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A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism
不需预知生物体生物信息学条件下将其mRNA转化为向导RNA(gRNA)库的方法

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Abstract

While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in most species, except for well-established model organisms, such as human, mouse, or yeast. A guide RNA (gRNA) library using the clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system can be used for the phenotypic screening of uncharacterized genes by forward genetics. The construction of a gRNA library usually requires an abundance of chemically synthesized oligos designed from annotated genes; if one wants to convert mRNA into gRNA without prior knowledge of the target DNA sequences, the major challenges are finding the sequences flanking the protospacer adjacent motif (PAM) and cutting out the 20-bp fragment. Recently, I developed a molecular biology-based technique to convert mRNA into a gRNA library (Arakawa, 2016) (Figure 1). Here I describe the detailed protocol of how to construct a gRNA library from mRNA.


Figure 1. A method to convert mRNA into a gRNA library construction (Sanjana et al., 2014). The scheme of the method is summarized. Each step of D-O is described in detail in the Procedure. Bg, BglII; Xb, XbaI; Bs, BsmBI; Aa, AatII. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2.

Keywords: CRISPR(CRISPR), Cas9(Cas9), gRNA(gRNA), Library(文库)

Background

The clustered regularly interspersed palindromic repeats (CRISPR) system is responsible for the acquired immunity of bacteria (Barrangou et al., 2007), which is shared among 40% of eubacteria and 90% of archaea (Grissa et al., 2007). While CRISPR/Cas9 is, physiologically, an endonuclease used to eliminate the infectious pathogen (Barrangou et al., 2007), CRISPR/Cas9 can be used to cleave any locus of the genome if a guide RNA (gRNA) is provided (Cong et al., 2013; Mali et al., 2013). By designing gRNA for the gene of interest, individual genes can be knocked out one-by-one by non-homologous end joining (NHEJ) (Cong et al., 2013; Mali et al., 2013); additionally, CRISPR/Cas9 can be utilized to make a gRNA library available for genetic screening (Zhou et al., 2001; Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014). The gRNA for Streptococcus pyogenes (Sp) Cas9 can be designed as a 20-bp sequence adjacent to the protospacer adjacent motif (PAM) NGG (Cong et al., 2013; Mali et al., 2013). Such a sequence can usually be identified from the coding sequence or locus of interest by bioinformatics techniques. Here, I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes cDNA from the extracted RNA using a semi-random primer containing a PAM-complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.

Materials and Reagents

  1. 1.5 ml microcentrifuge tube
  2. 0.2 ml PCR tube
  3. Disposable pipette tip
  4. OligodT column (QIAGEN, supplemented with the Oligotex mRNA Mini Kit [QIAGEN, catalog number: 70022 ])
  5. STBL4 electro-competent cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11635018 )
  6. Lentiviral vector
    lentiCRISPR v2 (Sanjana et al., 2014) (Addgene, catalog number: 52961 )
  7. Oligonucleotides
    Semi-random primer p NNNCCN
    5’ switching mechanism at RNA transcript (SMART) tag TGGTCAAGCTTCAGCAGATCTACACGGACGTCGCrGrGrG
    5’ SMART PCR primer TGGTCAAGCTTCAGCAGATCTACACG
    3’ linker I forward p CTGCTGACTTCAGTGGTTCTAGAGGTGTCCAA
    3’ linker I reverse GTTGGACACCTCTAGAACCACTGAAGTCAGCAGT
    5’ linker I forward GCATATAAGCTTGACGTCTCTCACCG
    5’ linker I reverse p NNCGGTGAGAGACGTCAAGCTTATATGC
    3’ linker II forward p GTTTGGAGACGTCTTCTAGATCAGCG
    3’ linker II reverse CGCTGATCTAGAAGACGTCTCCAAACNN
    3’ linker I PCR primer GTTGGACACCTCTAGAACCACTGAAGTCAGCAGTNNNCC
    3’ linker II PCR primer CGCTGATCTAGAAGACGTCTCCAAAC
    LentiCRISPR forward CTTGGCTTTATATATCTTGTGGAAAGGACG
    LentiCRISPR reverse CGGACTAGCCTTATTTTAACTTGCTATTTCTAG
  8. TRIzol reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596026 )
  9. Phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich, catalog number: P2069-100ML )
  10. Ethanol
  11. RNase-free water
  12. Oligotex mRNA Mini Kit (QIAGEN, catalog number: 70022 )
  13. T4 DNA ligase reaction buffer (New England Biolabs, catalog number: B0202S )
  14. SMART Scribe reverse transcriptase (Takara Bio, Clontech, catalog number: 639536 )
  15. DTT (Takara Bio, supplemented with SMART Scribe reverse transcriptase [Takara Bio, Clontech, catalog number: 639536 ])
  16. dNTP mix (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18427013 )
  17. RNaseOUT (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777019 )
  18. RNase H (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18021014 )
  19. MilliQ water
  20. Advantage 2 polymerase mix (Takara Bio, Clontech, catalog number: 639201 )
  21. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28104 )
  22. Quick Ligation Kit (New England Biolabs, catalog number: M2200S )
  23. EcoP15I (New England Biolabs, catalog number: R0646S )
  24. BglII (New England Biolabs, catalog number: R0144S )
  25. AcuI (New England Biolabs, catalog number: R0641S )
  26. XbaI (New England Biolabs, catalog number: R0145S )
  27. BsmBI (New England Biolabs, catalog number: R0580S )
  28. AatII (New England Biolabs, catalog number: R0117S )
  29. 10-bp ladder
  30. 1x CutSmart buffer (included in XbaI [New England Biolabs, catalog number: R0145S ])
  31. S-adenosylmethionine (SAM) (New England Biolabs, supplemented with AcuI [New England Biolabs])
  32. 3 M sodium acetate (pH 5.5) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9740 )
  33. Acrylamide/Bis solution (19:1) (40 % w/v, 5 % C) (SERVA Electrophoresis, catalog number: 10679.01 )
  34. Glycogen (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0561 )
  35. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32851 )
  36. TE buffer (pH 8.0) (see Recipes)

Equipment

  1. Pipettes
  2. Centifuge (Eppendorf, models: 5424 R , 5810 R )
  3. Heating block
  4. Glass beaker
  5. GeneAmp PCR System 9700 (Thermo Fisher Scientific, model: GeneAmp PCR System 9700 )
    Note: This product has been discontinued.
  6. Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratores, model: Mini-PROTEAN® Tetra Vertical Electrophoresis Cell )
  7. GenePulser II (Bio-Rad Laboratores, model: Gene Pulser II )
  8. High Performance Laboratory Incubator–Mod. 2800 (F.lli GALLI, model: MOD. 2800 )
  9. CLC Genomics Workbench (QIAGEN, model: CLC Genomics Workbench )
  10. Bioanalyzer (Agilent Technologies)
  11. Autoclave

Procedure

  1. Total RNA preparation
    1. Lyse 108 cells with 10 ml of TRIzol by repetitive pipetting. Incubate for 5 min at room temperature.
    2. Add 2 ml of chloroform. Shake by hand for 15 sec. Incubate for 2-3 min at room temperature. Centrifuge at 12,000 x g for 15 min at 4 °C.
    3. Transfer the aqueous phase to a fresh tube. Add 5 ml of isopropyl alcohol and mix well. Centrifuge at 12,000 x g for 10 min at 4 °C.
    4. Remove the supernatant. Wash the pellet once with 80% ethanol. Air dry the pellet for 5-10 min.
    5. Dissolve RNA in 1 ml of RNase-free water by pipetting, then incubate the sample for 10 min at 55-60 °C.

  2. PolyA RNA preparation (see Note 1)
    1. Use the Oligotex mRNA Midi Kit. Heat the Oligotex suspension to 37 °C. Heat buffer OEB to 70 °C on a heating block. Mix buffer OBB well.
    2. Pipet 500 µg of total RNA into an RNase-free 1.5-ml microcentrifuge tube and adjust the volume with RNase-free water to 1 ml.
    3. Add 500 µl of buffer OBB and 30 µl of the Oligotex suspension.
    4. Incubate for 3 min at 70 °C. Remove from heat.
    5. Incubate for 10 min at room temperature.
    6. Centrifuge for 2 min at 15,871 x g. Remove supernatant by pipetting.
    7. Add 250 µl of RNase-free water and 250 µl of buffer OBB. Mix the contents thoroughly by pipetting.
    8. Incubate for 3 min at 70 °C. Remove from heat.
    9. Incubate for 10 min at room temperature.
    10. Centrifuge for 2 min at 15,871 x g. Remove supernatant by pipetting.
    11. Resuspend the pellet in 400 µl of buffer OW2. Pipet the mixture onto a small spin column placed in a 1.5-ml tube. Centrifuge for 1 min at 15,871 x g.
    12. Transfer the spin column to a new RNase-free 1.5-ml tube. Apply 400 µl of buffer OW2. Pipet onto a small spin column placed in a 1.5-ml tube. Centrifuge for 1 min at 15,871 x g.
    13. Transfer the spin column to a new RNase-free 1.5-ml tube. Pipet 25 µl of hot (70 °C) buffer OEB onto the column. Pipet up and down 3-4 times to resuspend the resin. Centrifuge for 1 min at 15,871 x g.
    14. Pipet another 25 µl of hot (70 °C) buffer OEB onto the column. Pipet up and down 3-4 times to resuspend the resin. Centrifuge for 1 min at 15,871 x g.

  3. Linker preparation
    1. Combine the following reagents in a 1.5-ml microcentrifuge tube: 10 µl of 100 µM linker forward oligo, 10 µl of 100 µM linker reverse oligo, and 2.2 µl of 10x T4 DNA ligase buffer (New England Biolabs).
    2. Place the tubes in a glass beaker containing 2 L of boiled water and incubate the tubes until the water cools down to room temperature naturally.
    3. Dilute the annealed oligos with 77.8 µl of TE buffer (pH 8.0) and use as 10 µM linkers.

  4. First-strand cDNA synthesis
    1. Combine the following reagents in a 0.2 ml PCR tube: 200 ng of poly(A) RNA (from step A2), 0.6 µl of 25 µM semi-random primers, and RNase-free water in a 4.75 µl volume.
    2. Incubate the tube at 72 °C in a hot-lid thermal cycler for 3 min, cool on ice for 2 min, and further incubate at 25 °C for 10 min.
    3. Increase the temperature to 42 °C and add a 5.25 µl mixture of the following reagents: 0.5 µl of 25 µM 5’ SMART tag, 2 µl of 5x SMART Scribe buffer, 0.25 µl of 100 mM DTT, 1 µl of 10 mM dNTP mix, 0.5 µl of RNaseOUT (Invitrogen), and 1 µl SMART Scribe reverse transcriptase (100 U) (Clontech).
    4. Incubate the first-strand cDNA reaction mixture at 42 °C for 90 min and then at 68 °C for 10 min. To degrade RNA, add 1 µl of RNase H (Invitrogen) to the mixture and incubate the mixture at 37 °C for 20 min.

  5. Double-stranded (ds) cDNA synthesis by primer extension
    1. Mix 11 µl of the prepared first-strand poly(A) cDNA (from step C4) with 74 µl of MilliQ water, 10 µl of 10x Advantage 2 PCR buffer, 2 µl of 10 mM dNTP mix, 1 µl of 25 µM 5’ SMART PCR primer, and 2 µl of 50x Advantage 2 polymerase mix (Clontech).
    2. Incubate a 100 µl volume of the reaction mixture for primer extension at 95 °C for 1 min, 68 °C for 20 min, and then 70 °C for 10 min.
    3. Purify the prepared ds cDNA using a QIAquick PCR Purification Kit (QIAGEN) and elute the DNA with 40 µl of TE buffer (pH 8.0).

  6. 3’ linker I ligation
    1. Mix ds poly(A) cDNA (from step D3) with 0.5 µl of 10 µM 3’ linker I and 1 µl of Quick T4 DNA ligase (New England Biolabs; NEB) in 1x Quick ligation buffer.
    2. Incubate the ligation reaction mixture at room temperature for 15 min, then purify it using a QIAquick PCR Purification Kit and elute it with 80 µl of TE buffer.

  7. EcoP15I digestion
    1. Digest the 3’ linker I-ligated DNA (from step E2) with 1 µl EcoP15I (10 U/µl, NEB) in 1x NEBuffer 3.1 containing 1x ATP in a 100 µl volume at 37 °C overnight.
    2. Purify the EcoP15I-digested DNA using a QIAquick PCR Purification Kit and elute the DNA with 40 µl of TE buffer.

  8. 5’ linker I ligation and BglII digestion
    1. Mix the digested DNA (from step F2) with 0.5 µl of 10 µM 5’ linker I and 1 µl of Quick T4 DNA ligase (NEB) in 1x Quick ligation buffer.
    2. Incubate the ligation reaction mixture at room temperature for 15 min, purify it using a QIAquick PCR Purification Kit, and elute it with 80 µl of TE buffer.
    3. Digest the DNA with 1 µl of BglII (10 U/µl, NEB) (see Note 2) in 1x NEBuffer 3.1 in a 100 µl volume at 37 °C for 3 h.
    4. Purify the EcoP15I/BglII-digested DNA using a QIAquick PCR Purification Kit and elute the DNA with 50 µl of TE buffer.

  9. First PCR optimization (see Note 3)
    1. To determine the optimal number of PCR cycles, prepare a 0.2 ml PCR tube containing 5 µl of the ds cDNA ligated with 5’ linker I/3’ linker I (from step G4), 0.5 µl of 25 µM 5’ linker I forward primer, 0.5 µl of 25 µM 3’ linker I PCR primer, 5 µl of 1x Advantage 2 PCR buffer, 1 µl of 10 mM dNTP mix, 1 µl of 50x Advantage 2 polymerase mix, and MilliQ water in a 50 µl volume.
    2. Perform PCR with the following cycling parameters: 6 cycles of 98 °C for 10 sec and 68 °C for 10 sec.
    3. After the 6 cycles, transfer 5 μl of the reaction to a clean microcentrifuge tube. Perform PCR with the rest of the PCR reaction mixture for 3 additional cycles of 98 °C for 10 sec and 68 °C for 10 sec.
    4. After these additional 3 cycles, transfer 5 μl to a clean microcentrifuge tube.
    5. In the same way, repeat additional PCR until reaching 30 total cycles. Thus, prepare a series of PCR reactions of 6, 9, 12, 15, 18, 21, 24, 27, and 30 cycles and analyze by 20% polyacrylamide gel electrophoresis to compare the band patterns (Figure 2; PCR optimization 1).


      Figure 2. The first PCR cycle optimization and size fractionation (Arakawa, 2016). PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles. (see Note 4)

    6. Determine the optimal number of PCR cycles as the minimal number of PCR cycles yielding the greatest quantity of the 84-bp product (typically around 17 cycles).
    7. Repeat two 50-µl PCR reactions with the optimal number of PCR cycles.
    8. Purify the PCR product using a QIAquick PCR Purification Kit and elute with 50 µl of TE buffer.

  10. AcuI/XbaI digestion
    1. Digest the PCR product (from step H8) with 2 µl of AcuI (5 U/µl, NEB) and 2 µl of XbaI (20 U/µl, NEB) (see Note 2) in 1x CutSmart buffer containing 40 µM S-adenosylmethionine (SAM) in a 60 µl volume at 37 °C overnight.

  11. The crush and soak procedure
    1. Run the AcuI/XbaI-digested DNA (from step I1) on a 20% polyacrylamide gel (Figure 2; size fractionation 1).
    2. Cut the 45-bp fragment out of the gel and transfer it to a microfuge tube. Crush the gel slice against the wall of the microfuge tube with the disposable pipette tip.
    3. Add 1-2 volumes of TE to the gel slice. Incubate the tube at room temperature overnight on a rotating wheel.
    4. Centrifuge the sample at 21,130 x g for 1 min at 4 °C. Transfer the supernatant to a fresh microfuge tube, being careful to avoid transferring fragments of polyacrylamide.
    5. Add 0.5 volumes of TE to the polyacrylamide pellet. Vortex the tube briefly. Centrifuge the sample at 21,130 x g for 1 min at 4 °C.
    6. Combine the two supernatants and then centrifuge at 21,130 x g for 1 min at 4 °C. Transfer the supernatant to the new microcentrifuge tube in order to remove the small PAGE debris.
    7. Mix the sample with 0.1 volume of 3 M sodium acetate and 1/400 volume of 20 µg/µl of glycogen. Add 2.5 volumes of cold ethanol to the sample and mix briefly. Store the solution on ice for 30 min. Centrifuge the sample at 21,130 x g for 20 min at 4 °C.
    8. Discard the supernatant. Rinse the pellet once with 70% ethanol.
    9. Dissolve the pellet into 20 µl of TE buffer.

  12. 3’ linker II ligation
    1. Mix the digested DNA (from step J9) with 2 µl of 10 µM 3’ linker II and 1 µl of Quick T4 DNA ligase (NEB) in 1x Quick ligation buffer.
    2. Incubate the ligation reaction mixture at room temperature for 15 min, purify it using a QIAquick PCR Purification Kit, and elute it with 100 µl of TE buffer.

  13. Second PCR optimization (see Note 3)
    1. To determine the optimal number of PCR cycles, prepare a 0.2-ml PCR tube containing 5 µl of the ds cDNA ligated with 5’ linker I/3’ linker II (from step K2), 0.5 µl of 25 µM 5’ linker I forward primer, 0.5 µl of 25 µM 3’ linker II PCR primer, 5 µl of 1x Advantage 2 PCR buffer, 1 µl of 10 mM dNTP mix, 1 µl of 50x Advantage 2 polymerase mix, and MilliQ water in a 50 µl volume.
    2. Perform PCR with the following cycling parameters: 6 cycles of 98 °C for 10 sec and 68 °C for 10 sec. After the 6 cycles, transfer 5 μl of the reaction to a clean microcentrifuge tube.
    3. Perform PCR with the rest of the PCR reaction mixture for an additional 3 cycles of 98 °C for 10 sec and 68 °C for 10 sec.
    4. After these additional 3 cycles, transfer 5 μl of the reaction to a clean microcentrifuge tube.
    5. In the same way, repeat additional PCR cycles until 18 total cycles are reached.
    6. Thus, prepare a series of PCR reactions of 6, 9, 12, 15, and 18 cycles and analyze by 20% polyacrylamide gel electrophoresis to compare the band patterns (Figure 3; PCR optimization 2).


      Figure 3. The second PCR cycle optimization and size fractionation (Arakawa, 2016). PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles. (see Notes 4 and 5)

    7. Determine the optimal number of PCR cycles as the minimal number of PCR cycles yielding the greatest quantity of the 72-bp product (typically around 9 cycles).
    8. Repeat five PCR reactions, each containing 50 µl, with the optimal number of PCR cycles.
    9. Purify the PCR product using a QIAquick PCR Purification Kit and elute it with 100 µl of TE buffer.

  14. BsmBI/AatII digestion
    1. Digest the PCR product (from step L9) with 10 µl of BsmBI (10 U/µl, NEB) in 1x NEBuffer 3.1 in a 100 µl volume at 55 °C for 6 h, and then add 5 µl of AatII (20 U/µl, NEB) (see Note 2) to the solution; leave the solution at 37 °C overnight.
    2. Run the BsmBI/AatII digested DNA on a 20% polyacrylamide gel.
    3. Typically, 3 bands, corresponding to 25, 24, and 23 bp, are visible (Figure 2; size fractionation 2).
    4. Cut the 25-bp fragment out of the gel to purify by the crush and soak procedure as described in step K, and dissolve the fragment into 50 µl of TE buffer.
    5. Measure the concentration of the purified DNA by a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).

  15. Cloning
    1. Digest the lenti CRISPR ver. 2 (lentiCRISPR v2) (Sanjana et al., 2014) (Addgene) with BsmBI, treat with calf intestine phosphatase, extract with phenol/chloroform, and purify by ethanol precipitation.
    2. Mix 5 ng of the purified 25-bp guide sequence fragment (from step M5) with 3 µg of lentiCRISPR v2 and 1 µl of Quick T4 DNA ligase (NEB) in 1x Quick ligation buffer in a 40 µl volume.
    3. Incubate the ligation reaction mixture at room temperature for 15 min and then purify by ethanol precipitation.
    4. Electroporate the prepared gRNA library into STBL4 electro-competent cells (Invitrogen) using the following electroporator conditions: 1,200 V, 25 µF, and 200 Ω.

  16. Deep sequencing
    1. To amplify the guide sequences, prepare a 0.2-ml PCR tube containing 1 µl of 100 ng/µl of lentiviral plasmid library (from step N4), 0.5 µl of 25 µM lentiCRISPR forward primer, 0.5 µl of 25 µM lentiCRISPR reverse primer, 5 µl of 1x Advantage 2 PCR buffer, 1 µl of 10 mM dNTP mix, 1 µl of 50x Advantage 2 polymerase mix, and MilliQ water in a 50 µl volume.
    2. Perform PCR with the following cycling parameters: 15 cycles of 98 °C for 10 sec and 68 °C for 10 sec.
    3. Purify the 100-bp PCR fragment containing the guide sequence from the 2% agarose gel using a QIAquick Gel Extraction Kit (QIAGEN). Prepare the deep sequencing library using a TruSeq Nano DNA Library Preparation Kit (Illumina).
    4. Deep sequence using Miseq (Illumina) (see Note 6).

Data analysis

  1. Demultiplex FASTQ files by Illumina Miseq.
  2. Trim the sequence reads to exclude vector backbone sequences using the CLC Genomics Workbench (QIAGEN) (see Note 6) and add the PAM-sequence NGG to the sequence reads.
  3. Align these sequence reads with a reference genome using the RNA-seq analysis toolbox in the CLC Genomics Workbench (QIAGEN).

Notes

  1. The quality of the poly(A) RNA is one of the most important factors that affects the library’s quality (Procedure B). During setup of the methodology for gRNA library construction, rRNA contamination was observed in poly(A) RNA purified using an oligodT column, and rRNA-originated guide sequences sometimes occupied 40-50% of the total original library. Since rRNA occupies more than 90% of intracellular RNA, generally speaking, it is hard to avoid having some rRNA contamination. The levels of rRNA contamination could be tested by the Bioanalyzer (Agilent). If a high amount of rRNA is contaminated, the washing step with buffer OBB (steps B7-B10) must be repeated. Alternatively, an rRNA depletion kit could be incorporated into the protocol to reduce rRNA contamination.
  2. PCR artifacts amplifying the linker sequences were also observed during setup of the methodology. For this reason, the linker sequence was designed with additional restriction sites, namely BglII for the 5’ SMART tag (step H3), XbaI for the 3’ linker I (step J1), and AatII for the 5’ linker I and 3’ linker II (step N1). By cutting with these additional restriction enzymes, it was possible to remove most of the PCR artifacts amplifying the linker sequences.
  3. Because the PCR conditions are optimized for Advantage 2 polymerase (Clontech), I recommend using Advantage 2, which is optimal for efficient amplification of a complex cDNA library (Procedure I and Procedure M). PCR conditions, such as cycling number, primer concentration, or template amount, have to be optimized if other polymerases are used for PCR.
  4. PCR cycle number has to be carefully titrated because the desired PCR products will be reduced or lost by over-cycling of PCR (Figures 2 and 3).
  5. The BsmBI restriction digest of the final PCR reaction generated the right size of DNA fragment (25 bp) in addition to one- or two-bp shorter, unexpected DNA fragments (Figure 3). These shorter DNA fragments were probably due to the inaccuracy of the cleavage position of the type III and type IIS restriction enzymes. The 25-bp fragment should be carefully isolated in order to avoid contamination of the 24- or 23-bp fragment, which may not have a PAM in the proper position.
  6. The deep sequencer and sequence analysis software can be chosen by users depending on their purpose and lab environment (Procedure P, Data analysis). For example, deep sequencing can also be done by the Illumina HiSeq.

Recipes

  1. TE buffer (10 mM Tris HCl [pH 8], 1 mM Na2EDTA)
    For 100 ml:
    1 ml 1 M Tris HCl (pH 8)
    0.4 ml 250 mM Na2EDTA
    98.6 ml MilliQ water
    Autoclave and then store at room temperature

Acknowledgments

I am grateful to Giulia Bastianello for critical reading the manuscript. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. This protocol is adopted from Arakawa (2006).

References

  1. Arakawa, H. (2016). A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism. Sci Adv 2(8): e1600699.
  2. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D. A. and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819): 1709-1712.
  3. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. and Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121): 819-823.
  4. Grissa, I., Vergnaud, G. and Pourcel, C. (2007). The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8: 172.
  5. Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. and Yusa, K. (2014). Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32(3): 267-273.
  6. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E. and Church, G. M. (2013). RNA-guided human genome engineering via Cas9. Science 339(6121): 823-826.
  7. Sanjana, N. E., Shalem, O. and Zhang, F. (2014). Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8): 783-784.
  8. Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D. A., Mikkelsen, T. S., Heckl, D., Ebert, B. L., Root, D. E., Doench, J. G. and Zhang, F. (2014). Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343(6166): 84-87.
  9. Wang, T., Wei, J. J., Sabatini, D. M. and Lander, E. S. (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science 343(6166): 80-84.
  10. Zhou, X., Marks, P. A., Rifkind, R. A. and Richon, V. M. (2001). Cloning and characterization of a histone deacetylase, HDAC9. Proc Natl Acad Sci U S A 98(19): 10572-10577.

简介

虽然物种的多样性代表着特殊的生物学能力的多样性,但许多编码这些特殊能力的基因却是不变的,留下了未开发的遗传信息金矿;然而,尽管基因组生物信息学方面取得了进展,但除了成熟的模型生物体,如人,小鼠或酵母,遗传信息的注释在大多数物种中是不完全的。使用聚簇常规散布的回文重复序列(CRISPR)/ Cas9(CRISPR相关蛋白9)系统的引导RNA(gRNA)文库可用于通过正向遗传学对非特异性基因的表型筛选。 gRNA文库的构建通常需要从注释基因设计的大量化学合成寡核苷酸;如果想在没有目标DNA序列的先验知识的情况下将mRNA转换成gRNA,那么主要的挑战就是发现原始邻近基序(PAM)侧翼的序列并切出20-bp片段。最近,我开发了基于分子生物学技术将mRNA转化为gRNA文库(Arakawa,2016)(图1)。我在这里描述了如何从mRNA构建gRNA文库的详细协议。


图1.将mRNA转化为gRNA文库构建的方法(Sanjana等人,2014)。总结了该方法的方案。在步骤中详细描述了D-O的每个步骤。 Bg,Bgl II; Xb,Xba I; Bs,Bsm BI; Aa, II。 PCR,聚合酶链反应; lentiCRISPR v2,lentiCRISPR版本2。

背景 聚类定期散布的回文重复(CRISPR)系统负责获得的细菌免疫力(Barrangou等人,2007),其中40%的真细菌和90%的古细菌(Grissa < em> et al。,2007)。虽然CRISPR / Cas9在生理学上是用于消除感染性病原体的内切核酸酶(Barrangou等人,2007),但是如果引导RNA(例如, gRNA)(Cong等人,2013; Mali等人,2013)。通过设计目的基因的gRNA,可以通过非同源末端连接(NHEJ)逐个敲除单个基因(Cong等人,2013; Mali等人,2013);另外,CRISPR / Cas9可用于制备可用于遗传筛选的gRNA文库(Zhou等人,2001; Koike-Yusa等人,2014; Shalem et al。,2014; Wang等人,2014)。化脓性链球菌(Sp)Cas9的gRNA可以设计为邻近原始相邻基序(PAM)NGG的20bp序列(Cong等人,2013; Mali等人)。 ,2013)。这样的序列通常可以通过生物信息学技术从编码序列或感兴趣的基因座鉴定。在这里,我描述了一种通过分子生物学技术构建gRNA文库而不依靠生物信息学的方法。简言之,使用含有PAM-互补序列的半随机引物从提取的RNA合成cDNA,然后使用IIS和III型限制酶切除与PAM相邻的20聚体以产生gRNA文库。所描述的方法不需要关于靶DNA序列的先前知识,使其适用于任何物种。

关键字:CRISPR, Cas9, gRNA, 文库

材料和试剂

  1. 1.5 ml微量离心管
  2. 0.2 ml PCR管
  3. 一次性移液器吸头
  4. Oligo dT 列(QIAGEN,补充有Oligotex mRNA迷你试剂盒[QIAGEN,目录号:70022])
  5. STBL4电感受态细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:11635018)
  6. 慢病毒载体
    lentiCRISPR v2(Sanjana等人,2014)(Addgene,目录号:52961)
  7. 寡核苷酸
    半随机引物p NNNCCN
    RNA转录本(SMART)标签上的5'切换机制TGGTCAAGCTTCAGCAGATCTACACGGACGTCGCrGrGrG
    5'SMART PCR引物TGGTCAAGCTTCAGCAGATCTACACG
    3'接头I转发pCTGCTGACTTCAGTGGTTCTAGAGGTGTCCAA
    3'接头I逆转GTTGGACACCTCTAGAACCACTGAAGTCAGCAGT
    5'连接器我转发GCATATAAGCTTGACGTCTCTCACCG
    5'连接子I逆转NNCGGTGAGAGACGTCAAGCTTATATGC
    3'连接子II正向p GTTTGGAGACGTCTTCTAGATCAGCG
    3'接头II反向CGCTGATCTAGAAGACGTCTCCAAACNN
    3'连接子I PCR引物GTTGGACACCTCTAGAACCACTGAAGTCAGCAGTNNNCC
    3'接头II PCR引物CGCTGATCTAGAAGACGTCTCCAAAC
    LentiCRISPR转发CTTGGCTTTATATATCTTGTGGAAAGGACG
    LentiCRISPR反向CGGACTAGCCTTATTTTAACTTGCTATTTCTAG
  8. TRIzol试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:15596026)
  9. 苯酚:氯仿:异戊醇25:24:1(Sigma-Aldrich,目录号:P2069-100ML)
  10. 乙醇
  11. 无RNase的水
  12. Oligotex mRNA Mini Kit(QIAGEN,目录号:70022)
  13. T4 DNA连接酶反应缓冲液(New England Biolabs,目录号:B0202S)
  14. SMART Scribe逆转录酶(Takara Bio,Clontech,目录号:639536)
  15. DTT(Takara Bio,补充有SMART Scribe逆转录酶[Takara Bio,Clontech,目录号:639536])
  16. dNTP混合物(Thermo Fisher Scientific,Invitrogen TM,目录号:18427013)
  17. RNaseOUT(Thermo Fisher Scientific,Invitrogen TM,目录号:10777019)
  18. RNase H(Thermo Fisher Scientific,Invitrogen TM,目录号:18021014)
  19. MilliQ水
  20. Advantage 2聚合酶混合物(Takara Bio,Clontech,目录号:639201)
  21. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  22. 快速连接套件(New England Biolabs,目录号:M2200S)
  23. EcoP15I(New England Biolabs,目录号:R0646S)
  24. Bgl II(New England Biolabs,目录号:R0144S)
  25. Acu I(New England Biolabs,目录号:R0641S)
  26. Xba I(New England Biolabs,目录号:R0145S)
  27. Bsm BI(New England Biolabs,目录号:R0580S)
  28. II。(II New England Biolabs,目录号:R0117S)
  29. 10-bp梯形图
  30. 1x CutSmart缓冲区(包括在Xba I [New England Biolabs,目录号:R0145S])
  31. S-腺苷甲硫氨酸(SAM)(New England Biolabs,补充有Acu I [New England Biolabs])
  32. 3 M醋酸钠(pH 5.5)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9740)
  33. 丙烯酰胺/双溶液(19:1)(40%w/v,5%C)(SERVA电泳,目录号:10679.01)
  34. 糖原(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:R0561)
  35. Qubit dsDNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32851)
  36. TE缓冲液(pH 8.0)(参见食谱)

设备

  1. 移液器
  2. Centifuge(Eppendorf,型号:5424 R,5810 R)
  3. 加热块
  4. 玻璃烧杯
  5. GeneAmp PCR System 9700(Thermo Fisher Scientific,型号:GeneAmp PCR System 9700)
    注意:本产品已停产。
  6. Mini-PROTEAN四立式电泳池(Bio-Rad Laboratores,型号:Mini-PROTEAN Tetra Vertical Electrophoresis Cell)
  7. GenePulser II(Bio-Rad Laboratores,型号:Gene Pulser II)
  8. 高性能实验室孵化器2800(F.lli GALLI,型号:MOD。2800)
  9. CLC Genomics Workbench(QIAGEN,型号:CLC Genomics Workbench)
  10. 生物分析仪(Agilent Technologies)
  11. 高压灭菌器

程序

  1. 总RNA制备
    1. 通过重复移取,用10 ml的TRIzol溶解细胞。在室温下孵育5分钟。
    2. 加入2毫升氯仿。用手摇动15秒。在室温下孵育2-3分钟。在4℃下以12,000 x g离心15分钟。
    3. 将水相转移到新鲜管中。加入5ml异丙醇,充分混匀。在4℃下以12,000 x g离心10分钟。
    4. 去除上清液。用80%乙醇洗涤沉淀一次。空气干燥沉淀5-10分钟
    5. 通过移液将RNA溶解在1ml无RNase的水中,然后在55-60°C下将样品孵育10分钟。

  2. PolyA RNA制备(见注1)
    1. 使用Oligotex mRNA Midi Kit。将Oligotex悬浮液加热至37°C。加热块上加热缓冲液OEB至70°C。混合缓冲液OBB很好。
    2. 将500μg总RNA吸入无RNA酶的1.5 ml微量离心管中,并用无RNase的水调节体积至1 ml。
    3. 加入500μl缓冲液OBB和30μlOligotex悬浮液。
    4. 在70℃下孵育3分钟。从热中取出。
    5. 在室温下孵育10分钟。
    6. 在15,871 xg离心2分钟。通过移液去除上清液。
    7. 加入250μl无RNase的水和250μl缓冲液OBB。通过移液彻底混合内容。
    8. 在70℃下孵育3分钟。从热中取出。
    9. 在室温下孵育10分钟。
    10. 在15,871 xg离心2分钟。通过移液去除上清液。
    11. 将沉淀重悬于400μl缓冲液OW2中。将混合物吸入置于1.5 ml管中的小型旋转柱上。在15,871 xg离心1分钟。
    12. 将旋转柱转移到新的无RNA酶的1.5-ml管中。应用400μl缓冲液OW2。将其吸入置于1.5ml管中的小旋转柱上。在15,871 xg离心1分钟。
    13. 将旋转柱转移到新的无RNA酶的1.5-ml管中。将25μl热(70℃)缓冲液OEB吸入柱上。吸管上下3-4次以重悬树脂。在15,871 xg离心1分钟。
    14. 将另外25μl热(70℃)缓冲液OEB吸取到柱上。吸管上下3-4次以重悬树脂。在15,871 xg离心1分钟。

  3. 链接器准备
    1. 将以下试剂合并到1.5ml微量离心管中:将10μl100μM连接子前向寡核苷酸,10μl100μM连接子反向寡核苷酸和2.2μl10×T4DNA连接酶缓冲液(New England Biolabs)。
    2. 将管置于含有2升开水的玻璃烧杯中,孵育管直到水自然冷却至室温。
    3. 用77.8μlTE缓冲液(pH 8.0)稀释退火的寡核苷酸,并使用10μM接头。

  4. 第一链cDNA合成
    1. 将以下试剂合并在0.2ml PCR管中:200ng聚(A)RNA(来自步骤A2),0.6μl25μM半随机引物和4.75μl体积的无RNA酶的水。
    2. 在热盖热循环仪中将管孵育72分钟3分钟,在冰上冷却2分钟,并进一步在25℃下孵育10分钟。
    3. 将温度升至42℃,加入以下试剂的5.25μl混合物:0.5μl25μM5'SMART标签,2μl5x SMART Scribe缓冲液,0.25μl100mM DTT,1μl10mM dNTP混合物,0.5μlRNaseOUT(Invitrogen)和1μlSMART Scribe逆转录酶(100U)(Clontech)。
    4. 将第一链cDNA反应混合物在42℃孵育90分钟,然后在68℃孵育10分钟。要降解RNA,将1μlRNA酶H(Invitrogen)加入到混合物中,并将混合物在37℃下孵育20分钟。

  5. 通过引物延伸的双链(ds)cDNA合成
    1. 将11μl制备的第一链聚(A)cDNA(来自步骤C4)与74μlMilliQ水,10μl10x Advantage 2 PCR缓冲液,2μl10mM dNTP混合物,1μl25μM5' SMART PCR引物和2μl50x Advantage 2聚合酶混合物(Clontech)。
    2. 孵育100μl体积的反应混合物,在95℃下引物延伸1分钟,68℃20分钟,然后70℃10分钟。
    3. 使用QIAquick PCR纯化试剂盒(QIAGEN)纯化制备的ds cDNA,并用40μlTE缓冲液(pH 8.0)洗脱DNA。

  6. 3'连接子连接
    1. 在1x快速连接缓冲液中混合0.5μl10μM3'接头I和1μlQuick T4 DNA连接酶(New England Biolabs; NEB)的ds聚(A)cDNA(来自步骤D3)。
    2. 将连接反应混合物在室温下孵育15分钟,然后使用QIAquick PCR Purification Kit进行纯化,并用80μlTE缓冲液洗脱。

  7. EcoP15I消化
    1. 在含有1x ATP的1x NEBuffer 3.1中,在100μl体积的37℃下将3'连接体I连接的DNA(来自步骤E2)与1μlEcoP15I(10U /μl,NEB)一起消化过夜。
    2. 使用QIAquick PCR纯化试剂盒纯化EcoP15I消化的DNA,并用40μlTE缓冲液洗脱DNA。

  8. 5'连接子I连接和Bgl II消化
    1. 将消化的DNA(来自步骤F2)与0.5μl10μM5'接头I和1μlQuick T4 DNA连接酶(NEB)在1x快速连接缓冲液中混合。
    2. 在室温下孵育连接反应混合物15分钟,使用QIAquick PCR Purification Kit进行纯化,并用80μlTE缓冲液洗脱。
    3. 用1x NEBuffer 3.1中的1μlBgl II(10U /μl,NEB)(参见注释2)在37℃下将DNA消化3小时。
    4. 使用QIAquick PCR纯化试剂盒纯化EcoRI15I/Bgl II消化的DNA,并用50μlTE缓冲液洗脱DNA。

  9. 第一次PCR优化(见注3)
    1. 为了确定PCR循环的最佳数量,制备含有5μl连接于5'连接体I/3'连接体I(来自步骤G4)的ds cDNA的0.2ml PCR管,0.5μl25μM连接子I正向引物,0.5μl25μM3'接头I PCR引物,5μl1x Advantage 2 PCR缓冲液,1μl10mM dNTP混合物,1μl50x Advantage 2聚合酶混合物和50μl体积的MilliQ水。
    2. 使用以下循环参数进行PCR:6个循环,98℃10秒和68℃10秒。
    3. 6个循环后,将5μl反应转移到干净的微量离心管中。与PCR反应混合物的其余部分进行PCR,再循环3次,98℃10秒,68℃10秒。
    4. 经过这3个循环,将5μl转移到干净的微量离心管中。
    5. 以相同的方式,重复另外的PCR直到达到30个总循环。因此,制备6,9,12,15,18,21,24,27,30个循环的一系列PCR反应,并通过20%聚丙烯酰胺凝胶电泳进行分析,以比较条带模式(图2; PCR优化1)。


      图2.第一次PCR循环优化和大小分级(Arakawa,2016)。 PCR产物在20%聚丙烯酰胺凝胶上运行。使用10-bp的梯子作为大小标记。预期尺寸的条带用三角形标记。 (见注4)

    6. 确定PCR循环的最佳数量作为产生最大量的84-bp产物的PCR循环的最小数量(通常约17个循环)。
    7. 重复两次50μlPCR反应,最佳PCR循环次数。
    8. 使用QIAquick PCR纯化试剂盒纯化PCR产物,并用50μlTE缓冲液洗脱
  10. 我的消化我的Xba Acu
    1. 用2μl的AcuI(5U /μl,NEB)和2μl的XbaI(20U /μl,NEB)将PCR产物(来自步骤H8)消化)(见注2)在含有40μMS-腺苷甲硫氨酸(SAM)的1x CutSmart缓冲液中,在37℃下60μl体积过夜。

  11. 粉碎和浸泡的程序
    1. 在20%聚丙烯酰胺凝胶上运行I-digest DNA(来自步骤I1)的Xba I/XbaI(图2;大小分级1)。
    2. 将45-bp片段切割出凝胶,并将其转移到微量离心管中。用一次性移液管尖端将凝胶切片压在微量离心管壁上。
    3. 向凝胶切片中加入1-2体积的TE。在室温下将管子在旋转轮上孵育过夜。
    4. 在4℃下将样品以21,130×g离心1分钟。将上清液转移到新鲜的微量离心管中,小心避免转移聚丙烯酰胺片段。
    5. 向聚丙烯酰胺颗粒中加入0.5体积的TE。短暂旋转管。在4℃下将样品以21,130×g离心1分钟。
    6. 合并两个上清液,然后在4℃下以21,130×g离心1分钟。将上清液转移到新的微量离心管中,以除去小的PAGE残留物
    7. 将样品与0.1体积的3M乙酸钠和1/400体积的20μg/μl糖原混合。向样品中加入2.5体积的冷乙醇,并短暂混合。将溶液储存在冰上30分钟。在4℃下将样品以21,130×g离心20分钟
    8. 丢弃上清液。用70%乙醇冲洗沉淀一次
    9. 将沉淀溶解于20μlTE缓冲液中
  12. 3'连接子II连接
    1. 将消化的DNA(来自步骤J9)与2μl10μM3'接头II和1μlQuick T4 DNA连接酶(NEB)在1x快速连接缓冲液中混合。
    2. 将连接反应混合物在室温下孵育15分钟,使用QIAquick PCR Purification Kit进行纯化,并用100μlTE缓冲液洗脱。

  13. 第二次PCR优化(见注3)
    1. 为了确定PCR循环的最佳数量,制备含有5μl连接于5'连接子I/3'连接体II(来自步骤K2)的ds cDNA的0.2-ml PCR管,0.5μl25μM连接子I前向引物,0.5μl25μM3'接头II PCR引物,5μl1x Advantage 2 PCR缓冲液,1μl10mM dNTP混合物,1μl50x Advantage 2聚合酶混合物和50μl体积的MilliQ水。 br />
    2. 使用以下循环参数进行PCR:6个循环,98℃10秒和68℃10秒。 6个循环后,将5μl反应转移到干净的微量离心管中。
    3. 与PCR反应混合物的其余部分进行PCR,再循环9个循环,98℃10秒,68℃10秒。
    4. 经过这3个循环,将5μl反应转移到干净的微量离心管中。
    5. 以相同的方式,重复另外的PCR循环,直到达到18个总循环。
    6. 因此,制备6,9,12,15和18个循环的一系列PCR反应,并通过20%聚丙烯酰胺凝胶电泳进行分析以比较条带模式(图3; PCR优化2)。


      图3.第二次PCR循环优化和大小分级(Arakawa,2016)。 PCR产物在20%聚丙烯酰胺凝胶上运行。使用10-bp的梯子作为大小标记。预期尺寸的条带用三角形标记。 (见注4和5)

    7. 确定PCR循环的最佳数量作为产生最大数量的72-bp产物的PCR循环的最小数量(通常约9个循环)。
    8. 重复5次PCR反应,每次含有50μlPCR PCR循环次数。
    9. 使用QIAquick PCR Purification Kit纯化PCR产物,并用100μlTE缓冲液洗脱
  14. bsm BI/ Aat II消化
    1. 将来自步骤L9的PCR产物用10μl的Bax BI(10U /μl,NEB)在1x NEBuffer 3.1中以100μl体积在55℃下消化6小时,然后加入将5μl的IIA(20U /μl,NEB)(见注2)给予溶液;将溶液在37℃下放置过夜。
    2. 在20%聚丙烯酰胺凝胶上运行II消化DNA的 BI/ Aat
    3. 通常,对应于25,24和23bp的3个条带是可见的(图2;尺寸分级2)。
    4. 将25-bp片段从凝胶中切下,通过如步骤K所述的挤压和浸泡步骤纯化,并将该片段溶解在50μlTE缓冲液中。
    5. 通过Qubit dsDNA HS测定试剂盒(Thermo Fisher Scientific)测量纯化的DNA的浓度
  15. 克隆
    1. 摘要lenti CRISPR ver。 2(lentiCRISPR v2)(Sanjana等人,2014)(Addgene)与Bsm BI,用小牛肠磷酸酶处理,用酚/氯仿提取,并通过乙醇纯化沉淀。
    2. 将5ng纯化的25-bp引导序列片段(来自步骤M5)与3μglentiCRISPR v2和1μlQuick T4 DNA连接酶(NEB)在1×Quick连接缓冲液中以40μl体积混合。
    3. 将连接反应混合物在室温下孵育15分钟,然后通过乙醇沉淀纯化。
    4. 使用以下电穿孔器条件将制备的gRNA文库电穿孔至STBL4电感受态细胞(Invitrogen):1,200V,25μF和200Ω。

  16. 深度测序
    1. 为了扩增引导序列,制备含有1μl100ng /μl慢病毒质粒文库(来自步骤N4),0.5μl25μMlentiCRISPR正向引物,0.5μl25μMlentiCRISPR反向引物,5μl μl1x Advantage 2 PCR缓冲液,1μl10mM dNTP混合物,1μl50x Advantage 2聚合酶混合物和50μl体积的MilliQ水。
    2. 用以下循环参数进行PCR:98℃10秒和68℃10秒的15个循环。
    3. 使用QIAquick Gel Extraction Kit(QIAGEN),从2%琼脂糖凝胶纯化含有引导序列的100-bp PCR片段。使用TruSeq纳米DNA文库准备工具(Illumina)准备深度测序文库。
    4. 使用Miseq(Illumina)的深层次序列(见注6)

数据分析

  1. 由Illumina Miseq解复用FASTQ文件。
  2. 使用CLC Genomics Workbench(QIAGEN)(见注6)修剪序列读取以排除载体骨架序列,并将PAM序列NGG添加到序列读取中。
  3. 使用CLC Genomics Workbench(QIAGEN)中的RNA-seq分析工具箱,将这些序列读数与参考基因组对齐。

笔记

  1. 聚(A)RNA的质量是影响图书馆质量的最重要因素之一(程序B)。在建立gRNA文库构建方法时,在使用寡核苷酸柱纯化的poly(A)RNA中观察到rRNA污染,并且rRNA起始的引导序列有时占总原始数的40-50%图书馆。由于rRNA占细胞内RNA的90%以上,一般而言,很难避免出现一些rRNA污染。生物分析仪(Agilent)可以检测rRNA污染水平。如果大量的rRNA被污染,则必须重复用缓冲液OBB洗涤步骤(步骤B7-B10)。或者,rRNA耗尽试剂盒可以并入该方案以减少rRNA污染。
  2. 在设计方法时也观察到扩增接头序列的PCR伪影。为此,针对5'SMART标签(步骤H3),对于3'连接子I,具有另外的限制性位点,即Bgl II, (步骤J1)和对于5'连接体I和3'连接体II的IIA(步骤N1)。通过用这些额外的限制酶切割,可以去除扩增接头序列的大部分PCR伪影。
  3. 因为针对Advantage 2聚合酶(Clontech)对PCR条件进行了优化,我建议使用Advantage 2,这是对于复杂cDNA文库的有效扩增的最佳选择(方法I和程序M)。如果其他聚合酶用于PCR,必须优化PCR条件,如循环数,引物浓度或模板量。
  4. PCR周期数必须仔细滴定,因为所需的PCR产物将通过PCR的过度循环减少或丧失(图2和3)。
  5. 除了一个或两个bp短的意想不到的DNA片段之外,最终PCR反应的Bsm BI限制性消化产生正确大小的DNA片段(25bp)(图3)。这些较短的DNA片段可能是由于III型和IIS型限制酶的切割位置的不准确性。应仔细分离25-bp的片段,以避免24-或23-bp片段的污染,其可能没有PAM在适当的位置。
  6. 用户可根据用途和实验室环境选择深度序列分析软件和序列分析软件(程序P,数据分析)。例如,深度测序也可以由Illumina HiSeq完成。

食谱

  1. TE缓冲液(10mM Tris HCl [pH 8],1mM Na 2 EDTA) 100 ml:
    1 ml 1M Tris HCl(pH 8)
    0.4ml 250mM Na 2 EDTA
    98.6毫升MilliQ水
    高压灭菌,然后在室温下储存

致谢

我非常感谢朱利亚·巴斯蒂尼亚洛(Gulia Bastianello)批评阅读手稿。这项研究没有得到任何公共,商业或非营利部门的资助机构的具体拨款。该方案来自荒川(2006)。

参考

  1. Arakawa,H。(2016)。  转换方法mRNA转入用于任何生物体的CRISPR/Cas9编辑的gRNA文库。 2(8):e1600699。
  2. Barargou,R.,Fremaux,C.,Deveau,H.,Richards,M.,Boyaval,P.,Moineau,S.,Romero,DA and Horvath,P。(2007)。< a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/17379808"target ="_ blank"> CRISPR在原核生物中提供对病毒的获得性抵抗。 科学 315(5819):1709-1712。
  3. Cong,L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA and Zhang,F 。(2013)。使用CRISPR/Cas的多重基因组工程系统。 科学 339(6121):819-823。
  4. Grissa,I.,Vergnaud,G。和Pourcel,C。(2007)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/17521438" target ="_ blank"> CRISPRdb数据库和显示CRISPR的工具,并生成间隔符和重复的字典。 生物信息学(BMC Bioinformatics) 8:172.
  5. Koike-Yusa,H.,Li,Y.,Tan,EP,Velasco-Herrera Mdel,C.and Yusa,K。(2014)。  使用慢病毒CRISPR引导RNA文库的哺乳动物细胞中的全基因组隐性遗传筛选 Nat Biotechnol 32(3):267-273。
  6. Mali,P.,Yang,L.,Esvelt,KM,Aach,J.,Guell,M.,DiCarlo,JE,Norville,JE and Church,GM(2013)。  通过Cas9进行RNA指导的人类基因组工程 科学 339( 6121):823-826。
  7. Sanjana,NE,Shalem,O.和Zhang,F。(2014)。用于CRISPR筛选的改进的载体和全基因组库。 Nat方法 11(8):783-784。
  8. Shalem,O.,Sanjana,NE,Hartenian,E.,Shi,X.,Scott,DA,Mikkelsen,TS,Heckl,D.,Ebert,BL,Root,DE,Doench,JG and Zhang, )。人类基因组规模CRISPR-Cas9敲除筛选细胞。 科学 343(6166):84-87。
  9. Wang,T.,Wei,JJ,Sabatini,DM and Lander,ES(2014)。  使用CRISPR-Cas9系统的人类细胞中的遗传筛选。 343(6166):80-84。
  10. Zhou,X.,Marks,PA,Rifkind,RA and Richon,VM(2001)。  组蛋白脱乙酰酶HDAC9的克隆和表征。美国Proc Natl Acad Sci USA 98(19):10572-10577。
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引用:Arakawa, H. (2017). A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism. Bio-protocol 7(10): e2319. DOI: 10.21769/BioProtoc.2319.
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