参见作者原研究论文

本实验方案简略版
Mar 2019

本文章节


 

Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA
通过合成共价闭合环状(3Cs)DNA构建无偏和专用CRISPR / Cas gRNA文库   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning techniques, thus coupling library diversity with distribution. Here, we provide additional technical expertise to apply our covalently-closed-circular synthesized (3Cs) gRNA library generation technology for the generation of high-quality CRISPR/Cas gRNA libraries. F1-origin of replication-containing plasmid DNA is transformed into CJ236 bacteria for single colony outgrow followed by M13KO7 bacteriophage superinfection for the production and preparation of circular dU-containing ssDNA. dU-ssDNA is annealed with homology- and gRNA-encoding DNA oligonucleotides for their T7 DNA polymerase-mediated extension to form hetero-duplexed CCC-dsDNA (3Cs-dsDNA). 3Cs-dsDNA is electroporated for the selected amplification of the newly synthesized, gRNA-containing strand. To remove wild-type plasmid remnants, the purified plasmid DNA is digested with restriction enzymes targeting the gRNA-placeholder sequence in the template DNA. Undigested plasmid is electroporated for the extraction of the final 3Cs gRNA library. Due to the absence of PCR amplification and conventional cloning steps, the 3Cs technology uncouples sequence diversity from sequence distribution, thereby generating gRNA libraries with near-uniform distribution in diversities being only limited by electroporation efficiencies.

Keywords: CRISPR/Cas (CRISPR/Cas9), gRNA library (gRNA文库), 3Cs technology (3Cs技术), 3Cs gRNA (3Cs gRNA), Gene editing (基因编辑), CRISPR screen (CRISPR筛选)

Background

CRISPR/Cas gene editing has developed to be the gold standard for tailored and unbiased high-throughput screening (Doench, 2018). Unbiased pooled screening enables the simultaneous testing of thousands of individual genetic perturbations in a single experiment. However, a prerequisite is the presence of a gRNA library, in which each individual construct defines a specific genomic target site (gRNA sequence), at which the gene perturbation is desired, with a library’s gRNA distribution and diversity dictating the experimental scale (Sanson et al., 2018). Classically, protocols to generate CRISPR/Cas gRNA libraries for arrayed or pooled applications contain T4 ligase or recombination-based ligations steps, requiring DNA polymerase-dependent amplification of the gRNA-encoding oligonucleotides coupled with restriction enzyme-digested plasmid DNA for subsequent gRNA cloning (Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014; Schmidt et al., 2015; Vidigal and Ventura, 2015; Arakawa, 2016; Ong et al., 2017). Due to these technical limitations, the resulting gRNA libraries contain undesired PCR and cloning-dependent sequence biases in their gRNA sequence distribution that directly affects the experimental scale needed for robust hit calling (Shalem et al., 2014; Wang et al., 2014). Unbiased and tailored CRISPR/Cas gRNA screening is becoming more ubiquitous, highlighting the need for novel protocols to generate gRNA libraries that can readily be established in non-expert laboratories.

Our recently described protocol to generate covalently-closed-circular synthesized (3Cs) gRNAs avoids DNA polymerase-dependent amplification steps of gRNA-encoding oligonucleotides, as well as enzyme-digested plasmid DNA (Figure 1, Wegner et al., 2019), thereby circumventing the occurrence of PCR-dependent gRNA sequence bias, as well as cloning artifacts within the final gRNA library, resulting in the decoupling of sequence distribution from sequence diversity and leading to nearly uniformly distributed gRNA libraries. As a consequence, the quality and distribution of the gRNA sequences within the original gRNA-encoding oligonucleotide pool is very important and particular caution should be paid to only use high-quality oligonucleotide pools.



Figure 1. Covalently-closed-circular (3Cs) synthesized gRNA reagents. Amplification of f1-ori-containing plasmids in M13KO7-infected dut-/ung- E. coli CJ236 bacteria and subsequent purification yields uracilated single-stranded DNA (dU-ssDNA). Annealing of gRNA-encoding oligonucleotides, extension with T7 polymerase, and ligation with T4 ligase produces a heteroduplex dU-dsDNA plasmid library. Electroporation into dut-/ung- E. coli yields the final library. The gRNA-encoding oligonucleotide is designed to anneal 5′ and 3′ relative to the I-SceI placeholder site in the library backbone and acts as a primer for T7 polymerase in the synthesis step of the 3Cs protocol.

Materials and Reagents

  1. 2 mm electroporation cuvette (BTX, catalog number: 45-0125)
  2. Conical centrifuge tube, 50 ml (Falcon, catalog number: 352070)
  3. Safe-lock tubes, 1.5 ml (Eppendorf, catalog number: 0030120086)
  4. Bacteria (E. coli) CJ236 (TaKaRa, catalog number: 9053 or Lucigen, catalog number: 60701), storage -80 °C
  5. Bacteria (E. coli) 10-beta, electrocompetent (New England Biolabs, catalog number: C3020K), storage -80 °C
  6. Bacteria (E. coli) SS320 (MC1061 F’), electrocompetent (Lucigen, catalog number: 60512)
  7. dNTP mix (Carl Roth, catalog number: 0178), storage -20 °C
  8. T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number: M0201), storage -20 °C
  9. T4 DNA ligase (New England Biolabs, catalog number: M0202), storage -20 °C
  10. T7 DNA polymerase (unmodified) (New England Biolabs, catalog number: M0274), storage -20 °C
  11. Restriction enzyme I-SceI (New England Biolabs, catalog number: R0694), storage -80 °C
  12. Restriction enzyme EcoRV-HF (New England Biolabs, catalog number: R3195), storage -20 °C
  13. Helper phage M13KO7 (New England Biolabs, catalog number: N0315)
  14. LB broth with agar (Lennox) (Sigma-Aldrich, catalog number: L2897)
  15. LB medium (Lennox) (Carl Roth, catalog number: X964)
  16. 2x YT medium (Carl Roth, catalog number: 6676)
  17. S.O.C. medium (Thermo Fisher, catalog number: 15544034)
  18. Polyethylene glycol 8000 (PEG 8000) (Carl Roth, catalog number: 263)
  19. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434-M)
  20. Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D8662)
  21. E.Z.N.A. M13 DNA Mini Kit (Omega Bio-Tek, catalog number: D69001-01)
  22. GeneJET Gel Extraction Kit (Thermo Fisher, catalog number: K0692)
  23. GeneJET Plasmid Miniprep Kit (Thermo Fisher, catalog number: K0503)
  24. DNA Clean and Concentrator-5 Kit (Zymo Research, catalog number: D4013)
  25. Plasmid Midi Kit (Qiagen, catalog number: 12143)
  26. Plasmid Maxi Kit (Qiagen, catalog number: 12163 or Promega, catalog number: 12163)
  27. Ampicillin sodium salt (Amp) (Carl Roth, catalog number: K0292)
  28. Chloramphenicol (Cmp) (Carl Roth, catalog number: 3886)
  29. Kanamycin sulphate (Kan) (Carl Roth, catalog number: T832)
  30. Adenosine 5'-triphosphate (ATP) (New England Biolabs, catalog number: P0756), storage -20 °C
  31. Dithiothreitol (DTT) (Cell Signaling Technology, catalog number: 7016), storage -20 °C
  32. Agarose, standard (Applichem, catalog number: 732-2789)
  33. SYBR Safe DNA gel stain (Thermo Fisher, catalog number: S33102)
  34. KCl (Carl Roth, catalog number: P017)
  35. CaCl2 (Carl Roth, catalog number: CN93)
  36. MgCl2 (Carl Roth, catalog number: KK36)
  37. Tris base (Carl Roth, catalog number: A411)
  38. Tris-HCl (Carl Roth, catalog number: 9090)
  39. EDTA (Applichem, catalog number: A5097,0500)
  40. Acetic acid (Sigma-Aldrich, catalog number: 33209)
  41. KCM buffer (see Recipes)
  42. 20% PEG/NaCl (see Recipes)
  43. 10x TM buffer (see Recipes)
  44. 50x TAE buffer (see Recipes)

Equipment

  1. Bacterial shaker (Eppendorf, model: New Brunswick Innova 44/44R, catalog number: M1282-0012; or similar)
  2. Gene Pulser electroporation system (Bio-Rad, catalog number: 164-2076, or similar system for electroporation of bacteria)
  3. Mini-sub cell GT horizontal electrophoresis system, 7 x 10 cm (Bio-Rad, catalog number: 1704467, or similar)
  4. Centrifuge Avanti J-E (Beckman, catalog number: 369003, or similar)
  5. Rotor JA-12 (fixed-angle aluminum rotor- 12 x 50 ml, 12,000 rpm, 23,200 x g, Beckman, catalog number: 360993)
  6. NanoDrop (Thermo Fisher, catalog number: ND-1000, or similar)

Software

  1. bCl2fastq (Illumina, https://support.illumina.com/)
  2. cutadapt (https://cutadapt.readthedocs.io/en/stable/; Martin, 2011)
  3. Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml; Langmead and Salzberg, 2012)
  4. PinAPL-Py (http://pinapl-py.ucsd.edu/; Spahn et al., 2017)

Procedure

  1. Preparation of plasmid single-stranded DNA (ssDNA)
    1. Transform 100-200 ng of plasmid DNA into 25-50 μl competent E. coli CJ236 bacteria. Use either heat-shock (1 min, 42 °C, heat-block) or KCM buffer (use the same volume of bacteria and a mix of 5x KCM buffer diluted 1:5 with water, ice/15 min, room temperature/10 min) for transformation. Add 50-200 μl S.O.C. medium and plate on LB plates containing 25 μg/ml Cmp and additional antibiotic matching plasmid resistance (usually 100 μg/ml Amp*, depending on plasmid), leave overnight at 37 °C.
      *Note: In theory, any plasmid containing f1-origin of replication can be used, such as lentiCRISPR v2 (Addgene 52961). The plasmid should have an antibiotic resistance gene other than Amp, Cmp and Kan.
    2. Next day, pick several single colonies of E. coli CJ236 with a sterile pipette tip, resuspend each into 1 ml of 2x YT medium supplemented with Amp* (100 μg/ml), Cmp (25 μg/ml) and 1 μl helper phage. Shake for 2 h (37 °C, 200 rpm); add 100 μg/ml Kan and continue shaking for 6-10 h (37 °C, 200 rpm).
    3. Transfer 1 ml of initial culture into 30 ml of 2x YT medium containing Amp* (100 μg/ml) and Kan (100 μg/ml) and shake 16-20 h (37 °C, 200 rpm).
    4. Transfer 30 ml bacterial culture into a 50 ml Falcon tube; centrifuge (10 min, 12,000 x g, 4 °C).
    5. Transfer phage-containing supernatant (Figure 2A) into 6 ml of sterile-filtered PEG/NaCl buffer, invert 4-6 times, incubate for 30-60 min at room temperature; centrifuge (10 min at 12,000 x g, 4 °C).
    6. Decant supernatant and aspirate the remaining supernatant.
    7. Resuspend phage pellet (Figure 2B) in 1 ml 1x PBS and transfer to a new 1.5 ml reaction tube.


      Figure 2. Preparation of ssDNA from bacterial culture. E. coli CJ236 bacterial culture before (A) and after (B) centrifugation (pellet contains bacteria, whereas supernatant contains phage). C. Phage pellet following PEG/NaCl precipitation and subsequent centrifugation. D. Phage resuspended in 1x PBS buffer (transparent solution). E. After addition of ssDNA lysis buffer (turbid solution, turbidity reflects amount of phage).

    8. Centrifuge for 5 min at 16,100 x g to remove cell debris, transfer the supernatant to a new 1.5 ml reaction tube and store at 4 °C.
    9. Purify ssDNA from phage supernatant according to the Omega Bio-Tek E.Z.N.A M13 DNA Mini Kit protocol.
    10. Determine ssDNA concentration using NanoDrop.
    11. Analyze ssDNA in a 0.8% TAE/agarose gel using 500 ng ssDNA and 250 ng of the respective dsDNA. The ssDNA should appear as a predominant single band (Figure 3A).


      Figure 3. Quality control of ssDNA and 3Cs reaction products. A. ssDNA appears as a single band on an agarose gel and usually exhibits higher electromobility than its dsDNA plasmid counterpart. #1, #2, and #3 refer to example plasmids of different sizes. Low quality ssDNA exhibits multiple bands indicating phage-derived recombination products that will lead to poor performance during 3Cs (!). B. The 3Cs reaction product typically exhibits three bands on an agarose gel. All three electrophoreses were run on 0.8% 1x TAE agarose for 30 min at 110 V.

  2. Oligonucleotide phosphorylation with T4 polynucleotide kinase
    In a 1.5 ml reaction tube, combine 600 ng oligonucleotide pool with 2 μl 10x TM buffer, 1 mM ATP, 5 mM DTT and 20 units T4 polynucleotide kinase in a total volume of 20 μl; incubate for 2 h at 37 °C.
    Note: Oligonucleotide pools can be ordered from various commercial sources. Hand-pooled oligonucleotides generally lead to libraries with very variable gRNA distribution and should therefore be avoided.

  3. Annealing of the oligonucleotides to the template
    Mix 20 μg freshly prepared ssDNA with 25 μl 10x TM buffer (final concentration 1x TM) and 20 μl phosphorylated oligonucleotides in a total volume of 250 μl; incubate in a thermoblock as follows: 90 °C (3 min), 50 °C (5 min), room temperature (5 min).
    Note: Molar ratio between oligonucleotides and vector ssDNA should be determined experimentally and might be different for a particular plasmid. Usually, ratio ranges of 3:1, 5:1 and 7:1 are optimal for 3Cs reactions.

  4. Enzymatic synthesis of 3Cs-dsDNA
    Add the following components to the previous mixture: 10 μl of 10 mM ATP, 10 μl of 100 mM dNTPs, 15 μl of 100 mM DTT, 3 μl of T4 DNA ligase (stock: 400,000 units/ml) and 5 μl of T7 DNA polymerase (stock: 10,000 units/ml); mix carefully by pipetting up and down or flicking the tube; incubate for 16 h at 22 °C.

  5. Purification of 3Cs-dsDNA with DNA Clean and Concentrator-5 Kit
    1. Mix the 3Cs reaction with 2x volume of DNA binding buffer and incubate for 10 min at room temperature.
    2. Divide the 3Cs reaction on 2 columns.
    3. Spin down (1 min, 2,300 x g, room temperature); re-spin each flow through twice.
    4. Wash column twice each with 200 μl wash buffer (1 min, 2,300 x g, room temperature).
    5. Discard flowthrough and dry the column (1 min, 7,400 x g, room temperature).
    6. Incubate column with preheated 10 μl of elution buffer (10 min, 50 °C), spin down at maximum speed (2 min), re-spin each flow through twice.
    7. Repeat elution with 20 μl flow through (final volume will be around 40 μl).
    8. Run a gel to control the quality of dsDNA alongside to ssDNA (Figure 3B).
    9. Determine concentration by Nanodrop.

  6. Electroporation of electrocompetent E. coli bacteria with 3Cs dsDNA
    1. Prepare 20 ml pre-warmed S.O.C. medium at 37 °C in a small Erlenmeyer flask.
    2. Incubate the entire purified 3Cs dsDNA and a 2 mm electroporation cuvette on ice.
    3. Thaw 400 μl of electrocompetent E. coli strain SS320 or NEB 10-beta bacteria on ice (20 min).
    4. Add the cells to the pre-chilled and purified 3Cs DNA and mix slowly by pipetting, avoid generating bubbles.
    5. For electroporation, use the following settings: 2.5 kV field strength, 25 µF capacitance, 200 ohms resistance (optimal duration: 3.8-4.2 ms).
    6. Place the dried cuvette into the electroporator holder.
    7. Transfer the mixture gently (without bubbles) into the cuvette and tap the cuvette (avoid air bubbles that lead to arcing!).
    8. Tightly close the holder, put the lid on the cuvette.
    9. Electroporation: press both buttons on the power supply at once, release once beeping starts (usually 3.8-4.2 ms).
    10. Immediately rescue the electroporated cells by rinsing in 2x 1 ml pre-warmed S.O.C. medium. Any delay will significantly decrease the electroporation efficiency.
    11. Incubate Erlenmeyer flask (with 20 ml S.O.C. medium and electroporated bacteria) for 30 min (37 °C, 140 rpm).
    12. Remove 40 μl of bacterial culture, keep for preparing serial dilutions.
      Note: In a 96-well plate prepare 3 columns (i.e., 3 x 8 wells) with 90 μl 1x PBS per library. Add 10 μl of bacterial culture into the first 90 μl 1x PBS (prepare in triplicate), use it for subsequent serial dilutions (1:10). Plate 5 μl of diluted bacteria on Amp* LB plate, incubate at 37 °C overnight. Calculate library diversity as follows: count colonies in a dilution where separate colonies are nicely visible and calculate the mean number of colonies per 1 μl diluted bacterial culture. Knowing the final volume of the bacterial culture (20.4 ml) and dilution from which colonies were counted (for example, lane 3 dilution in a 96-well plate corresponds to 103 dilution), calculate the library diversity: Diversity = (number of bacterial colonies in 1 μl diluted bacterial culture) x 10(dilution) x bacterial culture volume (μl).
    13. Transfer the remaining content of the small Erlenmeyer flask into 180 ml of pre-warmed 2x YT medium (with 100 μg/ml Amp*), shake overnight (37 °C, 140 rpm).
    14. Next day prepare a mini-prep from 5 ml of the overnight culture and use it to check library quality (see Steps G1-G3).
    15. Spin the remaining culture down (4 °C, 6,000 x g, 20 min) for subsequent midi-prep.

  7. Analysis of the pre-library by restriction enzymes
    1. Digest 0.5 μg pre-library dsDNA with the appropriate restriction enzymes; always use original vector as cleavage control as well.
    2. Load undigested and digested pre-library and original vector on 0.5-0.8% 1x TAE agarose gel.
    3. Check the quality of the pre-library and compare it with the original vector (Figure 4).


      Figure 4. Removal of wild-type remnants from pre-library preparation and quality control of final library. Restriction enzyme digestion of the wild-type plasmid, the pre-library, and the final library indicates the ratio of wild-type and 3Cs plasmids at each step. As control, the wild-type plasmid (wt plasmid) is linearized with either I-SceI or EcoRV. A double digest with both enzymes removes a fragment of approximately 2,500 bp as both enzymes cut the plasmid (!). In the pre-library pool, the incomplete digestion with I-SceI and the faint band resulting from the double-digest indicate the majority of DNA being 3Cs products. The double digest reveals a band at ~2,500 bp with a significantly lower intensity than in the wild-type plasmid pool. In the final library, the I-SceI digest reveals no visible linearized fragment. The double digest linearizes the library and shows no visible band at ~2,500 bp, indicating the final library to be free of wild-type plasmid. Electrophoresis was run on 0.8% 1x TAE agarose for 30 min at 110 V.

  8. Digestion of pre-library library prior final electroporation
    1. Digest 10-20 μg of the pre-library with the appropriate enzyme and purify by Clean & Concentrator Kit.
    2. Transform and process 5-10 μg of the cleaved pre-library into 400 μl electrocompetent bacteria as described under Steps F1-F12.
    3. Grow 200-1,000 ml of 2x YT-Amp* (100 µg/ml) bacterial culture (volume depends on library size, future needs and amount of remaining oligonucleotides) at 37 °C overnight.
    4. Next day spin down bacteria (prepare multiple aliquots of bacterial pellets from either 50, 100 or 150 ml bacterial culture, depending on the maxi-prep kit that you are using and size of your library).
    5. Prepare a maxi-prep from one bacterial pellet, keep the remaining pellets at -20 °C until needed.

  9. Analysis of the final library by restriction enzymes
    Follow Steps G1-G3 (Figure 4).

  10. Sequence analysis of the final library by Sanger sequencing
    Send appropriate amount (usually 700-1,200 ng) of the final library for Sanger sequencing. Choose a sequencing primer that is near your gRNA cassette, for example U6 primer. Representative data is presented in Figure 5.


    Figure 5. SANGER sequencing of final library. As a first quality control step, SANGER sequencing of the final library with the U6-primer reveals randomization at the I-SceI placeholder side. In this example, digestion of the final optimized truly genome-wide library (oTGW) from Wegner et al. (2019) results in a randomized nucleotide pattern resembling the intended sequence composition of that library.

  11. Sequence analysis of the final library guide distribution by next-generation sequencing (NGS)
    For a detailed description of data processing and data analysis we refer to Wegner et al., 2019, a brief overview of the procedures is described below.

Data analysis

A detailed description of data processing and data analysis can be found in Wegner et al., 2019. To quality control 3Cs reagents, multiplexed Next Generation Sequencing (NGS) of several different plasmid libraries is possible when using barcoded Illumina adapter sequences. Raw NGS data can be demultiplexed using bCl2fastq from Illumina, preferably in the most recent version. To count individual reads of a library, use a designated tool like PinAPL-Py (Spahn et al., 2017) or design a customized pipeline that performs read trimming and sequence alignments according to your needs using, e.g., cutadapt and bowtie2.
  Adapter trimming can be performed using cutadapt by trimming the read to the 20 gRNA-encoding nucleotides (Martin, 2011, https://cutadapt.readthedocs.io/en/stable/). Alignment of the trimmed reads against the library can then be performed with bowtie2 or custom scripts. The final library should contain all desired gRNA sequences. To assess the distribution of the library, generate Lorenz curves of gRNA representation and compute the area under the resulting curve according to (Wegner et al., 2019) (Figure 6A). This will allow you to compare different libraries. A histogram of the frequencies of the gRNA reads and calculating the ratio between the 90% and 10% percentiles reveals the skew of a library (Figure 6B). For the analyses of gene perturbation screens, we refer to the relevant literature as the experimental settings and read out determine the analyses techniques.


Figure 6. NGS analysis and visualization of expected sequence distributions. A. gRNA counts of three gRNA libraries are visualized as Lorenz plots, displaying the cumulative fraction of represented NGS reads against the gRNAs ranked by abundance. The ideal distribution would be uniform and resembles a diagonal line with an area under the curve (AUC) of 0.5 (green curve, ideal). A well distributed library contains every intended gRNA sequence and exhibits an AUC that differs only slightly from 0.5 (blue curve, good). An increased AUC value indicates an unevenly distributed library (orange curve, bad). Note that the slope of this curve decreases to 0 near x = 1.0, indicating that there are gRNA sequences missing in that library. B. Plotting the gRNA distributions from (A) as histograms visualizes the skew or symmetry of a library. A well distributed library exhibits a low skew near 1 (blue, good), indicating symmetry. A skewed library is asymmetrically distributed. The bad example is skewed towards higher gRNA read counts as revealed by the long right tail in the histogram (orange, bad).

Notes

  1. Oligonucleotides are usually 55-65 nucleotides in length, having a gRNA-encoding region in the middle, and plasmid sequence homology regions (3Cs homology) at its 5′ and 3′ ends. Homology of 18 nucleotides (on each side of the gRNA) and annealing temperature above 45 °C are optional for 3Cs reaction. The oligonucleotides should not have any modifications at either end as the efficiency of the ligation will decrease.
    Important: The template plasmid must contain an f1-origin of replication. The E. coli CJ236 strain is Cmp-resistant and dut-/ung-.
  2. Phage suspended in PBS can be stored for months at 4 °C.
  3. Once isolated, ssDNA is stable up to a few days or weeks. We recommend using freshly prepared ssDNA.
  4. To confirm the quality of your ssDNA, you can mix 1 μl of phage prep with KCM-competent bacteria and 200 μl S.O.C. medium. Incubate at 37 °C for 1 h and then dilute to 5 ml LB medium complemented with Amp* (100 μg/ml). Shake overnight (37 °C, 200 rpm), isolate dsDNA using a mini-prep kit and analyse dsDNA by using the same approach as in Procedure (Steps G1-G3). Use original plasmid dsDNA as positive control (compare size and restriction pattern in agarose gel).
  5. Use only freshly phosphorylated oligonucleotides for 3C reaction.
  6. High-quality electrocompetent E. coli strain SS320 or NEB 10-beta bacteria can also be prepared in the lab.
  7. The removal of dU-containing DNA in dut+/ung+ E. coli is not 100% efficient, therefore, it can be beneficial to introduce restriction sites (such as I-SceI) in the area of plasmid where the gRNAs will be introduced (gRNA placeholder sequence). Such an approach ensures that all the original sequences (still containing I-SceI site) will be depleted upon pre-library cleavage by I-SceI and subsequent electroporation. Importantly, make sure that the restriction recognition sequence does not resemble an active gRNA sequence in the genome of interest to avoid unintended editing events. A guide sequence containing an I-SceI restriction site is safe to use in the human genome. Also ensure that the restriction site is not a part of any of the gRNA sequences in the library to avoid unintended removal of a gRNA from the final pool.
  8. For pLentiGuide (Addgene 52963) and pLentiCRISPRv2 (Addgene 52961) plasmids, use a forward primer (5′-GGGCCTATTTCCCATGATTCCTTCATATTTGC-3′) that binds in the U6-promoter for Sanger sequencing.

Recipes

  1. KCM buffer
    Note: KCM renders the dut-/ung- E. coli CJ236 cells competent.
    0.5 M KCl
    0.15 M CaCl2
    0.25 M MgCl2
  2. 20% PEG/NaCl
    Note: PEG/NaCl is used to precipitate phage particles containing dU-ssDNA.
    20 % polyethylene glycol
    2.5 M NaCl
  3. 10x TM buffer
    Note: Serves together with ATP and DTT as a reaction buffer for polynucleotide kinase T4 in the phosphorylation step of the 3Cs protocol.
    0.1 M MgCl2
    0.5 M Tris-HCl
    pH 7.5
  4. 50x TAE buffer
    2 M Tris base
    0.5 M EDTA
    1 M acetic acid

Acknowledgments

We thank Alkmini Kalousi for providing images related to bacteria and phage pellets. We further thank all members of the Frankfurt CRISPR/Cas Screening Center (FCSC) and the Kaulich laboratory who contributed to the original research paper from which this protocol is derived from (Wegner et al., 2019). This work was supported by the Hessian Ministry for Science and the Arts (HMWK, LOEWE-CGT, IIIL5-518/17.004), the German Research Foundation (DFG; CEF-MC - EXC115/2; ECCPS - EXC147/2) and in part by the LOEWE Center Frankfurt Cancer Institute (FCI) funded by the Hessen State Ministry for Higher Education, Research and the Arts (IIIL5-519/03/03.001-(0015)).

Competing interests

The Goethe University Frankfurt has filed a patent application related to this work on which Martin Wegner and Manuel Kaulich are inventors (WO2017EP84625). The Goethe University provides an exclusive license of the 3Cs technology to Vivlion GmbH for which Manuel Kaulich is co-founder, shareholder and chief scientific officer.

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. Doench, J. G. (2018). Am I ready for CRISPR? A user's guide to genetic screens. Nat Rev Genet 19(2): 67-80.
  3. 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.
  4. Langmead, B. and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4): 357-359.
  5. Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17(1): 10-12.
  6. Ong, S. H., Li, Y., Koike-Yusa, H. and Yusa, K. (2017). Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries. Sci Rep 7(1): 7384.
  7. Sanson, K. R., Hanna, R. E., Hegde, M., Donovan, K. F., Strand, C., Sullender, M. E., Vaimberg, E. W., Goodale, A., Root, D. E., Piccioni, F. and Doench, J. G. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9(1): 5416.
  8. Schmidt, T., Schmid-Burgk, J. L. and Hornung, V. (2015). Synthesis of an arrayed sgRNA library targeting the human genome. Sci Rep 5: 14987.
  9. Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D. A., Mikkelson, T., 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.
  10. Spahn, P. N., Bath, T., Weiss, R. J., Kim, J., Esko, J. D., Lewis, N. E. and Harismendy, O. (2017). PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens. Sci Rep 7(1): 15854.
  11. Vidigal, J. A. and Ventura, A. (2015). Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries. Nat Commun 6: 8083.
  12. 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.
  13. Wegner, M., Diehl, V., Bittl, V., de Bruyn, R., Wiechmann, S., Matthess, Y., Hebel, M., Hayes, M. G., Schaubeck, S., Benner, C., Heinz, S., Bremm, A., Dikic, I., Ernst, A. and Kaulich, M. (2019). Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome. Elife 8: 42549.

简介

CRISPR / Cas系统的简单性,效率和多功能性极大地促进了其在广泛领域中的快速使用。无偏CRISPR / Cas筛选的应用正在增加,因此对无偏和定制CRISPR / Cas gRNA文库的需求日益增长。gRNA文库生成的常规方法应用PCR和克隆技术,从而使文库多样性与分布相结合。在这里,我们提供了额外的技术专长,以将我们的共价闭合环状合成(3Cs)gRNA文库生成技术应用于高质量CRISPR / Cas gRNA文库的生成。将含有复制的质粒DNA的F1来源转化为CJ236细菌,以进行单菌落生长,然后将M13KO7噬菌体进行超级感染,以生产和制备含dU的环状ssDNA。dU-ssDNA与同源性和gRNA编码的DNA寡核苷酸退火,以进行T7 DNA聚合酶介导的延伸,形成异源双链CCC-dsDNA(3Cs-dsDNA)。将3Cs-dsDNA电穿孔,用于新合成的含gRNA链的选定扩增。为了去除野生型质粒残留物,用靶向模板DNA中gRNA占位符序列的限制酶消化纯化的质粒DNA。将未消化的质粒进行电穿孔,以提取最终的3Cs gRNA文库。由于没有PCR扩增和常规的克隆步骤,因此3Cs技术将序列多样性与序列分布脱钩,从而生成了gRNA文库,其多样性接近均匀分布,仅受电穿孔效率的限制。
【背景】CRISPR / Cas基因编辑已成为定制和无偏高通量筛选的金标准(Doench,2018)。无偏见合并筛选可在单个实验中同时测试成千上万的个体遗传扰动。但是,前提条件是存在一个gRNA文库,其中每个单独的构建体都定义了一个特定的基因组靶位点(gRNA序列),在该位点上需要进行基因扰动,文库的gRNA分布和多样性决定了实验规模(Sanson等等(2018)。传统上,为阵列或汇集应用生成CRISPR / Cas gRNA文库的方案包含T4连接酶或基于重组的连接步骤,需要DNA聚合酶依赖的gRNA编码寡核苷酸的扩增以及限制性内切酶消化的质粒DNA的结合用于后续gRNA克隆( Koike-Yusa等人,2014; Shalem等人,2014; Wang等人,2014; Schmidt等人,2015; Vidigal和Ventura,2015; Arakawa,2016; Ong等人,2017)。由于这些技术限制,所得的gRNA文库在其gRNA序列分布中包含不需要的PCR和依赖克隆的序列偏倚,直接影响稳健的命中调用所需的实验规模(Shalem等人,2014; Wang等人,2014) 。公正,量身定制的CRISPR / Cas gRNA筛选正变得越来越普遍,这凸显了对生成可在非专家实验室中轻松建立的gRNA文库的新方案的需求。

我们最近描述的生成共价闭合环状合成(3Cs)gRNA的方案避免了编码gRNA的寡核苷酸以及酶消化的质粒DNA的DNA聚合酶依赖性扩增步骤(图1,Wegner et al。,2019),从而避免了PCR依赖性gRNA序列偏向的发生以及最终gRNA库中的克隆伪像,从而导致序列分布与序列多样性脱钩,从而导致gRNA库几乎均匀分布。因此,原始gRNA编码寡核苷酸库中gRNA序列的质量和分布非常重要,应特别注意仅使用高质量的寡核苷酸库。

关键字:CRISPR/Cas9, gRNA文库, 3Cs技术, 3Cs gRNA, 基因编辑, CRISPR筛选



图1.共价闭合环状(3Cs)合成的gRNA试剂。在受M13KO7感染的 dut - / ung -大肠杆菌 CJ236细菌并进行后续纯化,产生尿嘧啶核苷单链DNA(dU-ssDNA)。编码gRNA的寡核苷酸的退火,T7聚合酶的延伸以及T4连接酶的连接产生异源双链dU-dsDNA质粒文库。电穿孔进入 dut - / ung -大肠杆菌产生最终文库。编码gRNA的寡核苷酸经设计可相对于文库骨架中的I-SceI占位符位点退火5'和3',并在3Cs方案的合成步骤中充当T7聚合酶的引物。

材料和试剂

  1. 2 mm电穿孔比色杯(BTX,目录号:45-0125)
  2. 锥形离心管50 ml(Falcon,目录号:352070)
  3. 1.5 ml安全锁管(Eppendorf,目录号:0030120086)
  4. 细菌(大肠杆菌)CJ236(TaKaRa,目录号:9053或Lucigen,目录号:60701),储存-80°C
  5. 细菌(大肠杆菌)10-beta,具有电子功能(New England Biolabs,目录号:C3020K),储存-80°C
  6. 细菌(大肠杆菌)SS320(MC1061 F'),具有电子功能(Lucigen,目录号:60512)
  7. dNTP mix(Carl Roth,目录号:0178),存储-20°C
  8. T4多核苷酸激酶(PNK)(New England Biolabs,目录号:M0201),储存-20°C
  9. T4 DNA连接酶(New England Biolabs,目录号:M0202),储存-20°C
  10. T7 DNA聚合酶(未修饰)(新英格兰生物实验室,目录号:M0274),存储
    -20°摄氏度
  11. 限制性酶I-SceI(New England Biolabs,目录号:R0694),储存-80°C
  12. 限制性酶EcoRV-HF(New England Biolabs,目录号:R3195),储存-20°C
  13. 辅助噬菌体M13KO7(新英格兰生物实验室,目录号:N0315)
  14. LB琼脂肉汤(Lennox)(Sigma-Aldrich,目录号:L2897)
  15. LB中号(Lennox)(Carl Roth,目录号:X964)
  16. 2x YT介质(卡尔·罗斯,目录号:6766)
  17. SOC介质(Thermo Fisher,目录号:15544034)
  18. 聚乙二醇8000(PEG 8000)(卡尔·罗斯,目录号:263)
  19. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434-M)
  20. Dulbecco的磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8662)
  21. EZNA M13 DNA迷你试剂盒(Omega Bio-Tek,目录号:D69001-01)
  22. GeneJET凝胶提取试剂盒(Thermo Fisher,目录号:K0692)
  23. GeneJET质粒微量制备试剂盒(Thermo Fisher,目录号:K0503)
  24. DNA Clean and Concentrator-5 Kit(Zymo Research,目录号:D4013)
  25. 质粒Midi试剂盒(Qiagen,货号:12143)
  26. 质粒最大试剂盒(Qiagen,目录号:12163或Promega,目录号:12163)
  27. 氨苄西林钠盐(Amp)(Carl Roth,目录号:K0292)
  28. 氯霉素(Cmp)(卡尔·罗斯,目录号:3886)
  29. 硫酸卡那霉素(Kan)(Carl Roth,目录号:T832)
  30. 腺苷5'-三磷酸(ATP)(新英格兰生物实验室,目录号:P0756),存储
    -20°摄氏度
  31. 二硫苏糖醇(DTT)(Cell Signaling Technology,目录号:7016),储存-20°C
  32. 琼脂糖,标准品(Applichem,目录号:732-2789)
  33. SYBR Safe DNA凝胶染料(Thermo Fisher,目录号:S33102)
  34. KCl(卡尔·罗斯,目录号:P017)
  35. CaCl 2 (Carl Roth,目录号:CN93)
  36. MgCl 2 (卡尔·罗斯,目录号:KK36)
  37. Tris base(卡尔·罗斯,目录号:A411)
  38. Tris-HCl(Carl Roth,目录号:9090)
  39. EDTA(Applichem,目录号:A5097,0500)
  40. 乙酸(Sigma-Aldrich,目录号:33209)
  41. KCM缓冲区(请参阅食谱)
  42. 20%PEG / NaCl(请参阅食谱)
  43. 10x TM缓冲区(请参阅食谱)
  44. 50x TAE缓冲区(请参阅食谱)

设备

  1. 细菌摇床(Eppendorf,型号:New Brunswick Innova 44 / 44R,目录号:M1282-0012;或类似编号)。
  2. Gene Pulser电穿孔系统(Bio-Rad,目录号:164-2076,或用于细菌电穿孔的类似系统)。
  3. Mini-sub cell GT水平电泳系统,7 x 10 cm(Bio-Rad,目录号:1704467或类似产品)。
  4. 离心Avanti JE(贝克曼,目录号:369003或类似产品)。
  5. 转子JA-12(定角铝转子-12 x 50毫升,12,000 rpm,23,200 x g ,Beckman,目录号:360993)。
  6. NanoDrop(Thermo Fisher,目录号:ND-1000或类似产品)

软件

  1. bCl 2 fastq(Illumina, https://support.illumina.com/ )
  2. cutadapt( https://cutadapt.readthedocs.io/en/stable/ ;马丁,2011年)
  3. Bowtie2( http://bowtie-bio.sourceforge.net/bowtie2/index.shtml; Langmead和Salzberg,2012年)
  4. PinAPL-Py( http://pinapl-py.ucsd.edu/ ; Spahn et等,2017)

程序

  1. 质粒单链DNA(ssDNA)的制备
    1. 将100-200 ng质粒DNA转化为25-50μl感受态E。大肠杆菌CJ236细菌。使用热休克(1分钟,42°C,加热块)或KCM缓冲液(使用相同体积的细菌和5x KCM缓冲液以1:5的比例稀释的混合物,用水,冰/ 15分钟,室温/ 10分钟)进行转化。添加50-200μlSOC培养基,并在含有25μg/ ml Cmp和其他抗生素匹配质粒抗性(通常为100μg/ ml Amp *,取决于质粒)的LB平板上平板,在37°C下放置过夜。
      *注:从理论上讲,可以使用任何包含f1-origin复制的质粒,例如lentiCRISPR v2(Addgene 52961)。该质粒应具有Amp,Cmp和Kan以外的抗生素抗性基因。
    2. 第二天,挑选几个 E单菌落。带有无菌移液器吸头的CJ236,重悬于1 ml的2x YT培养基中,其中添加了Amp *(100μg/ ml),Cmp(25μg/ ml)和1μl辅助噬菌体。摇动2 h(37°C,200 rpm); 加入100μg/ ml Kan并继续振摇6-10 h(37°C,200 rpm)。
    3. 将1 ml初始培养物转移到30 ml的含有Amp *(100μg/ ml)和Kan(100μg/ ml)的2x YT培养基中,并摇动16-20 h(37°C,200 rpm)。
    4. 将30 ml细菌培养物转移到50 ml Falcon管中;离心(10分钟,12,000 x g ,4°C)。
    5. 将含有噬菌体的上清液(图2A)转移到6 ml无菌过滤的PEG / NaCl缓冲液中,颠倒4-6次,在室温下孵育30-60分钟;离心(4分钟,在12,000 x g 下10分钟)。
    6. 倒出上清液并吸出剩余的上清液。
    7. 在1 ml 1x PBS中重悬噬菌体沉淀(图2B),并转移到新的1.5 ml反应管中。


      图2.从细菌培养物中制备ssDNA。 E。离心前(A)和离心后(B)进行大肠杆菌CJ236细菌培养(丸中含有细菌,而上清液中含有噬菌体)。C. PEG / NaCl沉淀并随后离心后的噬菌体沉淀。D.噬菌体重悬于1x PBS缓冲液(透明溶液)中。E.加入ssDNA裂解缓冲液(混浊溶液,浊度反映噬菌体的数量)。

    8. 以16,100 x g 离心5分钟以去除细胞碎片,将上清液转移至新的1.5 ml反应管中并在4°C下储存。
    9. 根据Omega Bio-Tek EZNA M13 DNA Mini Kit方案从噬菌体上清液中纯化ssDNA。
    10. 使用NanoDrop确定ssDNA浓度。
    11. 使用500 ng ssDNA和250 ng相应的dsDNA在0.8%TAE /琼脂糖凝胶中分析ssDNA。ssDNA应该显示为主要的单条带(图3A)。


      图3. ssDNA和3Cs反应产物的质量控制。 A。ssDNA在琼脂糖凝胶上显示为一条条带,通常比dsDNA质粒对应物具有更高的电迁移率。#1,#2和#3是指不同大小的示例质粒。低质量的ssDNA会显示多个条带,表明噬菌体衍生的重组产物会导致3C期间的性能下降(!)。B. 3Cs反应产物通常在琼脂糖凝胶上显示三个条带。所有三种电泳均在110 V的0.8%1x TAE琼脂糖上电泳30分钟。

  2. T4多核苷酸激酶将寡核苷酸磷酸化
    在1.5 ml反应管中,将600 ng寡核苷酸池与2μl10x TM缓冲液,1 mM ATP,5 mM DTT和20单位T4多核苷酸激酶合并,总体积为20μl;在37°C下孵育2小时。
    注意:寡核苷酸库可从各种商业渠道订购。手工汇集的寡核苷酸通常会导致文库的gRNA分布变化很大,因此应避免使用。

  3. 寡核苷酸与模板的退火
    将20μg新鲜制备的ssDNA与25μl10x TM缓冲液(终浓度1x TM)和20μl磷酸化寡核苷酸混合,总体积为250μl;在热块中孵育如下:90°C(3分钟),50°C(5分钟),室温(5分钟)。
    注意:寡核苷酸和载体ssDNA之间的摩尔比应通过实验确定,对于特定质粒可能有所不同。通常,3:3、5:1和7:1的比率范围对于3Cs反应是最佳的。

  4. 酶法合成3Cs-dsDNA
    将以下成分添加到之前的混合物中:10μl的10 mM ATP,10μl的100 mM dNTP,15μl的100 mM DTT,3μlT4 DNA连接酶(库存:400,000单位/ ml)和5μlT7 DNA聚合酶(库存:10,000单位/毫升); 上下吹打或轻弹试管小心混合;在22°C下孵育16小时。

  5. 使用DNA Clean和Concentrator-5试剂盒纯化3Cs-dsDNA
    1. 将3Cs反应液与2x体积的DNA结合缓冲液混合,并在室温下孵育10分钟。
    2. 将3Cs反应分为2根色谱柱。
    3. 降速旋转(1分钟,2,300 x g ,室温);将每个流重新旋转两次。
    4. 用200μl洗涤缓冲液洗涤两次,每次两次(1分钟,2,300 x g ,室温)。
    5. 丢弃流通液并干燥色谱柱(1分钟,室温7,400 x g )。
    6. 将柱子与预热的10μl洗脱缓冲液(10分钟,50°C)一起孵育,以最大速度旋转(2分钟),将每个流重新旋转两次。
    7. 用20μl流通液重复洗脱(最终体积约为40μl)。
    8. 运行凝胶以控制dsDNA和ssDNA的质量(图3B)。
    9. 通过Nanodrop测定浓度。

  6. 有电能力的Eem的电穿孔。3Cs dsDNA的大肠杆菌细菌
    1. 在小锥形瓶中,在37°C下准备20 ml预热的SOC介质。
    2. 在冰上孵育整个纯化的3Cs dsDNA和2 mm电穿孔比色杯。
    3. 解冻400μl电活性E. 冰上的大肠杆菌 菌株 SS320或NEB 10-beta细菌(20分钟)。
    4. 将细胞添加到预冷并纯化的3Cs DNA中,并通过移液缓慢混合,避免产生气泡。
    5. 对于电穿孔,请使用以下设置:2.5 kV场强,25 µF电容,200欧姆电阻(最佳持续时间:3.8-4.2 ms)。
    6. 将干燥的比色皿放入电穿孔仪支架中。
    7. 将混合物轻轻地(无气泡)转移到比色皿中,然后轻按比色皿(避免气泡引起电弧!)。
    8. 拧紧支架,将盖子盖在比色皿上。
    9. 电穿孔:立即按下电源上的两个按钮,一旦蜂鸣声开始(通常3.8-4.2毫秒)就松开。
    10. 通过在2x 1 ml预热的SOC培养基中冲洗,立即拯救电穿孔的细胞。任何延迟将显着降低电穿孔效率。
    11. 将锥形瓶(含20 ml SOC培养基和电穿孔细菌)孵育30分钟(37°C,140 rpm)。
    12. 取出40μl细菌培养物,准备制备系列稀释液。
      注意:在96孔板中,每个文库用90μl1x PBS制备3列(即3 x 8孔)。向最初的90μl1x PBS(一式三份制备)中加入10μl细菌培养物,用于随后的系列稀释(1:10)。在Amp * LB平板上平板接种5μl稀释的细菌,在37°C下孵育过夜。计算文库多样性的方法如下:在稀释液中计数菌落,在其中可以清楚看到单独的菌落,并计算每1μl稀释的细菌培养物的平均菌落数。知道细菌培养物的最终体积(20.4 ml)以及从中计算菌落的稀释度(例如,在96孔板中的第3道稀释度对应于103稀释度),计算文库多样性:多样性=(细菌菌落数在1μl稀释细菌培养物中)x 10(稀释)x细菌培养体积(μl)。
    13. 将小锥形瓶的剩余内容物转移到180 ml预热的2x YT培养基(含100μg/ ml Amp *)中,振荡过夜(37°C,140 rpm)。
    14. 第二天,从5毫升的过夜培养物中制备微量制备物,并用其检查文库质量(请参阅步骤G1-G3)。
    15. 旋转剩余的培养物(4°C,6,000 x g ,20分钟)以进行随后的Midi准备。

  7. 限制酶分析图书馆前库
    1. 用适当的限制酶消化0.5μg的文库dsDNA; 始终将原始载体也用作切割控制。
    2. 将未消化和消化的文库前和原始载体上样至0.5-0.8%1x TAE琼脂糖凝胶中。
    3. 检查预图书馆的质量,并将其与原始载体进行比较(图4)。


      图4.从文库制备前除去野生型残留物和最终文库的质量控制。野生型质粒,文库和最终文库的限制性酶切表明比例每个步骤中检测野生型和3Cs质粒。作为对照,将野生型质粒(wt质粒)用I-SceI或EcoRV线性化。当两种酶都切割质粒时,两种酶的双重消化会去除大约2500 bp的片段(!)。在图书馆前库中,I-SceI的不完全消化和双重消化产生的微弱带表明大多数DNA是3Cs产物。双重消化揭示了一条〜2,500 bp的条带,其强度明显低于野生型质粒库中的条带。在最终的文库中,I-SceI摘要没有发现可见的线性化片段。双酶切消化使文库线性化,在〜2,500 bp处无可见条带,表明最终文库不含野生型质粒。在0.8%1x TAE琼脂糖上于110 V电泳30分钟。

  8. 最终电穿孔前对文库的消化
    1. 用适当的酶消化10-20μg的预库并通过Clean& 集中器套件。
    2. 如步骤F1-F12所述,将5-10μg裂解的文库转化并处理为400μl具有电感受态的细菌。
    3. 在37°C下生长200-1,000 ml的2x YT-Amp *(100 µg / ml)细菌培养物(体积取决于文库大小,未来需求和剩余寡核苷酸的量)。
    4. 第二天旋转细菌(根据您使用的最大制备试剂盒和文库大小,从50、100或150 ml细菌培养物中制备多个细菌沉淀等分试样)。
    5. 用一种细菌沉淀物制备最大量的制备物,将其余的沉淀物保持在-20°C直至需要。

  9. 用限制酶分析最终文库
    遵循步骤G1-G3(图4)。

  10. 通过Sanger测序对最终文库进行序列分析
    发送适当量(通常为700-1,200 ng)的最终文库用于Sanger测序。选择靠近您的gRNA盒的测序引物,例如U6引物。代表性数据如图5所示。


    图5.最终库的SANGER测序。作为第一步质量控制步骤,使用U6-primer对最终库的SANGER测序揭示了I-SceI占位符侧的随机性。在此示例中,从Wegner等(2019)对最终优化的真正全基因组全文库(oTGW)的消化会产生类似于该文库预期序列组成的随机核苷酸模式。 >
  11. 下一代测序(NGS)对最终文库指南分布的序列分析
    有关数据处理和数据分析的详细说明,请参阅Wegner等人(2019年),以下是对过程的简要概述。

数据分析

有关数据处理和数据分析的详细说明,请参见Wegner等人,,2019年。为了质量控制3Cs试剂,使用条形码扫描时,可以对几种不同质粒文库进行多重下一代测序(NGS) Illumina适配器序列。可以使用Illumina的bCl 2 fastq对NGS原始数据进行多路分解,最好使用最新版本。要计算单个库的读数,请使用诸如PinAPL-Py(Spahn et al。,2017年)之类的指定工具,或设计定制的管道,根据您的需要执行读取修整和序列比对,例如,cutadapt和bowtie2。
可以使用cutadapt通过将读数修整为20个gRNA编码核苷酸来进行衔接子修整(Martin,2011,https://cutadapt.readthedocs.io/en/stable/)。然后,可以使用bowtie2或自定义脚本执行将修饰的读段与库进行比对。最终文库应包含所有所需的gRNA序列。为了评估库的分布,根据(Wegner等人,2019)(图6A)生成gRNA表示的洛伦兹曲线并计算所得曲线下的面积。这将允许您比较不同的库。gRNA读取频率的直方图和计算90%和10%百分位数之间的比率揭示了文库的偏斜(图6B)。对于基因扰动筛选的分析,我们参考相关文献作为实验设置,并读出确定分析技术的方法。


图6. NGS分析和预期序列分布的可视化。A.三个gRNA库的gRNA计数以Lorenz图的形式可视化,显示了代表的NGS读数相对于按丰度排序的gRNA的累积分数。理想分布将是均匀的,并且类似于对角线,曲线下的面积(AUC)为0.5(绿色曲线,理想)。分布良好的文库包含每个预期的gRNA序列,并且展示的AUC与0.5仅略有不同(蓝色曲线,良好)。AUC值增加表示库分布不均匀(橙色曲线,不良)。请注意,该曲线的斜率在x = 1.0附近减小至0,表明该文库中缺少gRNA序列。B.从(A)中绘制gRNA分布,作为直方图可视化文库的偏斜或对称性。分布良好的库在1(蓝色,良好)附近显示低偏斜,表示对称。倾斜的库是不对称分布的。坏的例子偏向于更高的gRNA读取计数,如直方图中的长右尾所示(橙色,坏)。

笔记

  1. 寡核苷酸的长度通常为55-65个核苷酸,在中间具有gRNA编码区,在其5'和3'末端具有质粒序列同源区(3Cs同源性)。对于3Cs反应,18个核苷酸的同源性(在gRNA的每一侧)和高于45°C的退火温度是可选的。寡核苷酸的两端均不应有任何修饰,因为连接效率会降低。
    重要提示:模板质粒必须包含复制的f1来源。 E。大肠杆菌 CJ236菌株对Cmp具有抗性并且是dut - / ung -。
  2. 悬浮在PBS中的噬菌体可以在4°C下保存几个月。
  3. 一旦分离,ssDNA可以稳定几天或几周。我们建议使用新鲜制备的ssDNA。
  4. 为了确认您的ssDNA的质量,您可以将1μl的噬菌体制备液与具有KCM功能的细菌和200μlSOC培养基混合。在37°C下孵育1小时,然后稀释到5 ml LB培养基中,并补充Amp *(100μg/ ml)。摇动过夜(37°C,200 rpm),使用微型制备试剂盒分离dsDNA,并使用与步骤(步骤G1-G3)相同的方法分析dsDNA。使用原始质粒dsDNA作为阳性对照(比较琼脂糖凝胶的大小和限制模式)。
  5. 仅将新鲜磷酸化的寡核苷酸用于3C反应。
  6. 高质量的电子能力 E。还可以在实验室中制备大肠杆菌 菌株 SS320或NEB 10-beta细菌。
  7. dut + / ung + 大肠杆菌中含dU的DNA去除效率不是100%,因此对在将引入gRNA的质粒区域(gRNA占位符序列)中引入限制位点(例如I-SceI)。这样的方法可确保在通过I-SceI进行文库切割和随后的电穿孔后,所有原始序列(仍包含I-SceI位点)都将被清除。重要的是,请确保限制性识别序列与目标基因组中的活性gRNA序列不相似,以避免意外的编辑事件。包含I-SceI限制性酶切位点的指导序列可安全用于人类基因组。还要确保限制位点不是文库中任何gRNA序列的一部分,以避免意外地从最终库中去除gRNA。
  8. 对于pLentiGuide(Addgene 52963)和pLentiCRISPRv2(Addgene 52961)质粒,请使用正向引物(5'-GGGCCTATTTCCCATGATTCCTTCATATTTGC-3'),该引物与U6-启动子结合以进行Sanger测序。

菜谱

  1. KCM缓冲区
    注意:KCM渲染 dut- / ung- E。大肠菌 CJ236细胞。
    0.5 M氯化钾
    0.15 M CaCl 2
    0.25 M MgCl 2
  2. 20%PEG / NaCl
    注意:PEG / NaCl用于沉淀含有dU-ssDNA的噬菌体颗粒。
    20%聚乙二醇
    2.5 M氯化钠
  3. 10x TM缓冲区
    注意:在3Cs方案的磷酸化步骤中,与ATP和DTT一起用作多核苷酸激酶T4的反应缓冲液。
    0.1 M MgCl 2
    0.5 M Tris-HCl
    pH值7.5
  4. 50x TAE缓冲区
    2 M Tris基地
    0.5 M EDTA
    1 M乙酸

致谢

感谢Alkmini Kalousi提供有关细菌和噬菌体沉淀物的图像。我们还要感谢法兰克福CRISPR / Cas筛选中心(FCSC)和Kaulich实验室的所有成员为该协议所源自的原始研究论文做出了贡献(Wegner et al。,2019)。这项工作得到了黑森州科学技术部(HMWK,LOEWE-CGT,IIIL5-518 / 17.004),德国研究基金会(DFG; CEF-MC-EXC115 / 2; ECCPS-EXC147 / 2)和部分由黑森州立高等教育,研究和艺术部资助的LOEWE中心法兰克福癌症研究所(FCI)(IIIL5-519 / 03 / 03.001-(0015))。

利益争夺

法兰克福歌德大学已提交了与此工作相关的专利申请,Martin Wegner和Manuel Kaulich是其发明者(WO2017EP84625)。歌德大学向Vivlion GmbH提供3Cs技术的独家许可,Manuel Kaulich是其共同创始人,股东和首席科学官。

参考文献

  1. 荒川(2016)。一种将mRNA转换为gRNA库的方法,以便对任何生物进行CRISPR / Cas9编辑。 Sci Adv 2(8):e1600699。
  2. Doench,JG(2018)。我准备好接受CRISPR吗?遗传筛选用户指南。 Nat Rev Genet 19(2):67-80。
  3. Koike-Yusa,H.,Li,Y.,Tan,EP,Velasco-Herrera Mdel,C.和Yusa,K.(2014)。使用慢病毒CRISPR-guide RNA库在哺乳动物细胞中进行全基因组隐性遗传筛选。 Nat Biotechnol 32(3):267-273。
  4. Langmead,B.和Salzberg,SL(2012)。与Bowtie 2的快速阅读缺口比对。 自然方法 9(4):357-359。
  5. 马丁,米(2011)。 Cutadapt从高通量测序读段中除去了适配器序列。 EMBnet J 17(1):10-12。
  6. Ong,SH,Li,Y.,Koike-Yusa,H.和Yusa,K.(2017)。带有第二代gRNA库的CRISPR-KO筛选的优化指标。 科学(Sci Rep) 7(1):7384。
  7. Sanson,KR,Hanna,RE,Hegde,M.,Donovan,KF,Strand,C.,Sullender,ME,Vaimberg,EW,Goodale,A.,Root,DE,Piccioni,F.和Doench,JG(2018) 。具有多种模式的CRISPR-Cas9遗传筛选的优化文库。 Nat Commun 9(1):5416。
  8. Schmidt,T.,Schmid-Burgk,JL和Hornung,V.(2015)。合成了针对人类基因组的阵列化sgRNA文库。 Sci Rep < 5:14987。
  9. O.Shalem,NE,Sanjana,NE,Hartenian,Shi,X.,Scott,DA,Mikkelson,T.,Heckl,D.,Ebert,BL,Root,DE,Doench,JG和Zhang,F.( 2014)。在人类细胞中进行基因组规模的CRISPR-Cas9基因敲除筛选。 Science < 343(6166):84-87。
  10. Spahn,PN,Bath,T.,Weiss,RJ,Kim,J.,Esko,JD,Lewis,NE and Harismendy,O.(2017年)。 PinAPL-Py:用于分析CRISPR / Cas9筛选的综合网络应用。 Sci Rep 7(1):15854。
  11. Vidigal,JA and Ventura,A.(2015年)。快速,高效地一步生成配对的gRNA CRISPR-Cas9文库。 Nat Commun 6:8083。
  12. Wang,T.,Wei,JJ,Sabatini,DM and Lander,ES(2014)。 使用CRISPR-Cas9系统在人细胞中进行基因筛选。 Science < 343(6166):80-84。
  13. Wegner,M.,Diehl,V.,Bittl,V.,de Bruyn,R.,Wiechmann,S.,Matthess,Y.,Hebel,M.,Hayes,MG,Schaubeck,S.,Benner,C., Heinz,S.,Bremm,A.,Dikic,I.,Ernst,A. and Kaulich,M.(2019年)。环状合成CRISPR / Cas gRNA,用于编码和非编码基因组中的功能询问。 Elife 8:42549.
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Wegner et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wegner, M., Husnjak, K. and Kaulich, M. (2020). Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA. Bio-protocol 10(1): e3472. DOI: 10.21769/BioProtoc.3472.
  2. Wegner, M., Diehl, V., Bittl, V., de Bruyn, R., Wiechmann, S., Matthess, Y., Hebel, M., Hayes, M. G., Schaubeck, S., Benner, C., Heinz, S., Bremm, A., Dikic, I., Ernst, A. and Kaulich, M. (2019). Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome. Elife 8: 42549.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。