence is the first base of the 3' linker immediately adjacent to the 5' linker, the target region of the sgRNA being close to the 5' linker
  • the 'linker side is composed of 15 to 25 bases, or consists of a fragment of 15 to 25 bases of the 5' linker which is separated from the first base of the PAM sequence by 8 bases.
  • the first base of the PAM sequence is the m-th base of the 3' linker near the 5' linker, m ⁇ 2, and the target region of the sgRNA is The 5' linker and the 3' linker are composed of fragments.
  • At least half of the base sequences of the fragments are sequences of the 5' linker.
  • step (3) is carried out under conditions suitable for cleavage of the 5' and 3' linker byproducts by the Cas enzyme.
  • step (1) comprises:
  • step (1b) adding the complementary sequence of the 3' linker, annealing, binding the complementary sequence to the 3' linker, and inactivating the ligase used in step (1a);
  • a third aspect of the invention provides a method of constructing an RNA sequencing library, the method comprising: removing the RNA-DNA: cDNA hybrid duplex or 5' produced during the construction of the library using the method of the first aspect or the second aspect herein The step of the 3' linker connecting the byproducts.
  • the method comprises:
  • the 3' linker comprises a anterior region sequence proximity motif (PAM) recognized by the Cas enzyme; the sgRNA is capable of specifically binding to a portion of a cDNA strand produced by reverse transcription; and the Cas enzyme is specific for sexual identification
  • PAM anterior region sequence proximity motif
  • the sgRNA sequence consists of a target region and a Cas recognition region, wherein the base sequence of the target region is 15 to 25 bases long by the 5' and 3' linker ligation byproducts
  • the fragment consists of a length of 18 to 22 bases, the fragment is immediately adjacent to the first base of the PAM sequence, or the last base of the fragment is separated from the first base of the PAM by 8 or less, Preferably, the bases are within 5 or less.
  • the Cas enzyme is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2 , Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2 , Csf3, Csf4, homologs thereof or modified forms thereof.
  • the Cas enzyme is a Cas9 enzyme, including but not limited to Cas9 (SpCas9) from S. pyogenes, Cas9 (SaCas9) from S. aureus, and Cas9 from S. thermophilus (St1Cas9).
  • the 1st base of the PAM sequence is the 3' linker immediately adjacent to the 1st base of the 5' linker
  • the target region of the sgRNA is by the 5' linker It consists of 15 to 25 bases on the side of the 3' linker, or consists of fragments of 15 to 25 bases of the 5' linker within 8 bases of the first base of the PAM sequence.
  • the first base of the PAM sequence is m bases of the 3' linker near the 5' linker side, m ⁇ 2, and the target region of the sgRNA is The 5' linker and the fragment of the 3' linker are composed.
  • At least half of the base sequences of the fragments are sequences of the 5' linker.
  • the step (1) comprises:
  • step (1b) adding the complementary sequence of the 3' linker, annealing, binding the complementary sequence to the 3' linker, and inactivating the ligase used in step (1a);
  • step (3) is carried out under conditions suitable for cleavage of the 5' and 3' linker byproducts by the Cas enzyme.
  • a fourth aspect of the invention provides a kit comprising: a Cas enzyme, a sgRNA, a 3' linker and a 5' linker; wherein the 3' linker comprises a prostagland region adjacent sequence recognized by the Cas enzyme Order (PAM sequence);
  • the sgRNA is capable of specifically binding to a portion of a cDNA strand produced by reverse transcription of 5' and 3' linker ligation byproducts; and the Cas enzyme is capable of specifically recognizing the sgRNA and cleaving the 5' and 3' linkers Connect by-products.
  • the fifth aspect herein provides the use of the kit in the construction of an RNA deep sequencing library.
  • the constructing an RNA deep sequencing library comprises constructing a small RNA sequencing library, a transcriptome library, a CLIP library, a RIP library, or a GRO library.
  • the constructing an RNA deep sequencing library comprises constructing a small RNA sequencing library, a transcriptome library, a CLIP library, a RIP library, or a GRO library.
  • FIG. 1 The Cas9/sgRNA complex effectively removes the by-products of the 5' and 3' junctions in the small RNA building process.
  • the white lines in the figure are small RNA molecules derived from a small amount of cells or total RNA; the gray lines are 5' linker sequences; the black lines are 3' linker sequences; the shaded lines are reverse transcription (RT) primers; The cDNA sequence; the triangle indicates the cleavage position of Cas9/sgRNA; the ellipse is the Cas9/sgRNA complex.
  • Figure 2 Schematic diagram of the 5' and 3' linker ligation products of spCas9/sgRNA.
  • the self-ligated by-product of the linker is composed of a 5' adaptor (5'adapter, RNA) and a 3' adaptor (3'adapter, DNA) and a contralateral reverse transcription (RT) product; the target (19 nt) region
  • the cDNA sequence of the sgRNA target; the three base "TGG" indicated by PAM is the PAM sequence of Cas9; the position of Cas9/sgRNA cleavage of cDNA in the figure is indicated by a triangle.
  • Figure 3 6% PAGE gel electrophoresis map of small RNA library.
  • the experiment used HEK293 1ng total RNA as Input, water as a negative control of the template, PCR amplification 26 cycles according to the method, 6% PAGE gel separation; loading maker selected Takara 20bp ladder, the dotted arrow refers to the connection of small RNA
  • the target product is 140 bp in size, and the solid arrow indicates the by-product produced by the linkage between the 5' and 3' junctions in the library, and the size is 120 bp.
  • Figure 4 Association analysis of miRNA expression profiles in a technical replicate of 1 ng HEK293 total RNA library sequencing.
  • Figure 5 Cas9/sgRNA complex cleaves different types of substrate efficiencies.
  • 1, 2, 3 and 4 show different substrate structures, respectively.
  • 1 indicates a ssDNA sequence which mimics a cDNA substrate, and has a CY5 fluorescent label (closed circle) at the 3' end.
  • the ssDNA is complementary to the 3' linker, wherein the gray box in the 3' linker represents the PAM sequence and the gray broad line on the ssDNA strand is the sgRNA target region.
  • 3 shows the double-stranded hybrid sequence (ssDNA+ssDNA-RC) formed by the complementary pairing of ssDNA and ssDNA-RC, wherein ssDNA-RC is the reverse complementary strand of ssDNA, gray box represents PAM, and gray broad line on ssDNA strand is sgRNA target region.
  • Panel B shows the results of denatured gels and the results demonstrate that Cas9 has a good cutting effect on both classical and non-classical substrates (sequences shown in Figures 1, 2, 3 and 4 of Figure A).
  • Panel C shows a non-denaturing gel map showing the binding of Cas9/sgRNA to different substrates (sequences shown in Figures 1, 2, 3 and 4 of Figure A).
  • RNA based on ligation of 5' and 3' linkers is one of the important methods for constructing deep sequencing libraries of small RNA or RNA fragments.
  • the present invention uses CRISPR to specifically remove linker ligation by-products of non-classical CRISPR substrate formats, specifically using the CRISPR system to remove 5' and 3' linker ligation by-products in sequencing library construction.
  • Figure 1 illustrates the mechanism by which CRISPR removes by-products produced by the linkage between the 5' and 3' junctions formed in the library construction.
  • the by-product produced by the ligation between the 5' linker (RNA) and the 3' linker (DNA) during the construction of the small RNA library is an RNA-DNA hybrid strand.
  • the hybrid strand is reverse transcribed by reverse transcriptase to produce a cDNA strand, which together form an RNA-DNA: cDNA hybrid duplex.
  • the Cas enzyme recognizes and cleaves a non-canonical RNA-DNA: cDNA hybrid double-stranded substrate.
  • the TGG sequence at the beginning of the 3' linker sequence is the PAM sequence recognized by spCas9
  • the target recognition region of the sgRNA is designed to target the sequence of the 5' linker
  • the spCas9/sgRNA complex recognizes the RNA-
  • the DNA: cDNA hybridizes to the double strand and is cleaved at a specific position of the cDNA strand, thereby effectively preventing amplification of the cDNA sequence of the byproduct produced by the 5' and 3' linker ligation in a subsequent PCR reaction.
  • the main features of the present invention are: 1) recognition and cleavage of non-canonical RNA-DNA:DNA hybridization substrates using the CRISPR system complex Cas/sgRNA; 2) application of this feature in RNA deep sequencing library construction, specificity The by-products produced by the connection between the 5' and 3' linkers are removed.
  • the contents of the CRISPR system in addition to the description herein, see also CN 201380049665.5 and CN 201380072752.2, the entire contents of which are hereby incorporated by reference.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats
  • the system has been modified and optimized and is now widely used in in vitro biochemical reactions, cell and individual gene editing [Hsu, P.D., E.S.Lander and F. Zhang, Development and applications of CRISPR-Cas9 for genome engineering, Cell, 2014, 157(6): p.1262-1278].
  • a CRISPR system suitable for use herein typically contains a Cas protein with a restriction endonuclease activity and a single-stranded sgRNA, or consists of a Cas protein with endonuclease activity and a single-stranded sgRNA.
  • the Cas/sgRNA complex is complementary paired with the template strand in the target DNA by the pairing region of the sgRNA, and the double-stranded DNA is cleaved by Cas at a specific position.
  • the Cas protein suitable for use in the present invention may be various Cas proteins having endonuclease activity and variants thereof well known in the art.
  • the Cas protein can direct cleavage of one or both strands at the position of the target sequence, for example, directing cleavage within the target sequence and/or within the complement of the target sequence.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1.
  • a Type II CRISPR system that contains a Cas9 enzyme and a single-stranded sgRNA.
  • the Cas9 enzyme may be a Cas9 enzyme from a different species including, but not limited to, Cas9 (SpCas9) from S. pyogenes, Cas9 (SaCas9) from S. aureus, and Cas9 (St1 Cas9) from Streptococcus thermophilus, and the like.
  • Various variants of the Cas9 enzyme can be used as long as the variant has endonuclease activity, directing the cleavage of one or both strands at the position of the target sequence.
  • sgRNA usually consists of two parts: the target region and the Cas protein recognition region.
  • the target region and the Cas protein recognition region are usually joined in the 5' to 3' direction.
  • the target region is typically 15 to 25 bases in length, more typically 18 to 22 bases.
  • the target region is used to specifically bind to a portion of the 5' and 3' linker byproduct (RNA-DNA) reverse transcription product (cDNA).
  • the base sequence of the target region may consist entirely of a 5' linker fragment, or a 5' and 3' linker may be used to link a byproduct across a 5' linker and a 3' linker (ie, a 3' end sequence containing a 5' linker and 3 The composition of the base sequence at the 5' end of the linker.
  • the target region consists of a 5' and 3' linker ligation byproduct spanning a 5' linker and a 3' linker fragment
  • the 5' and 3' linker ligations correspond to the portion of the target region sequence, the last base of the 3' end is usually immediately adjacent to the 1st base of the PAM on the 3' linker, or the base may be separated by up to 10 bases.
  • Base for example, up to 8 bases apart, or up to 5 bases apart, or up to 3 bases apart, or 5-10 bases apart, or 1-8 bases apart, or 1-5 apart Base.
  • the first base of the PAM is the a' a base of the 5' and 3' linker ligated byproducts
  • the sequence of the target region may be before the a-base of the 5' and 3' linker ligated byproducts
  • the a-base interval is 0 to 10, or 0 to 8, or 0 to 5, or a fragment of 0 to 25 bases having a length of 15 to 25, preferably 18 to 22 bases.
  • sgRNA can be designed such that the minimum distance between the sgRNA and the cDNA after binding to the first base of the PAM complementary sequence on the cDNA is in the range of a dozen bases, for example, in the range of 15 bases. Or in the range of 8 bases, for example, 0 to 15 bases, 0 to 10 bases, 0 to 8 bases, 0 to 5 bases, or 1 to 5 bases, and the like.
  • the sgRNA recognized by each Cas enzyme is different. Therefore, the corresponding Cas protein recognition region needs to be designed for different Cas enzymes.
  • the Cas protein recognition region for different Cas enzymes is well known in the art.
  • sgRNA sequence of spCas9 in Fig. 2 2 to 20 nt at the 5' end is the target region sequence of the sgRNA, and the sequence after the 20th nt of the sgRNA sequence is the recognition region sequence of the Cas protein.
  • the target region of the sgRNA can be designed according to the 5' linker specifically used, ie, at least a partial sequence of the target region and the 5' linker (eg, 15-25 bases in length, or a continuous portion 18 to 22 bases in length) ) identical; the sequence recognized by the Cas protein can then be selected based on the Cas protein used.
  • the sgRNA can then be synthesized using conventional nucleic acid synthesis methods, including the preparation of sgRNA using chemical synthesis and in vitro transcription.
  • the function of the Cas/sgRNA complex requires a protospacer adjacent motif (PAM) in the non-template strand of DNA.
  • PAM protospacer adjacent motif
  • DNA sequence 3' linker
  • the PAM for the SaCas9 enzyme is NNGRRT. In certain preferred embodiments, the PAM for SpCas9 is TGG.
  • a 3' link suitable for use herein typically has a PAM at its 5' end.
  • the base sequence of the remainder of the 3' linker any base sequence may be employed, or may be designed according to methods conventional in the art.
  • the 3' linker sequence can be:
  • underline indicates PAM, N is A, T, G or C, and R is G or A.
  • the PAM can be calculated from the second base of the 3' linker, including, for example, the positions of the 3rd, 4th, and 5th positions.
  • the 3' linker sequence can be, for example:
  • underline indicates PAM, N is A, T, G or C, and R is G or A.
  • the target region of the sgRNA needs to move to the corresponding position in the PAM region.
  • Number of bases For example, if the PAM is calculated from the second base of the 3' linker, the target region of the sgRNA will consist of the corresponding base sequence at the 3' end of the 5' linker and the first base at the 5' end of the 3' linker. If the target region of the sgRNA is 19 bases in length, the target region will consist of the last 18 bases at the 3' end of the 5' linker and the first base at the 5' end of the 3' linker in the 5' to 3' direction.
  • the target region sequence of sgRNA is 5'-XY-3', where X is the 3' end of the 5' linker (ie, the end near the 3' linker) and the sequence is L-(m-1) bases in length, Y It is the 1st to m-1th base of the 5' end of the 3' linker, and L represents the length of the target region.
  • the cleavage site of the Cas enzyme on the cDNA strand is preferably near the 5' and 3' linker junctions, for example, 15 bases upstream and downstream of the 5' and 3' linker junctions, for example 10 Within the base, or within 8 bases, or within 5 bases, or within 3 bases, such that the ligation product between the 5' and 3' linkers is cleaved by the Cas enzyme and is difficult to be amplified by PCR. Therefore, when designing the 3' linker, the position of the PAM can be set, and the sgRNA can be designed accordingly so that the cleavage site of the Cas enzyme satisfies the above requirements.
  • the sequence length of the 3' linker is also not particularly limited, and its length is a conventional length, for example, 12 to 30 bases, or 15 to 25 bases, such as 18 to 23 bases.
  • the length of the linker region after the 3' linker PAM sequence i.e., the sequence near the 3' end of the 3' linker PAM
  • the length of the linker region after the 3' linker PAM sequence is preferably greater than 10 bases, more preferably greater than 15 bases, at this time
  • Cas enzyme Cutting efficiency is higher (O Connell, MR, et al., Programmable RNA Recognition and cleavage by CRISPR/Cas9.Nature, 2014.516(7530): p.263-266).
  • the characteristics of other different Cas proteins may be different and need to be flexibly designed according to the specific situation.
  • the sequence (RNA) of the 5' linker is not particularly limited.
  • the base composition may be a conventional base composition of a 5' linker used in constructing a sequencing library through a 5' linker and a 3' linker.
  • a 5' linker is typically an RNA sequence that is in the range of 15 to 35 bases in length, e.g., in the range of 20 to 30 bases.
  • the ends of the 5' linker are not modified at all.
  • the 5' end of the 3' linker typically has modifications that facilitate attachment of the 3' linker to other base sequences, such as App modifications.
  • a truncated RNA ligase 2 (K227Q) can be used which recognizes only the 5' end-applied linker as a substrate for ligation, thereby increasing the specificity of the reaction.
  • the 3' end of the 3' linker may contain any modification that prevents the 3' end from being linked to other sequences, such as blocking attachment to other linkers (including the 5' end of the 3' linker itself) and the 3' end of the 3' linker. Modification. Such modifications include, but are not limited to ddC, C3 NH 2 modification and modifications and the like.
  • a sequencing library can be constructed using a 3' linker and a 5' linker with the structural features described herein.
  • the library is typically an RNA library.
  • the construction of the sequencing library is the construction of an RNA deep sequencing library, including but not limited to the construction of a small RNA sequencing library, the construction of a transcriptome library, the construction of a CLIP library, the construction of a RIP library, and the construction of a GRO library. Wait.
  • RNA-DNA ligation product RNA-3' linker ligated to the 3' linker.
  • the amount of starting RNA, the amount of 3' linker, denaturing conditions (such as temperature, time, etc.), and the ligase, buffer, RNase inhibitor, and other suitable reagents used in the ligation and their amounts are Conventional technical means in the art.
  • the starting RNA used in the construction of small RNA libraries is a small RNA naturally occurring in vivo. These naturally occurring small RNAs can be isolated by conventional methods. The resulting RNA sequences can also be prepared using other sources or methods. For example, by encapsulating the transcriptome RNA to break it, a starting RNA suitable for transcriptome RNA library construction can be obtained; by subjecting the cell sample to UV cross-linking, the specific RNA-binding protein is pulled down by the antibody and digested with RNase, The starting RNA suitable for the construction of the CLIP library can be obtained; the cells are lysed, the target RNA binding protein is immunoprecipitated with a specific antibody, washed, RNA is digested with RNase, and the starting RNA suitable for RIP library construction can be obtained; the nucleus is isolated and isolated in vitro. Br-UTP was incorporated into the transcription, and after transcription for a while, the transcribed fragments were pulled down with magnetic beads coupled with Br-UTP antibody, recovered, and interrupted to obtain
  • the starting RNA is first dephosphorylated and then added to the 5' end of the phosphoric acid and purified for mixing with the 3' linker described herein.
  • RNA-3' linker product the complement of the 3' linker is added and annealed to bind the complement to the 3' linker while denaturation of the ligase.
  • a ligation reaction system of a 5' linker was added for ligation.
  • the ligation reaction system of the 5' linker comprises the 5' linker sequences described herein, as well as reagents such as ligases, ATP, RNase inhibitors, and the like, which are conventional in the art.
  • the product thus obtained is a 5' linker-RNA-3' linker, and the 3' end of the product is a duplex linker which complements the complementary sequence of the 3' linker.
  • a linker attachment by-product is present in the product, i.e., a linker by-product formed by the attachment of a 5' link to a 3' linker.
  • the 3' end of the byproduct is a duplex linker that complements the complementary sequence of the 3' linker. As shown in Figure 1.
  • the primers used in the reverse transcription can be designed according to, for example, a 3' linker sequence, usually a sequence complementary to the 3' linker. As previously described, the sequence complementary to the 3' linker has been complementary conjugated to the 3' linker prior to the 5' linker ligation.
  • Reverse transcription can be carried out by a conventional method, for example, using M-MuLV Reverse Transcriptase (available from NEB Corporation) and formulating the reaction system for reverse transcription according to the instructions. Of course, other reverse transcriptases can also be used depending on the actual situation.
  • the temperature of the reverse transcription can be determined depending on the optimum temperature of the reverse transcriptase used.
  • the CRISPR system described herein can then be added to the reverse transcription product.
  • a mixture containing Cas and sgRNA can be added to the reverse transcription product.
  • the sgRNA comprises a target region and a Cas protein recognition region.
  • the sequence of the target region is identical to at least a partial contiguous sequence of the 5' linker used in the ligation reaction, and the Cas protein recognition region is the sequence recognized by the Cas protein used.
  • the target region of the sgRNA will specifically bind to the reverse transcription product of the 5' linker, and the Cas protein binds to the reverse transcription product through the Cas protein recognition region of the sgRNA and is present on the reverse transcription product.
  • the cleavage function is performed corresponding to the PAM sequence of the Cas protein.
  • Cas/sgRNA does not cleave since the desired 5' linker-RNA-3' linker is ligated into the other sequence (ie, the RNA sequence of interest) between the 3' linker and the 5' linker. effect.
  • by-products formed by the attachment of the 5' linker to the 3' linker will be cleaved by the Cas/sgRNA complex and the target product will be retained.
  • the treatment using the CRISPR system can set specific processing conditions depending on the Cas enzyme used in the system.
  • the treatment temperature can be set to the optimum reaction temperature of the Cas enzyme used.
  • the reaction time can also be set according to the actual situation (for example, the amount of the reactants, etc.).
  • a mixed system of Cas enzyme and sgRNA complex may be prepared first, and the system may contain, for example, Cas enzyme, sgRNA, water (preferably MiliQ pure water treated with pyrocarbonate and autoclaved, ie DEPC Water), buffer of Cas enzyme, and the like. After incubation of the mixed system at a suitable temperature for a period of time, it is added to the reverse transcription product.
  • Cas enzyme, sgRNA, The amount of water, buffer, and the like can be easily determined depending on the actual situation, for example, the amount of the reverse transcription product, the activity of the Cas enzyme, and the like.
  • the PCR procedure comprises: denaturation at 94 °C for 2 minutes, denaturation at 98 °C for 10 seconds, annealing at 60 °C for 25 seconds, extension at 68 °C for 15 seconds, adjusting the number of cycles according to specific needs, and extending at 68 °C for 5 minutes.
  • the polymerase used for PCR can also be selected depending on the product to be amplified. This completes the construction of the library.
  • the Cas enzyme is irreversibly inactivated, releasing the substrate. Therefore, the product obtained by the CRISPR system treatment can usually be directly subjected to PCR.
  • the PCR amplification product can be recovered by a conventional method, for example, by conventional gel electrophoresis.
  • the recovered product can then be sequenced, for example, on a conventional sequencing platform.
  • RNA-DNA: cDNA hybrid duplex comprising the step of treating the RNA-DNA: cDNA hybrid duplex using Cas and sgRNA, wherein the 5' end of the DNA comprises The PAM sequence recognized by the Cas enzyme, the sgRNA can specifically bind to a portion of the cDNA strand complementary to the RNA, and can be specifically recognized by the Cas enzyme.
  • cDNA hybrid duplexes provided herein can be used in the construction of RNA libraries, particularly in RNA library construction based on ligation reactions of 5' and 3' linkers or similar ligation reactions.
  • Such library construction includes not only the construction of libraries for small RNA sequencing, but also the construction of libraries such as transcriptome libraries, CLIP libraries, RIP libraries, and GRO libraries.
  • the invention thus provides a method of removing 5' and 3' linker ligation products produced upon construction of an RNA sequencing library, the method comprising:
  • the sgRNA specifically binds to the reverse transcription product of the 5' linker and is specifically recognized by the Cas enzyme; the 5' end of the 3' linker contains the PAM sequence recognized by the Cas enzyme.
  • the application also provides a method of constructing an RNA sequencing library, the method comprising:
  • the sgRNA specifically binds to the reverse transcription product of the 5' linker and is specifically recognized by the Cas enzyme; the 5' end of the 3' linker contains the PAM sequence recognized by the Cas enzyme.
  • RNA suitable for library construction using the methods described herein can be RNA from a variety of species, including biological endogenous RNA, interrupted and biochemically treated RNA, or synthetic RNA.
  • the RNA carries a phosphate at the 5' end and a hydroxyl group at the 3' end or other RNA modification that is capable of a ligation reaction.
  • reaction conditions of the respective steps in the above methods such as temperature and time, may be carried out by reaction conditions well known in the art or as described herein, or may be adjusted depending on the reaction conditions with reference to the reaction conditions described herein.
  • kits comprising: a Cas enzyme, an sgRNA, a 3' linker and a 5' linker; wherein the sgRNA is capable of specifically binding to a reverse transcript of the 5' linker and is capable of being subjected to the Cas Enzyme-specific recognition; the 5' end of the 3' linker contains the PAM sequence recognized by the Cas enzyme.
  • the 3' linker and the 5' linker are located in separate containers, while the Cas enzyme and sgRNA can be located in the same or different containers.
  • Reagents for formulating Cas/sgRNA complexes including but not limited to water (such as DEPC water) and buffers suitable for Cas enzyme, may also be included in the kit. Buffers suitable for use in the Cas enzyme are well known in the art.
  • the Cas enzyme, sgRNA, 3' linker and 5' linker contained in the kit should satisfy the various conditions described above or a combination thereof.
  • the kit may further contain one or more of a reagent for extracting RNA, a reagent for performing reverse transcription, and a reagent for performing PCR.
  • the kit further includes instructions for directing the skilled person to perform one or more of the methods described herein using the ingredients contained in the kit.
  • RNA sequencing is taken as an example to describe the process and characteristics of the technology based on linker-linked reverse transcription sequencing.
  • the following examples are merely illustrative and are not intended to limit the scope of the invention.
  • the methods and materials used in the examples, unless otherwise indicated, are routine methods and materials in the art.
  • ssDNA RC (SEQ ID NO: 4):
  • RT Primer (SEQ ID NO: 6):
  • PCR upstream primer (RP1) (SEQ ID NO: 7):
  • PCR downstream primer SEQ ID NO: 8; RPI, RNA PCR primer, 6N is the indicator sequence, which can be designed by NEB or Illumina;
  • sgRNA in vitro transcription upstream PCR primer SEQ ID NO: 9
  • sgRNA in vitro transcription downstream PCR primer SEQ ID NO: 10.
  • the Cas9/sgRNA in vitro cleavage assay used in the present invention uses 15 pmol of different types of sequences (including 3' linker, ssDNA RC, and linker chimera) and 10 pmol of CY5-ssDNA (Shanghai Biosynthesis) under 5 ul of 250 mM sodium chloride. Annealing (70 degrees 30 seconds, 60 degrees 30 seconds, 50 degrees 30 seconds, 40 degrees 30 seconds) was performed, and the sequences of the structures shown in items 2, 3 and 4 of Fig. A of Fig. 5 were prepared, respectively.
  • the HEK 293 cells used in this example were all grown in DMEM medium (purchased from GIBCO) containing 10% fetal calf serum and cultured at 37 ° C, 5% CO 2 .
  • PCR was first performed using the sgRNA upstream and downstream primers with the lentiGuide puro plasmid (purchased from Addgene cat #1000000049) as the sgRNA template, and the PCR product was purified using 2% agarose gel and in vitro transcribed and recovered according to the instructions of the MEGAshortscript T7Transcription Kit. Quantify RNA.
  • Linker sequence ligation 1 ng of the starting RNA was mixed with 0.25 pmol of the 3' linker sequence, and the RNA was denatured using a 5 ul system at 70 °C for 2 minutes and quickly placed on ice. Additional ligase 2 buffer (purchased from NEB, final concentration °C), PEG 8000 (purchased from NEB, final concentration 10%), RNase inhibitor (purchased from Thermo Fisher, final concentration 2 U/ul ), and added RNL2tr K227Q (4 U/ul, purchased from NEB Corporation), operated according to the instructions of NEB, and connected at 22 ° C for 2 hours. Add 1 ul of 5 picomoles of RTP and react for 5 minutes at 75 ° C Heat inactivated.
  • RT was used to prepare a 25 ul reaction system using M-MuLV Reverse Transcriptase (available from NEB Corporation) at 44 ° C for 1 hour.
  • Cas9/sgRNA complex treatment According to NEB's instructions, a 10 ul Cas9/sgRNA complex system containing 5 ul of DEPC water (purchased from Ambion), 10 x NEB cas9 buffer 1 ul, in vitro transcribed sgRNA 60 ng, Cas9 2 ul (purchased from NEB, 1000 nM concentration), incubated at 37 ° C for 15 minutes. 1 ul of the incubated Cas9/sgRNA complex was added to the RT product and reacted at 37 ° C for 30 minutes.
  • PCR amplification PCR using KOD Neo plus enzyme (purchased from Toyobo Co., Ltd.), 30 ul system, using RP1 and RPI primers, using 6 ul of RT product with or without Cas9/sgRNA treatment.
  • the reaction system was prepared according to the instructions. Denaturation at 94 ° C for 2 minutes, denaturation at 98 ° C for 10 seconds, annealing at 60 ° C for 25 seconds, extension at 68 ° C for 15 seconds, adjusting the number of cycles according to specific needs, extending at 68 ° C for 5 minutes.
  • the library product was electrophoresed using 6% non-deformed acrylamide gel, developed using GeneGreen dye (purchased from Tiangen), and the 130-160 bp product was recovered by tapping and recovered. Illumina Truseq small RNA library preparation). Samples were quantified using Qubit 2.0 (available from Invitrogen) and operated according to its instructions.
  • Panel B shows the results of denatured gels and the results demonstrate that Cas9 has a good cutting effect on both classical and non-classical substrates (sequences shown in Figures 1, 2, 3 and 4 of Figure A).
  • Panel C shows a non-denaturing gel map showing the binding of Cas9/sgRNA to different substrates (sequences shown in Figures 1, 2, 3 and 4 of Figure A).
  • the present invention uses 1 ng of HEK293 cell line RNA as a starting point, and after completing the reverse transcription reaction, separately Library construction with Cas9/sgRNA treatment and no treatment was performed.
  • the negative control was selected as DEPC water without RNA template, and two technical replicates were designed for the experimental group, as shown in Figure 3.
  • the experimental results show that the experimental samples without Cas9/sgRNA treatment will produce a large number of by-products produced by the connection between the 5' and 3' joints under the same building conditions, as indicated by the solid arrows in the figure, and in the dotted arrows.
  • the desired strip is not visible in the position shown.
  • the band at the 120 bp position indicated by the solid arrow was effectively reduced.
  • the 140 bp target band (mainly miRNA) indicated by the dotted arrow is clearly visible.
  • the Cas9/sgRNA complex designed according to Figure 2 was shown to effectively reduce by-products so that the target band in the PCR was efficiently enriched.
  • the Cas9/sgRNA complex is used to specifically remove by-products produced by the linkage between the 5' and 3' linkers in the library, greatly increasing the amplification efficiency of the target product in the PCR reaction.
  • This design can be extended to other linker-based library designs, such as CLIP-seq, RIP-seq, GRO-seq, etc., which can greatly reduce the amount of starting samples, improve the sensitivity of the method, and make some rare and rare samples.
  • RNA analysis is possible. The construction of a variety of RNA deep sequencing libraries based on this method provides a new method for future scientific research and diagnosis, and has a very broad market application prospect.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

基于测序文库构建中5'和3'接头连接副产物的去除方法。具体而言，提供一种切割RNA-DNA：cDNA杂交双链体的方法，所述方法包括混合Cas酶、sgRNA和所述RNA-DNA：cDNA杂交双链体的步骤；其中，该杂交双链体中的DNA部分包含该Cas酶识别的前间区序列邻近基序(PAM序列)；该sgRNA能特异性结合该cDNA链的一部分；和该Cas酶能特异性识别该sgRNA，并切割所述杂交双链体；任选地，所述RNA-DNA：cDNA杂交双链体产生于RNA测序文库的构建过程中；以及任选地，所述RNA为RNA测序文库构建过程中使用的5'接头，所述DNA为RNA测序文库构建过程中使用的3'接头。

Description

测序文库构建中5’和3’接头连接副产物的去除方法 技术领域
本发明属于核酸测序领域，尤其是基于接头连接的RNA测序，具体涉及测序文库构建中5’和3’接头连接副产物的去除方法。
背景技术
RNA深度测序(deep sequencing)的cDNA文库构建方法主要包括随机引物或oligo-dT引物反转录法和两步接头连接反转录法〔Reuter,J.A.，D.V.Spacek和M.P.Snyder，High-Throughput Sequencing Technologies，Molecular Cell，2015，58(4):p.586-597〕。前者通常用于mRNA等长转录本RNA的建库，而后者的应用更为广泛，适用于包括打断的长转录本RNA在内的任何适合连接反应的RNA深度测序文库的构建，例如小RNA测序〔Munafo,D.B.和G.B.Robb，Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA，RNA，2010，16(12):p.2537-52〕、CLIP测序〔Licatalosi,D.D.等，HITS-CLIP yields genome-wide insights into brain alternative RNA processing，Nature，2008，456(7221):p.464-469〕、RIP测序〔Helwak,A.等，Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding，Cell，2013，153(3):p.654-665〕、GRO测序〔Core,L.J.，J.J.Waterfall和J.T.Lis，Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters，Science，2008，322(5909):p.1845-8〕等。
动植物细胞中普遍存在大量非编码小RNA(small non-coding RNA，ncRNA)。虽然小RNA的发生机制各不相同，但它们几乎参与所有生理及病理的基因表达调控过程，展现尤为丰富的表达类型，和高度的组织特异性。基于这类具有调控作用的功能分子的发现，近些年已有一些研究成功地将小RNA的谱系作为特定疾病诊断的标志物。未来小RNA的检测将会在疾病的早期诊断，分型和个体化检测治疗中得到广泛地应用。常用的小RNA定量检测技术包括深度测序技术、芯片技术(microArray)和qRT-PCR技术。后两者需要合成特异性探针，因此只能检测已知种类的小RNA。而深度测序技术，不仅可以从头发现一些新的小RNA序列，而且可以准确地区分仅有微小差异的同源序列，因而在小RNA检测中有无可比拟的技术优势。
针对长转录本的深度测序技术已经能达到普通单细胞的水平〔Kolodziejczyk, A.A.等，The Technology and Biology of Single-Cell RNA Sequencing，Molecular Cell，2015，58(4):p.610-620〕，而小RNA测序由于技术上的限制，现有小RNA或RNA片段的文库构建方法仍较难对微量样品(低于100ng总RNA)中的小RNA进行检测。小RNA或RNA片段的文库构建的步骤首先需要在小RNA 3’端连接上3’接头(3’adapter)序列，然后对反应进行灭活，同时使用反转录引物与3’接头序列进行互补配对，接着在小RNA或RNA片段的的5’端连接上5’接头(5’adapter)序列，如图1所示。这部分产物经过反转录和PCR扩增就能获得用于深度测序的文库。在连接反应过程中，过量的5’接头和3’接头间会发生连接反应，产生无用的副产物。对于起始量非常低的小RNA或RNA片段的进行连接反应，5’和3’接头间连接产生的副产物占绝大多数，严重阻碍文库的后续PCR扩增。
因此，如何去除5’和3’接头间连接产生的副产物是实现微量小RNA或RNA片段建库的关键。
发明内容
本文第一方面提供一种切割RNA-DNA：cDNA杂交双链体的方法，该方法包括混合Cas酶、sgRNA和该RNA-DNA：cDNA杂交双链体的步骤；其中，该DNA包含该Cas酶所识别的前间区序列邻近基序(protospacer adjacent motif，PAM)；该sgRNA能特异性结合该cDNA链的一部分；和该Cas酶能特异性识别该sgRNA，并切割所述杂交双链体。
在一个或多个实施方案中，所述sgRNA序列由靶标区和Cas识别区组成，其中，靶标区的碱基序列由所述RNA-DNA序列上长15～25个碱基、优选长18～22个碱基的片段组成，该片段紧邻该PAM序列的第1个碱基，或该片段的最后1个碱基与该PAM序列第1个碱基之间隔开8个以内、优选5个以内的碱基。
在一个或多个实施方案中，所述Cas酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、其同源物或其修饰形式。
在一个或多个实施方案中，所述Cas酶为Cas9酶，包括但不限于来自化脓链球菌的Cas9(SpCas9)、来自金黄色葡萄球菌的Cas9(SaCas9)，以及来自嗜热链球菌的Cas9(St1Cas9)。
在一个或多个实施方案中，所述PAM的第1个碱基为所述DNA紧邻所述RNA 的第1个碱基，所述sgRNA的靶标区由所述RNA靠近所述DNA一侧的15～25个碱基组成，或由与该PAM序列第1个碱基隔开8个碱基以内的所述RNA的长15～25个碱基的片段组成。
在一个或多个实施方案中，所述PAM序列的第1个碱基为所述DNA靠近所述RNA一侧的第m个碱基，m≥2，所述sgRNA的靶标区由跨所述RNA和所述DNA的片段组成。
在一个或多个实施方案中，所述片段的碱基序列中至少一半以上为所述RNA的序列。
在一个或多个实施方案中，所述RNA-DNA：cDNA杂交双链体产生于RNA测序文库的构建过程中。
在一个或多个实施方案中，所述RNA为RNA测序文库构建过程中使用的5’接头，所述DNA为RNA测序文库构建过程中使用的3’接头。
在一个或多个实施方案中，所述混合在适于所述Cas酶切割RNA-DNA：cDNA杂交双链体的条件下进行。
本文第二方面提供一种去除RNA测序文库构建时产生的5’和3’接头连接副产物的方法，该方法包括：
(1)使用3’接头和5’接头与待测序RNA进行连接反应，获得连接反应的产物；
(2)对步骤(1)获得的产物进行反转录，获得反转录产物；和
(3)使步骤(2)获得的反转录产物与Cas酶和sgRNA混合，从而除去反转录产物中5’和3’接头连接副产物；
其中，所述3’接头含有所述Cas酶所识别的前间区序列邻近基序(PAM)；所述sgRNA能特异性结合反转录产生的cDNA链的一部分；和所述Cas酶能特异性识别所述sgRNA，并切割所述5’和3’接头连接副产物。
在一个或多个实施方案中，所述sgRNA序列由靶标区和Cas识别区组成，其中，靶标区的碱基序列由所述5’和3’接头连接副产物上长15～25个碱基、优选长18～22个碱基的片段组成，该片段紧邻该PAM序列的第1个碱基，或该片段的最后1个碱基与该PAM序列第1个碱基之间隔开8个以内、优选5个以内的碱基。
在一个或多个实施方案中，所述Cas酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、其同源物或其修饰形式。
在一个或多个实施方案中，所述Cas酶为Cas9酶，包括但不限于来自化脓链球菌的Cas9(SpCas9)、来自金黄色葡萄球菌的Cas9(SaCas9)，以及来自嗜热链球菌的Cas9(St1Cas9)。
在一个或多个实施方案中，所述PAM序列的第1个碱基为所述3’接头紧邻5’接头的第1个碱基，所述sgRNA的靶标区由所述5’接头靠近3’接头一侧的15～25个碱基组成，或由与该PAM序列第1个碱基隔开8个碱基以内的所述5’接头的长15～25个碱基的片段组成。
在一个或多个实施方案中，所述PAM序列的第1个碱基为所述3’接头靠近5’接头一侧的第m个碱基，m≥2，所述sgRNA的靶标区由跨所述5’接头和所述3’接头的片段组成。
在一个或多个实施方案中，所述片段的碱基序列中至少一半以上为所述5’接头的序列。
在一个或多个实施方案中，步骤(3)在在适于所述Cas酶切割5’和3’接头连接副产物的条件下进行。
在一个或多个实施方案中，步骤(1)包括：
(1a)混合3’接头与待测RNA序列，进行3’接头连接反应；
(1b)加入3’接头的互补序列，退火，使互补序列与3’接头结合，并使步骤(1a)使用的连接酶变性失活；
(1c)混合5’接头与步骤(1b)获得的反应产物，进行5’接头连接反应；
从而获得含有5’和3’接头连接副产物和5’接头-RNA-3’接头的连接反应产物。
本文第三方面提供一种构建RNA测序文库的方法，所述方法包括采用本文第一方面或第二方面所述方法去除构建文库过程中产生的RNA-DNA：cDNA杂交双链体或5’和3’接头连接副产物的步骤。
在一个或多个实施方案中，该方法包括：
(1)使用3’接头和5’接头与待测序RNA进行连接反应，获得连接反应的产物；
(2)对步骤(1)获得的产物进行反转录，获得反转录产物；
(3)使步骤(2)获得的反转录产物与Cas酶和sgRNA混合，从而除去反转录产物中5’和3’接头连接副产物；和
(4)对步骤(3)所获得的5’接头-RNA-3’接头：cDNA双链杂交体进行PCR扩增，从而构建RNA测序文库；
其中，所述3’接头含有所述Cas酶所识别的前间区序列邻近基序(PAM)；所述sgRNA能特异性结合反转录产生的cDNA链的一部分；和所述Cas酶能特异性识别所 述sgRNA，并切割所述5’和3’接头连接副产物。
在一个或多个实施方案中，所述sgRNA序列由靶标区和Cas识别区组成，其中，靶标区的碱基序列由所述5’和3’接头连接副产物上长15～25个碱基、优选长18～22个碱基的片段组成，该片段紧邻该PAM序列的第1个碱基，或该片段的最后1个碱基与该PAM第1个碱基之间隔开8个以内、优选5个以内的碱基。
在一个或多个实施方案中，所述Cas酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、其同源物或其修饰形式。
在一个或多个实施方案中，所述Cas酶为Cas9酶，包括但不限于来自化脓链球菌的Cas9(SpCas9)、来自金黄色葡萄球菌的Cas9(SaCas9)，以及来自嗜热链球菌的Cas9(St1Cas9)。
在一个或多个实施方案中，所述PAM序列的第1个碱基为所述3’接头紧邻所述5’接头的第1个碱基，所述sgRNA的靶标区由所述5’接头靠近3’接头一侧的15～25个碱基组成，或由与该PAM序列第1个碱基隔开8个碱基以内的所述5’接头的长15～25个碱基的片段组成。
在一个或多个实施方案中，所述PAM序列的第1个碱基为所述3’接头靠近5’接头一侧的m个碱基，m≥2，所述sgRNA的靶标区由跨所述5’接头和所述3’接头的片段组成。
在一个或多个实施方案中，所述片段的碱基序列中至少一半以上为所述5’接头的序列。
在一个或多个实施方案中，所述步骤(1)包括：
(1a)混合3’接头与待测RNA序列，进行3’接头连接反应；
(1b)加入3’接头的互补序列，退火，使互补序列与3’接头结合，并使步骤(1a)使用的连接酶变性失活；
(1c)混合5’接头与步骤(1b)获得的反应产物，进行5’接头连接反应；
从而获得含有5’和3’接头连接副产物和5’接头-RNA-3’接头的连接反应产物。
在一个或多个实施方案中，步骤(3)在在适于所述Cas酶切割5’和3’接头连接副产物的条件下进行。
本文第四方面提供一种试剂盒，所述试剂盒包括：Cas酶，sgRNA，3’接头和5’接头；其中，所述3’接头含有所述Cas酶所识别的前间区序列邻近基序(PAM序列)； 所述sgRNA能特异性结合由5’和3’接头连接副产物反转录产生的cDNA链的一部分；和所述Cas酶能特异性识别所述sgRNA，并切割所述5’和3’接头连接副产物。
本文第五方面提供所述试剂盒在构建RNA深度测序文库中的应用。
在一个或多个实施方案中，所述构建RNA深度测序文库包括构建小RNA测序文库、转录组文库、CLIP文库、RIP文库或GRO文库。
本文第六方面提供本文第一和二方面所述的方法在构建RNA深度测序文库中的应用。
在一个或多个实施方案中，所述构建RNA深度测序文库包括构建小RNA测序文库、转录组文库、CLIP文库、RIP文库或GRO文库。
附图说明
图1：Cas9/sgRNA复合物有效去除小RNA建库流程中5’和3’接头间连接副产物。图中白色线条为来自于少量细胞或总RNA的小RNA分子；灰色线条为5’接头序列；黑色线条为3’接头序列；阴影线条为反转录(RT)引物；虚线为RT反应后产生的cDNA序列；三角形指示Cas9/sgRNA的切割位置；椭圆为Cas9/sgRNA复合物。
图2：spCas9/sgRNA切割5’和3’接头连接产物原理图。图中接头自连副产物由5’接头(5’adapter，RNA)和3’接头(3’adapter，DNA)和对侧的反转录(RT)产物组成的双链；靶标(19nt)区为sgRNA靶标的cDNA序列；PAM指示的三个碱基“TGG”为Cas9的PAM序列；图中Cas9/sgRNA对cDNA的切割位置由三角形所示。
图3：小RNA文库6％PAGE胶电泳图。实验使用HEK293 1ng总RNA作为Input，水作为模板的阴性对照，按照方法所述PCR扩增26个循环，6％PAGE胶分离；loading maker选择Takara 20bp ladder，虚线箭头所指的为连接了小RNA的目的产物，大小为140bp，实线箭头所指的为建库中5’和3’接头间连接产生的副产物，大小为120bp。
图4：1ng HEK293总RNA建库测序的技术重复中miRNA表达谱关联分析。
图5：Cas9/sgRNA复合物切割不同类型底物效率。图A中，1、2、3和4分别显示不同的底物结构。其中，1表示模拟cDNA底物的ssDNA序列，其3’端有CY5荧光标记(实心圆)。2中，ssDNA与3’接头互补结合，其中，3’接头中的灰色方框代表PAM序列，ssDNA链上灰色宽线为sgRNA靶标区。3显示ssDNA与ssDNA-RC互补配对形成的双链杂交体序列(ssDNA+ssDNA-RC)，其中，ssDNA-RC为ssDNA的反向互补链，灰色方框代表PAM，ssDNA链上灰色宽线为sgRNA靶标区。4指示 ssDNA+接头嵌合体序列，该接头嵌合体为3’接头和5’接头连接后的RNA-DNA杂合链，其中该杂合链上的灰色实线表示RNA序列，灰色方框为PAM，ssDNA链上的灰色宽线表示sgRNA靶标区，实心圆代表CY5荧光标记。图B显示变性胶结果，结果证明Cas9对于经典和非经典底物(图A的1、2、3和4所示的序列)均具有良好的切割效果。图C显示非变性胶图，结果显示Cas9/sgRNA与不同底物(图A的1、2、3和4所示的序列)的结合情况。
具体实施方式
基于5’和3’接头的连接反应克隆RNA是构建小RNA或RNA片段的深度测序文库的重要方法之一。但5’和3’接头间连接产生的大量副产物限制了该方法在微量RNA文库构建中的应用。本发明将CRISPR用于特异性去除非经典CRISPR底物形式的接头连接副产物，具体是利用CRISPR系统除去测序文库构建中5’和3’接头连接副产物。图1对用CRISPR去除文库构建中形成的5’和3’接头间连接产生的副产物的机制进行了说明。与经典的CRISPR系统识别和切割双链DNA底物不同，小RNA文库构建过程中5’接头(RNA)与3’接头(DNA)间连接后产生的副产物为RNA-DNA杂合链，该杂合链经过反转录酶反转录产生cDNA链，两者共同组成了RNA-DNA：cDNA杂交双链。因此，本发明中Cas酶识别和切割的是一种非经典的RNA-DNA：cDNA杂交双链底物。作为一个例子，如图2所示，3’接头序列起始端的TGG序列为spCas9所识别的PAM序列，sgRNA的靶标识别区域设计为针对5’接头的序列，spCas9/sgRNA复合物识别该RNA-DNA：cDNA杂交双链，并在cDNA链的特定位置进行切断，从而有效防止在后续的PCR反应中对该5’和3’接头连接产生的副产物的cDNA序列进行扩增。
因此，本发明的主要特征是：1)使用CRISPR系统复合物Cas/sgRNA识别并切割非经典的RNA-DNA：DNA杂交底物；2)将该特征应用在RNA深度测序文库构建中，特异性地去除5’和3’接头间连接产生的副产物。关于CRISPR系统的内容，除本文下文所述外，还可参见CN 201380049665.5和CN 201380072752.2，本文将其全部内容以引用的方式纳入本文。
CRISPR系统
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)是细菌抵御病毒侵袭或躲避哺乳动物免疫反应的基因编辑系统。该系统经过改造和优化，目前已被广泛应用在体外生化反应、细胞与个体的基因编辑中〔Hsu,P.D.，E.S.Lander和 F.Zhang，Development and applications of CRISPR-Cas9for genome engineering，Cell，2014，157(6):p.1262-1278〕。
适用于本文的CRISPR系统通常含有具有核酸内切酶活性的Cas蛋白和单链sgRNA，或由具有核酸内切酶活性的Cas蛋白和单链sgRNA组成。Cas/sgRNA复合物通过sgRNA的配对区与靶标DNA中的模板链进行互补配对，由Cas在特定位置将双链DNA切断。
Cas蛋白
适用于本发明的Cas蛋白可以是本领域周知的各种具有核酸内切酶活性的Cas蛋白及其变异体。该Cas蛋白能指导在靶序列的位置处的一条或两条链的切割，例如指导在靶序列内和/或在靶序列的互补序列内的切割。
Cas蛋白质的非限制性例子包括Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、其同源物或其修饰形式。
在一些实施方案中，使用II型CRISPR系统，该系统含有一种Cas9酶和单链sgRNA。Cas9酶可以是来自不同物种的Cas9酶，包括但不限于来自化脓链球菌的Cas9(SpCas9)、来自金黄色葡萄球菌的Cas9(SaCas9)，以及来自嗜热链球菌的Cas9(St1Cas9)等。可以使用Cas9酶的各种变体，只要该变体具有核酸内切酶活性、指导在靶序列的位置处的一条或两条链的切割即可。
sgRNA
sgRNA通常包括两部分：靶标区和Cas蛋白识别区。靶标区与Cas蛋白识别区通常以5’到3’的方向连接。
靶标区的长度通常为15～25个碱基，更通常为18～22个碱基。靶标区用于与5’和3’接头连接副产物(RNA-DNA)的反转录产物(cDNA)的一部分特异性结合。靶标区的碱基序列可完全由5’接头的片段组成，也可由5’和3’接头连接副产物跨5’接头和3’接头的片段(即含有5’接头的3’端序列与3’接头的5’端的碱基序列)组成。当靶标区由5’和3’接头连接副产物跨5’接头和3’接头的片段组成时，优选的是，该片段的碱基序列中至少30％以上、优选40％以上(例如50％以上、60％以上、70％以上、80％以上)更优选至少90％以上的序列为5’接头的序列。
5’和3’接头连接副产物上对应于靶标区序列的部分，其3’端最后一个碱基通常紧邻3’接头上的PAM的第1个碱基，或者两者可相隔最多10个碱基，例如相隔最多8个碱基，或相隔最多5个碱基，或最多相隔3个碱基，或者相隔5－10个碱基，或相隔1－8个碱基，或相隔1－5个碱基。例如，PAM第1个碱基为5’和3’接头连接副产物的第a个碱基，那么靶标区的序列可以是该5’和3’接头连接副产物第a个碱基前与该第a个碱基间隔0～10，或者0～8，或者0～5，或者0～3个碱基的长15～25、优选长18～22个碱基的片段。
此外，已知Cas酶在cDNA链上的切割位点通常为PAM前十几个碱基的范围内，大部分Cas酶在PAM前的1～5个碱基进行切割。因此，可根据此原则设计sgRNA，使sgRNA与cDNA互补结合后离cDNA上PAM互补序列的第1个碱基的最小距离在十几个碱基的范围内，例如在15个碱基范围内，或在8个碱基范围内，例如间隔0～15个碱基，0～10个碱基，0～8个碱基，0～5个碱基或1～5个碱基等。
通常，每种Cas酶识别的sgRNA都不一样。因此，针对不同Cas酶需设计相应的Cas蛋白识别区。而针对不同Cas酶的Cas蛋白识别区是本领域周知的。
以图2中spCas9的sgRNA序列为例，其中5’端的2～20nt是sgRNA的靶标区序列，sgRNA序列第20nt之后的序列是Cas蛋白的识别区序列。
可根据所具体使用的5’接头设计sgRNA的靶标区，即，使该靶标区与5’接头的至少部分序列(例如长15～25个碱基，或长18～22个碱基的连续部分)完全相同；然后可根据所使用的Cas蛋白选择该Cas蛋白识别的序列。之后可采用常规的核酸合成方法合成所述sgRNA，包括采用化学合成和体外转录的方法制备sgRNA。
接头
Cas/sgRNA复合物行使功能需要在DNA的非模板链有前间区序列邻近基序(protospacer adjacent motif，PAM)。本文中，通过3’接头(DNA序列)引入PAM。应理解的是，不同Cas酶，其对应的PAM并不完全相同。例如，针对SpCas9的PAM通常是NGG；针对SaCas9酶的PAM通常是NNGRR；针对St1Cas9酶的PAM通常是NNAGAA；其中，N为A、C、T或G，R为G或A。
在某些优选的实施方式中，针对SaCas9酶的PAM是NNGRRT。在某些优选的实施方式中，针对SpCas9的PAM是TGG。
适用于本文的3’接头通常在其5’端具有PAM。对于3’接头其余部分的碱基序列并无特殊要求，可采用任意碱基序列，或可根据本领域常规的方法设计。因此，例如，3’接头序列可以是：
Figure PCTCN2017087678-appb-000001
5’-NNGRRNNNNNNNNNNNNNNN-3’(SEQ ID NO:13)，或
Figure PCTCN2017087678-appb-000002
其中，下划线表示PAM，N为A、T、G或C，R为G或A。
PAM可从3’接头的第2个碱基起算，包括例如从第3位、第4位、第5位等位置起算。因此，3’接头序列可以是例如：
Figure PCTCN2017087678-appb-000003
5’-NNNNGRRNNNNNNNNNNNNN-3’(SEQ ID NO:19)，或
5’-NNNNAGAANNNNNNNNNNNN-3’(SEQ ID NO:20)，等等，
其中，下划线表示PAM，N为A、T、G或C，R为G或A。
因此，在sgRNA的靶标区的对侧区域紧邻PAM的情况下，如果PAM的第一个碱基不是3’接头序列的第一个碱基，则sgRNA的靶标区就需要向PAM区域移动相应位数的碱基。例如，如果PAM从3’接头的第2个碱基起算，那么sgRNA的靶标区将由5’接头3’端相应的碱基序列以及3’接头5’端的第1个碱基组成。如果sgRNA的靶标区长19个碱基，那么该靶标区将由5’接头3’端的最后18个碱基与3’接头5’端的第1个碱基按5’到3’的方向组成。
因此，在具体实施方案中，如果3’接头中PAM的第1个碱基为3’接头5’端(即靠近5’接头的那一端)的第m个碱基(m≥2)，则sgRNA的靶标区序列为5’-X-Y-3’，其中X为5’接头的3’端(即靠近3’接头的那一端)长度为L-(m-1)个碱基的序列，Y为3’接头5’端的第1位到第m-1位碱基，L表示靶标区的长度。
除此之外，Cas酶在cDNA链上的切割位点最好在5’和3’接头连接处附近，例如在5’和3’接头连接处的上下游15个碱基以内，例如10个碱基以内，或8个碱基以内，或5个碱基以内，或3个碱基以内，这样5’和3’接头之间的连接产物被Cas酶切断后就难以被PCR所扩增。因此，在设计3’接头时，可设置PAM所处的位置，并依此设计sgRNA，以使Cas酶的切割位点满足上述要求。
对3’接头的序列长度也无特殊限制，其长度为常规的长度，例如12～30个碱基，或者15～25个碱基，如18～23个碱基等。以spCas9为例，3’接头PAM序列之后的接头区长度(即3’接头PAM后靠近其3’端的序列)最好大于10个碱基，更优选大于15个碱基，此时Cas酶的切割效率更高(O Connell,M.R.,et al.,Programmable RNA  recognition and cleavage by CRISPR/Cas9.Nature,2014.516(7530):p.263-266)。其它不同的Cas蛋白的特性可能不同，需要根据具体情况灵活设计。
对5’接头的序列(RNA)无特殊限制。其碱基组成可以是常规的通过5’接头和3’接头构建测序文库时使用的5’接头的碱基组成。例如，5’接头通常是一条RNA序列，其长度在15～35个碱基的范围内，例如在20～30个碱基的范围内。
通常，5’接头的两端可没有任何修饰。3’接头的5’端通常有能促进该3’接头与其它碱基序列连接的修饰，例如App修饰。可使用截短型RNA连接酶2(K227Q)，该连接酶仅能识别5’端有App的接头作为连接的底物，从而提高反应的特异性。3’接头的3’端可含有任何可阻止该3’端与其它序列连接的修饰，例如阻止与其它接头(包括3’接头本身的5’端)与该3’接头的3’端连接的修饰。这类修饰包括但不限于ddC、C3修饰和NH2修饰等。
文库构建
可使用具有本文所述结构特征的3’接头和5’接头构建测序文库。本文中，文库通常为RNA文库。在某些实施方案中，测序文库的构建为RNA深度测序文库的构建，包括但不限于小RNA测序文库的构建，转录组文库的构建，CLIP文库的构建，RIP文库的构建以及GRO文库的构建等。
小RNA文库的构建方法为本领域常规的方法。例如，通常，先进行3’接头的连接。具体而言，使起始RNA与本文所述的3’接头混合，将混合物中的RNA变性后，使用连接酶进行连接，获得与3’接头连接的RNA-DNA连接产物(RNA-3’接头)。对此，起始RNA的量、3’接头的用量、变性条件(例如温度、时间等)、以及连接所使用的连接酶、缓冲剂、RNA酶抑制剂及其它合适的试剂及其用量都是本领域常规的技术手段。
小RNA文库构建中使用的起始RNA为生物体内天然存在的小RNA。可采用常规的方法分离得到这些天然存在的小RNA。也可使用其它来源或方法制备得到的RNA序列。例如，通过富集转录组RNA将其打断，可获得适用于转录组RNA文库构建的起始RNA；通过将细胞样品进行紫外交联，用抗体拉下特异性RNA结合蛋白并用RNA酶消化，可获得适用于CLIP文库构建的起始RNA；裂解细胞，用特异性抗体免疫沉淀目的RNA结合蛋白，洗涤，RNA酶消化RNA，可获得适用于RIP文库构建的起始RNA；分离细胞核，在体外转录中掺入Br-UTP，转录一段时间后用偶联Br-UTP抗体的磁珠拉下这些转录片段，回收，打断，可获得适用于GRO文库构建的起始RNA。
通常，起始RNA需先去磷酸化，然后在其5’端加磷酸，纯化后即可用于与本文所述的3’接头混合。
获得RNA-3’接头产物后，加入3’接头的互补序列，退火使该互补序列与3’接头结合，同时使连接酶变性失活。然后加入5’接头的连接反应体系进行连接。同样地，所述5’接头的连接反应体系包括本文所述的5’接头序列，以及本领域常规的连接酶、ATP、RNA酶抑制剂等试剂。由此获得的产物为5’接头-RNA-3’接头，该产物的3’端为双链体接头，互补结合有3’接头的互补序列。
如前文所述，应理解的是，产物中存在接头连接副产物，即5’接头与3’接头连接形成的接头副产物。同样地，该副产物的3’端为双链体接头，互补结合有3’接头的互补序列。如图1所示。
将包括接头副产物在内的所有产物进行反转录。反转录中使用到的引物可根据例如3’接头序列进行设计，通常是与3’接头互补的序列。如前所述，与3’接头互补的序列在进行5’接头连接之前已互补结合到3’接头上。可采用常规的方法进行反转录，例如使用M-MuLV Reverse Transcriptase(购自NEB公司)并按照其说明配制反应体系进行反转录。当然也可根据实际情况使用其它反转录酶。反转录的温度可根据所使用的反转录酶的最适温度确定。
之后，可将本文所述的CRISPR系统加到反转录产物中。具体而言，可将含Cas及sgRNA的混合物加到反转录产物中。如前所述，sgRNA包含靶标区和Cas蛋白识别区。靶标区的序列与连接反应中所使用的5’接头的至少部分连续序列相同，Cas蛋白识别区是所使用的Cas蛋白识别的序列。
如图2所示，sgRNA的靶标区将与5’接头的反转录产物特异性结合，Cas蛋白通过sgRNA的Cas蛋白识别区而与反转录产物结合，并通过反转录产物上存在的对应于该Cas蛋白的PAM序列而行使切割功能。如图1所示，由于期望的产物5’接头-RNA-3’接头在3’接头和5’接头间连接入了其它序列(即感兴趣的RNA序列)，Cas/sgRNA就不会发生切割作用。因此，5’接头与3’接头连接形成的副产物将被Cas/sgRNA复合物切割，而目标产物将得以保留。
使用CRISPR系统进行的处理可根据该系统中所用的Cas酶而设置具体的处理条件。例如，可将处理温度设置为所用的Cas酶的最适反应温度。反应时间也可根据实际情况(例如反应物的量等)设置。通常，可先配制Cas酶和sgRNA复合物的混合体系，该体系可含有例如Cas酶、sgRNA、水(优选为经焦碳酸二乙酯处理过并经高温高压灭菌的MiliQ纯水，即DEPC水)、Cas酶的缓冲液等。在合适的温度下温育所述混合体系一段时间后，再将其加入反转录产物中。该混合体系中Cas酶、sgRNA、 水以及缓冲液等的用量可根据实际情况，例如反转录产物的量、Cas酶的活性等容易确定。
经CRISPR系统处理后，将处理所得产物进行PCR扩增。PCR扩增的条件为常规的PCR扩增条件，可根据不同的目标产物做出适当的调整。例如，在某些实施方案中，PCR程序包括：94℃变性2分钟，98℃变性10秒，60℃退火25秒，68℃延伸15秒，按照具体需求调整循环数，68℃延伸5分钟。PCR使用的聚合酶也可根据待扩增的产物来选择。由此完成文库的构建。通常，在PCR的第一步变性时，Cas酶即可被不可逆失活，释放出底物。因此，通常可将CRISPR系统处理所得的产物直接进行PCR。
可采用常规的方法回收PCR扩增产物，例如，可采用常规的凝胶电泳回收。
之后，可将回收所得产物进行测序，例如在常规的测序平台上进行测序。
方法
因此，本文提供一种切割RNA-DNA：cDNA杂交双链体的方法，该方法包括使用Cas酶和sgRNA处理该RNA-DNA：cDNA杂交双链体的步骤，其中，该DNA的5’端包含该Cas酶所识别的PAM序列，该sgRNA能特异性结合该cDNA链上与该RNA互补的部分，并能被所述Cas酶特异性识别。
本文提供的切割RNA-DNA：cDNA杂交双链体的方法可用于RNA文库的构建中，尤其用于基于5’和3’接头的连接反应或类似的连接反应的RNA文库构建中。这类文库构建不仅包括用于小RNA测序文库的构建，还包括转录组文库，CLIP文库，RIP文库，和GRO文库等文库的构建。
本文因此提供一种去除RNA测序文库构建时产生的5’和3’接头连接副产物的方法，该方法包括：
(1)使用3’接头和5’接头与待测序RNA进行连接反应，获得连接反应的产物；
(2)对步骤(1)获得的产物进行反转录，获得反转录产物；和
(3)使步骤(2)获得的反转录产物与Cas酶和sgRNA混合，从而除去反转录产物中5’和3’接头连接副产物；
其中，所述sgRNA能特异性结合5’接头的反转录产物，并能被所述Cas酶特异性识别；所述3’接头的5’端含有所述Cas酶所识别的PAM序列。
本申请还提供一种构建RNA测序文库的方法，该方法包括：
(1)使用3’接头和5’接头与待测序RNA进行连接反应，获得连接反应的产物；
(2)对步骤(1)获得的产物进行反转录，获得反转录产物；
(3)使步骤(2)获得的反转录产物与Cas酶和sgRNA混合，从而除去反转录产物中5’和3’接头连接副产物；和
(4)对步骤(3)所获得的5’接头-RNA-3’接头：cDNA双链杂交体进行PCR扩增，从而构建RNA测序文库；
其中，所述sgRNA能特异性结合5’接头的反转录产物，并能被所述Cas酶特异性识别；所述3’接头的5’端含有所述Cas酶所识别的PAM序列。
适用于本文所述方法进行文库构建的RNA可以是来自各种物种的RNA，包括生物内源性RNA，经过打断及生化处理的RNA，或者合成的RNA。通常，该RNA的5’端带有磷酸，3’端为羟基或者其它能发生连接反应的RNA修饰。
上述方法中的各步骤的反应条件，例如温度和时间等，可采用本领域周知或本文所述的反应条件，或者可参照本文所述的反应条件依据不同的反应情况而加以调整。
试剂盒
本文提供一种试剂盒，该试剂盒包括：Cas酶，sgRNA，3’接头和5’接头；其中，所述sgRNA能特异性结合该5’接头的反转录产物，并能被所述Cas酶特异性识别；所述3’接头的5’端含有所述Cas酶所识别的PAM序列。
通常，3’接头和5’接头位于不同的容器中，而Cas酶与sgRNA可位于相同或不同的容器中。试剂盒中还可包括用于配制Cas/sgRNA复合物的试剂，包括但不限于水(如DEPC水)和适用于Cas酶的缓冲液。适用于Cas酶的缓冲液为本领域所周知。
试剂盒所含的Cas酶，sgRNA，3’接头和5’接头应满足前文所述的各种条件或其组合。
试剂盒中还可含有用于提取RNA的试剂、用于进行反转录的试剂、以及用于进行PCR的试剂中的一种或多种。
优选的是，试剂盒中还包括说明书，用以指导技术人员使用试剂盒中所含有的成分实施本文所述的一种或多种方法。
本文将以小RNA测序为例简述基于接头连接反转录法测序建库技术的过程和特性。下述实施例仅仅是阐述性的，并非限制本发明的保护范围。实施例中所用的方法和材料，除非另有说明，否则为本领域常规的方法和材料。
一、材料与方法
1、Cas9/sgRNA切割底物实验及文库构建所需的DNA/RNA序列
3’接头序列(SEQ ID NO:1)：
Figure PCTCN2017087678-appb-000004
5’接头序列(SEQ ID NO:2)：
Figure PCTCN2017087678-appb-000005
CY5-ssDNA(SEQ ID NO:3)：
Figure PCTCN2017087678-appb-000006
ssDNA RC(SEQ ID NO:4)：
Figure PCTCN2017087678-appb-000007
接头嵌合体(SEQ ID NO:5)：
Figure PCTCN2017087678-appb-000008
RT引物(RTP)(SEQ ID NO:6)：
Figure PCTCN2017087678-appb-000009
PCR上游引物(RP1)(SEQ ID NO:7)：
Figure PCTCN2017087678-appb-000010
PCR下游引物(SEQ ID NO:8；RPI，RNA PCR引物，6N是指标序列，可参照NEB或者illumina公司设计)；
Figure PCTCN2017087678-appb-000011
sgRNA体外转录上游PCR引物(SEQ ID NO:9)：
Figure PCTCN2017087678-appb-000012
sgRNA体外转录下游PCR引物(SEQ ID NO:10)：
Figure PCTCN2017087678-appb-000013
体外转录的sgRNA序列(SEQ ID NO:11，由T7体外转录制备的RNA，5’端都带有三磷酸的修饰)：
Figure PCTCN2017087678-appb-000014
注：“r”表示序列骨架为核糖核酸。
2、体外切割实验
本发明所用Cas9/sgRNA体外切割实验使用15pmol的不同类型序列(包括3’接头、ssDNA RC、和接头嵌合体)与10pmol的CY5-ssDNA(上海生工合成)在5ul 250mM氯化钠的条件下进行退火(70度30秒，60度30秒，50度30秒，40度30秒)，分别制备得到图5的图A中第2、3和4项所示结构的序列。
然后使用DEPC水稀释到250ul。取2.5ul稀释液，加入2.5ul含有1pmol spCas9/sgRNA复合物(spCas9购自NEB公司)，37度反应1小时。之后加入5ul去离子甲酰胺(购自Amasco公司)上12％聚丙烯酰胺8M尿素变性胶，或者加入5ul20％甘油上12％聚丙烯酰胺非变性胶，使用1xTBE缓冲体系180伏特，45分钟。使用FLA-9000同位素成像仪，按照其操作说明进行检测。
3、细胞培养，RNA抽提及定量
本实施例所用到的HEK 293细胞都是生长在含10％胎牛血清的DMEM培养基(购自GIBCO公司)中并于37℃、5％CO2的环境下培养。
细胞总RNA的提取都是使用Trizol试剂(购自Takara公司)并按其说明进行操作。提纯的RNA使用Nanodrop进行定量(Thermo公司)，并使用DEPC水(购自Ambion公司)稀释待用。
4、体外转录sgRNA
首先使用sgRNA上下游引物以lentiGuide puro质粒(购自Addgene cat#1000000049)为sgRNA模板进行PCR，使用2％琼脂糖凝胶纯化回收PCR产物，并使用MEGAshortscript T7Transcription Kit按照其说明书要求进行体外转录并回收定量RNA。
5、文库构建流程
接头序列连接：起始RNA 1ng与0.25皮摩尔的3’接头序列混合，使用5ul体系70℃2分钟变性RNA，并迅速置于冰上。另加入连接酶2缓冲液(购自NEB公司，终浓℃为1x)、PEG8000(购自NEB公司，终浓度为10％)，RNA酶抑制剂(购自Thermo Fisher，终浓度为2U/ul)，并加入RNL2tr K227Q(4U/ul，购自NEB公司)，按照NEB的说明进行操作，22℃连接2小时。加入1ul 5皮摩尔的RTP，并对反应75℃5分钟 热失活。加入含有5’接头的连接反应体系3.5ul，包含5皮摩尔5’接头序列、10皮摩尔ATP、10U T4RNA连接酶1(购自NEB公司)和20U RiboLock RNA酶抑制剂(购自Thermo Fisher公司)，22℃连接1小时。
反转录：RT使用M-MuLV Reverse Transcriptase(购自NEB公司)，并按照其说明配制25ul反应体系，44℃1小时。
Cas9/sgRNA复合物处理：按照NEB公司的说明进行操作，配制10ul Cas9/sgRNA复合物的混合体系，包含5ul DEPC水(购自Ambion公司)、10xNEB cas9缓冲液1ul、体外转录的sgRNA 60ng，Cas9 2ul(购自NEB公司，1000nM浓度)，37℃孵育15分钟。并吸取1ul孵育后的Cas9/sgRNA复合物加入RT产物中，37℃反应30分钟。
PCR扩增：PCR使用KOD Neo plus酶(购自东洋纺公司)，30ul体系，使用RP1和RPI引物，使用6ul经过或未经过Cas9/sgRNA处理的RT产物。并按照其说明书配制反应体系。94℃变性2分钟，98℃变性10秒，60℃退火25秒，68℃延伸15秒，按照具体需求调整循环数，68℃延伸5分钟。
文库产物纯化回收：使用6％非变形丙烯酰胺胶对文库产物进行电泳，使用GeneGreen dye(购自天根公司)进行显色，割胶回收130-160bp的产物，并进行回收(具体的回收方法参见Illumina公司Truseq small RNA library preparation)。样品定量使用Qubit2.0(购自Invitrogen公司)，并按照其说明书进行操作。
最后，文库于Illumina测序平台进行测序。
二、结果
1、体外切割实验结果
Cas9/sgRNA复合物切割不同类型底物的效率如图5所示。图B显示变性胶结果，结果证明Cas9对于经典和非经典底物(图A的1、2、3和4所示的序列)均具有良好的切割效果。图C显示非变性胶图，结果显示Cas9/sgRNA与不同底物(图A的1、2、3和4所示的序列)的结合情况。
图5的结果证明，Cas9可识别并切割非经典底物(ssDNA+接头嵌合体)，其效率与已有报道的底物(ssDNA+3’接头)，以及经典底物(ssDNA+ssDNA-RC)相近(Sternberg,S.H.等，DNA interrogation by the CRISPR RNA-guided endonuclease Cas9，Nature，2014，507(7490):p.62-67)。
2、文库构建结果
本发明使用1ng HEK293细胞系RNA作为起始，在完成反转录反应后，分别进 行加入Cas9/sgRNA处理和不进行处理的文库构建。实验选择阴性对照为不含RNA模板的DEPC水，并对实验组设计了两个技术重复，如图3所示。实验结果表明，不加Cas9/sgRNA处理的实验样本在相同的建库条件下会产生大量的5’和3’接头间连接产生的副产物，如图中实线箭头所示，并且在虚线箭头所示的位置看不见目的条带。而在加入Cas9/sgRNA组中，实线箭头所示的120bp位置的条带有效地减少。而虚线箭头所示的140bp目的条带(主要为miRNA)则清晰可见。说明依照图2中设计的Cas9/sgRNA复合物对于副产物进行了有效的降低，使PCR中目的条带被有效富集。
将以上的两个1ng HEK 293总RNA Cas9/sgRNA处理的技术重复样本进行二代测序，并对文库中miRNA表达谱系进行关联分析，R2＝0.9996，如图4，显示了该建库方法的鲁棒性。
在文库构建的过程中，使用Cas9/sgRNA复合物特异性地去除文库中5’和3’接头间连接产生的副产物，大大提高了目的产物在PCR反应中的扩增效率。该种设计方案能够扩展应用到其它基于接头连接的文库设计，如CLIP-seq，RIP-seq，GRO-seq等，可大量降低起始样本的用量，提升方法灵敏度，使得一些珍贵稀有的样本的RNA解析成为可能。基于此法开发的多种RNA深度测序文库构建，为未来的科学研究及诊断领域提供新的方法，具有十分广阔的市场应用前景。

Claims (10)

  1. 一种切割RNA-DNA：cDNA杂交双链体的方法，其特征在于，所述方法包括混合Cas酶、sgRNA和所述RNA-DNA：cDNA杂交双链体的步骤；其中，该杂交双链体中的DNA部分包含该Cas酶识别的前间区序列邻近基序(PAM)；该sgRNA能特异性结合该cDNA链的一部分；和该Cas酶能特异性识别该sgRNA，并切割所述杂交双链体；
    任选地，所述RNA-DNA：cDNA杂交双链体产生于RNA测序文库的构建过程中；以及
    任选地，所述RNA为RNA测序文库构建过程中使用的5’接头，所述DNA为RNA测序文库构建过程中使用的3’接头。
  2. 一种去除RNA测序文库构建时产生的5’和3’接头连接副产物的方法，或构建RNA测序文库的方法，其特征在于，所述方法包括：
    (1)使用3’接头和5’接头与待测序RNA进行连接反应，获得连接反应的产物；
    (2)对步骤(1)获得的产物进行反转录，获得反转录产物；和
    (3)使步骤(2)获得的反转录产物与Cas酶和sgRNA混合，从而除去反转录产物中5’和3’接头连接副产物；
    其中，所述3’接头含有所述Cas酶所识别的PAM序列；所述sgRNA能特异性结合反转录产生的cDNA链的一部分；和所述Cas酶能特异性识别所述sgRNA，并切割所述5’和3’接头连接副产物。
  3. 如权利要求1所述的方法，其特征在于，
    所述sgRNA序列由靶标区和Cas识别区组成，其中，靶标区的碱基序列由所述RNA-DNA序列上长15～25个碱基、优选长18～22个碱基的片段组成，该片段紧邻该PAM序列的第1个碱基，或该片段的最后1个碱基与该PAM序列第1个碱基之间隔开8个以内、优选5个以内的碱基；
    优选地，所述PAM序列的第1个碱基为所述DNA紧邻所述RNA的第1个碱基，所述sgRNA的靶标区由所述RNA靠近所述DNA一侧的15～25个碱基组成，或由与所述PAM序列的第1个碱基隔开8个碱基以内的所述RNA的长15～25个碱基的片段组成；
    或者，所述PAM序列的第1个碱基为所述DNA靠近所述RNA一侧的第m个 碱基，m≥2，所述sgRNA的靶标区由跨所述RNA和所述DNA的片段组成。
  4. 如权利要求2所述的方法，其特征在于，
    所述sgRNA序列由靶标区和Cas识别区组成，其中，靶标区的碱基序列由所述5’和3’接头连接副产物上长15～25个碱基、优选长18～22个碱基的片段组成，该片段紧邻该PAM序列的第1个碱基，或该片段的最后1个碱基与该PAM第1个碱基之间隔开8个以内、优选5个以内的碱基；
    优选地，所述PAM序列的第1个碱基为所述3’接头紧邻所述5’接头的第1个碱基，所述sgRNA的靶标区由所述5’接头靠近3’接头一侧的15～25个碱基组成，或由与该PAM第1个碱基隔开8个碱基以内的所述5’接头的长15～25个碱基的片段组成；
    或者，所述PAM序列的第1个碱基为所述3’接头靠近5’接头一侧的第m个碱基，m≥2，所述sgRNA的靶标区由跨所述5’接头和所述3’接头的片段组成。
  5. 如权利要求1－4中任一项所述的方法，其特征在于，所述Cas酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、其同源物或其修饰形式；
    优选地，所述Cas酶为Cas9酶；更优选地，所述Cas9酶选自化脓链球菌的Cas9、金黄色葡萄球菌的Cas9，以及嗜热链球菌的Cas9。
  6. 如权利要求2或3所述的方法，其特征在于，所述步骤(1)包括：
    (1a)混合3’接头与待测RNA序列，进行3’接头连接反应；
    (1b)加入3’接头的互补序列，退火，使互补序列与3’接头结合，并使步骤(1a)使用的连接酶变性失活；
    (1c)混合5’接头与步骤(1b)获得的反应产物，进行5’接头连接反应；
    从而获得含有5’和3’接头连接副产物和5’接头-RNA-3’接头的连接反应产物。
  7. 一种试剂盒，所述试剂盒包括：Cas酶，sgRNA，3’接头和5’接头；其中，所述3’接头含有所述Cas酶所识别的PAM序列；所述sgRNA能特异性结合由5’和3’ 接头连接副产物反转录产生的cDNA链的一部分；和所述Cas酶能特异性识别所述sgRNA，并切割所述5’和3’接头连接副产物。
  8. 如权利要求7所述的试剂盒，其特征在于，所述sgRNA序列由靶标区和Cas识别区组成，其中，靶标区的碱基序列由所述5’和3’接头连接副产物上长15～25个碱基、优选长18～22个碱基的片段组成，该片段紧邻该PAM序列的第1个碱基，或该片段的最后1个碱基与该PAM序列第1个碱基之间隔开8个以内、优选5个以内的碱基；
    优选地，所述PAM序列的第1个碱基为所述3’接头紧邻所述5’接头的第1个碱基，所述sgRNA的靶标区由所述5’接头靠近3’接头一侧的15～25个碱基组成，或由与该PAM序列第1个碱基隔开8个碱基以内的所述5’接头的长15～25个碱基的片段组成；
    或者，所述PAM序列的第1个碱基为所述3’接头靠近5’接头一侧的第m个碱基，m≥2，所述sgRNA的靶标区由跨所述5’接头和所述3’接头的片段组成。
  9. 如权利要求8所述的试剂盒，其特征在于，所述Cas酶选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、其同源物或其修饰形式；
    优选地，所述Cas酶为Cas9酶；更优选地，所述Cas9酶选自化脓链球菌的Cas9、金黄色葡萄球菌的Cas9，以及嗜热链球菌的Cas9。
  10. 权利要求1－6中任一项所述的方法和权利要求7－9中任一项所述的试剂盒在构建RNA深度测序文库中的应用，优选地，所述构建RNA深度测序文库包括构建小RNA测序文库、转录组文库、CLIP文库、RIP文库或GRO文库。
PCT/CN2017/087678 2016-06-12 2017-06-09 测序文库构建中5'和3'接头连接副产物的去除方法 WO2017215517A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610406827.1 2016-06-12
CN201610406827.1A CN107488655B (zh) 2016-06-12 2016-06-12 测序文库构建中5’和3’接头连接副产物的去除方法

Publications (1)

Publication Number Publication Date
WO2017215517A1 true WO2017215517A1 (zh) 2017-12-21

Family

ID=60642755

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/087678 WO2017215517A1 (zh) 2016-06-12 2017-06-09 测序文库构建中5'和3'接头连接副产物的去除方法

Country Status (2)

Country Link
CN (1) CN107488655B (zh)
WO (1) WO2017215517A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652861A (zh) * 2018-12-22 2019-04-19 阅尔基因技术（苏州）有限公司 一种生化试剂盒及其应用方法
WO2023116681A1 (zh) * 2021-12-21 2023-06-29 翌圣生物科技(上海)股份有限公司 靶序列随机sgRNA全覆盖组的制备方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817804B (zh) * 2021-09-22 2024-03-08 上海金匙医学检验实验室有限公司 一种测序文库自连接头消除的方法及应用
WO2023046163A1 (zh) * 2021-09-26 2023-03-30 杭州诺辉健康科技有限公司 核酸文库构建和测序的方法和试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102534813A (zh) * 2011-11-15 2012-07-04 杭州联川生物信息技术有限公司 构建中小片段rna测序文库的方法
CN104630211A (zh) * 2013-11-15 2015-05-20 苏州吉玛基因股份有限公司 一种Small RNA cDNA文库的构建方法
CN104805078A (zh) * 2014-01-28 2015-07-29 北京大学 用于高效基因组编辑的rna分子的设计、合成及其应用
CN105177110A (zh) * 2015-09-11 2015-12-23 中国科学院微生物研究所 核酸的检测方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102212612A (zh) * 2011-03-23 2011-10-12 上海美吉生物医药科技有限公司 一种用于高通量454测序的双末端文库的构建方法
EP2765200A1 (en) * 2013-02-07 2014-08-13 Bayer CropScience LP Process for producing gougerotin employing Streptomyces microflavus strains
US10604802B2 (en) * 2014-02-04 2020-03-31 Jumpcode Genomics, Inc. Genome fractioning

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102534813A (zh) * 2011-11-15 2012-07-04 杭州联川生物信息技术有限公司 构建中小片段rna测序文库的方法
CN104630211A (zh) * 2013-11-15 2015-05-20 苏州吉玛基因股份有限公司 一种Small RNA cDNA文库的构建方法
CN104805078A (zh) * 2014-01-28 2015-07-29 北京大学 用于高效基因组编辑的rna分子的设计、合成及其应用
CN105177110A (zh) * 2015-09-11 2015-12-23 中国科学院微生物研究所 核酸的检测方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
FANG, RUI ET AL.: "CRISPR/Cas9-Mediated Genome Point Editing Technology", PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS, vol. 40, no. 8, 31 December 2013 (2013-12-31), pages 691 - 702 *
HSU, P.D. ET AL.: "Development and Applications of CRISPR-Cas9 for Genome Engineering", CELL, vol. 157, 5 June 2014 (2014-06-05), pages 1262 - 1278, XP028849523 *
JIANG, F.G. ET AL.: "Structures of a CRISPR-Cas9 R-Loop Complex Primed for DNA Cleavage", SCIENCE, vol. 351, 19 February 2016 (2016-02-19), pages 867 - 871, XP055450334 *
MUNAFO, D.B. ET AL.: "Optimization of Enzymatic Reaction Conditions for Generating Representative Pools of cDNA from Small RNA", RNA, vol. 16, no. 12, 31 December 2010 (2010-12-31), pages 2537 - 2552, XP055450328 *
REUTER, J.A. ET AL.: "High-Throughput Sequencing Technologies", MOLECULAR CELL, vol. 58, 21 May 2015 (2015-05-21), pages 586 - 597, XP029129112 *
STERNBERG, S.H. ET AL.: "DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9", NATURE, vol. 507, 6 March 2014 (2014-03-06), pages 62 - 67, XP055161285 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652861A (zh) * 2018-12-22 2019-04-19 阅尔基因技术（苏州）有限公司 一种生化试剂盒及其应用方法
WO2023116681A1 (zh) * 2021-12-21 2023-06-29 翌圣生物科技(上海)股份有限公司 靶序列随机sgRNA全覆盖组的制备方法

Also Published As

Publication number Publication date
CN107488655B (zh) 2021-07-09
CN107488655A (zh) 2017-12-19

Similar Documents

Publication Publication Date Title
US11692213B2 (en) Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using CRISPR/Cas system proteins
BR112020026306A2 (pt) Métodos de amplificação, sistemas e diagnósticos baseados em sistema efeitor crispr
KR20230116944A (ko) 고온 내성 Cas 단백질의 용도, 표적 핵산 분자의 검출방법 및 시약 키트
WO2017215517A1 (zh) 测序文库构建中5'和3'接头连接副产物的去除方法
EP3098324A1 (en) Compositions and methods for preparing sequencing libraries
US11761037B1 (en) Probe and method of enriching target region applicable to high-throughput sequencing using the same
US20120316075A1 (en) Sequence preserved dna conversion for optical nanopore sequencing
WO2020219838A1 (en) Methods and enzymatic compositions for forming libraries of adapter ligated nucleic acid molecules
JP2010514452A (ja) ヘテロ二重鎖による濃縮
BR112020025319A2 (pt) Composições, sistemas e métodos de amplificação baseada em crispr/cas e transposase
US20150087556A1 (en) COMPOSITIONS AND METHODS FOR MAKING cDNA LIBRARIES FROM SMALL RNAs
WO2009072972A1 (en) A method for enzymatic joining of a dsrna adapter to a dsrna molecule
CN109971843B (zh) 一种单细胞转录组的测序方法
CN116590392A (zh) 一种在全基因组水平鉴定植物R-loop位点的方法
CN116287124A (zh) 单链接头预连接方法、高通量测序文库的建库方法及试剂盒
CN115960987A (zh) 一种mRNA 3`末端测序文库快速构建方法及应用
CN116179652A (zh) 基于LAMP联合Cas13a核酸酶的检测痕量核酸的方法以及应用
JP7333171B2 (ja) Ｒｎａ検出方法、ｒｎａ検出用核酸及びｒｎａ検出用キット
JP5129498B2 (ja) 核酸クローニング法
WO2023116490A1 (zh) 小rna的新型检测方法及其应用
US11814689B2 (en) Nucleic acid detection using type III CRISPR complex
CN113355389B (zh) 利用CRISPR/Cas12a系统靶向富集核酸目标区域的方法及其应用
JP5048915B2 (ja) 整長ｃＤＮＡ由来両鎖ｃＲＮＡサブトラクション方法
WO2022094863A1 (zh) 一种全转录组水平rna结构检测方法及其应用
US20230122979A1 (en) Methods of sample normalization

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17812628

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17812628

Country of ref document: EP

Kind code of ref document: A1