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DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis
利用CRISPR技术对莱茵衣藻进行DNA-free的基因组编并对产生突变进行分析   

Jihyeon  YuJihyeon Yu*Kwangryul  BaekKwangryul Baek*EonSeon  JinEonSeon Jin Sangsu  BaeSangsu Bae  (*contributed equally to this work)
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Abstract

We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow covers from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.

Keywords: Genome editing(基因组编辑), CRISPR-Cas9( CRISPR-Cas9), Microalgae( 微藻), Ribonucleoproteins( 核糖核蛋白), Chlamydomonas reinhardtii( 莱茵衣藻), DNA-free transformation( DNA-free转化)

Background

We recently reported (Baek et al., 2016) a one-step transformation of the model green microalga Chlamydomonas reinhardtii (Harris, 2001) using preassembled Cas9 protein-guide RNA ribonucleoproteins (RNPs). The manner of DNA-free CRISPR-Cas9 delivery has several advantages such as no need for codon optimization and specific promoters in different species of microalgae. Furthermore, it reduces off-target effects and may also be less cytotoxic in cells because the Cas9 protein is transiently active and then degraded by endogenous proteases in cells (Kim et al., 2014). In addition, the resulting gene-edited microalgae could be exempt from genetically modified organism (GMO) regulations due to the absence of foreign DNA sequences. In this protocol, the detailed procedures of an entire workflow is contained from the initial target selection of CRISPR to the mutant analysis using NGS technology (Bae et al., 2014a and 2014b; Park et al., 2015 and 2017).

Materials and Reagents

  1. Cas9 protein purification
    1. 1.5 ml Eppendorf tubes
    2. Sterile 50 ml conical tubes
    3. 10 ml syringe
    4. 0.45 μm syringe filter
    5. Protein concentrator–100K MWCO (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88533 )
    6. Poly-Prep Chromatography columns (Bio-Rad Laboratories, catalog number: 7311550 )
    7. BL21-Pro cells
    8. pET28 plasmid containing 6xHis-SpCas9 (Addgene)
    9. Kanamycin
    10. Lysozyme
    11. PMSF
    12. Ni-NTA agarose beads (QIAGEN, catalog number: 30210 )
    13. Bradford reagent (Bio-Rad Laboratories, catalog number: 5000205 )
    14. Bovine serum albumin (BSA)
    15. Sodium chloride (NaCl)
    16. Tryptone
    17. Yeast extract
    18. IPTG
    19. Sodium phosphate monobasic (NaH2PO4)
    20. Imidazole
    21. Sodium hydroxide (NaOH)
    22. HEPES
    23. Ethylenediaminetetraacetic acid (EDTA)
    24. DL-dithiothreitol (DTT)
    25. Sucrose
    26. Glycerol
    27. LB medium (see Recipes)
    28. 1 M IPTG stock (see Recipes)
    29. Lysis buffer (see Recipes)
    30. Wash buffer (see Recipes)
    31. Elution buffer (see Recipes)
    32. Cas9 storage buffer (see Recipes)

  2. In vitro transcription and library PCR
    1. 1.5 ml Eppendorf tubes
    2. Forward and reverse oligos (see Tables 1 and 2)

      Table 1. Pre-index primers
      Pre-index
      forward primer
      5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT gDNA target-3’
      Pre-index
      reverse primer 
      5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT gDNA target-3’

      Table 2. Index primer sequences

      index
      sequence [i5]
      Index forward primer sequence
      D501
      tatagcct
      AATGATACGGCGACCACCGAGATCTACACtatagcctACACTCTTTCCCTACACGAC
      D502
      atagaggc
      AATGATACGGCGACCACCGAGATCTACACatagaggcACACTCTTTCCCTACACGAC
      D503
      cctatcct
      AATGATACGGCGACCACCGAGATCTACACcctatcctACACTCTTTCCCTACACGAC
      D504
      ggctctga
      AATGATACGGCGACCACCGAGATCTACACggctctgaACACTCTTTCCCTACACGAC
      D505
      aggcgaag 
      AATGATACGGCGACCACCGAGATCTACACaggcgaagACACTCTTTCCCTACACGAC
      D506
      taatctta
      AATGATACGGCGACCACCGAGATCTACACtaatcttaACACTCTTTCCCTACACGAC
      D507
      caggacgt
      AATGATACGGCGACCACCGAGATCTACACcaggacgtACACTCTTTCCCTACACGAC
      D508
      gtactgac
      AATGATACGGCGACCACCGAGATCTACACgtactgacACACTCTTTCCCTACACGAC

      index
      sequence [i7]
      Index reverse primer sequence
      D701
      cgagtaat
      CAAGCAGAAGACGGCATACGAGATcgagtaatGTGACTGGAGTTCAGACGTGT
      D702
      tctccgga
      CAAGCAGAAGACGGCATACGAGATtctccggaGTGACTGGAGTTCAGACGTGT
      D703
      aatgagcg
      CAAGCAGAAGACGGCATACGAGATaatgagcgGTGACTGGAGTTCAGACGTGT
      D704
      ggaatctc
      CAAGCAGAAGACGGCATACGAGATggaatctcGTGACTGGAGTTCAGACGTGT
      D705
      ttctgaat
      CAAGCAGAAGACGGCATACGAGATttctgaatGTGACTGGAGTTCAGACGTGT
      D706
      acgaattc
      CAAGCAGAAGACGGCATACGAGATacgaattcGTGACTGGAGTTCAGACGTGT
      D707
      agcttcag
      CAAGCAGAAGACGGCATACGAGATagcttcagGTGACTGGAGTTCAGACGTGT
      D708
      gcgcatta
      CAAGCAGAAGACGGCATACGAGATgcgcattaGTGACTGGAGTTCAGACGTGT
      D709
      catagccg
      CAAGCAGAAGACGGCATACGAGATcatagccgGTGACTGGAGTTCAGACGTGT
      D710
      ttcgcgga 
      CAAGCAGAAGACGGCATACGAGATttcgcggaGTGACTGGAGTTCAGACGTGT
      D711
      gcgcgaga
      CAAGCAGAAGACGGCATACGAGATgcgcgagaGTGACTGGAGTTCAGACGTGT
      D712
      ctatcgct
      CAAGCAGAAGACGGCATACGAGATctatcgctGTGACTGGAGTTCAGACGTGT

    3. dNTP mix
    4. Phusion DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F530L )
    5. PCR purification kit
    6. ATP, CTP, GTP, UTP, 100 mM MgCl2, DEPC-treated water
    7. T7 RNA polymerase (New England Biolabs, catalog number: M0251L )
    8. DNase I (RNase-free) (New England Biolabs, catalog number: M0303L )
    9. RNase inhibitor murine (New England Biolabs, catalog number: M0314L )
    10. RNeasy MinElute Cleanup Kit (QIAGEN, catalog number: 74204 )
    11. Illumina Miseq Reagent Kit (v2) 

  3. Transfection
    1. 1.5 ml Eppendorf tubes
    2. Sterile 15 ml and 50 ml conical tubes
    3. Purified Cas9 protein and sgRNA
    4. Chlamydomonas reinhardtii strains CC-4349 cw15 mt-(Chlamydomonas Resource Center)
    5. PCR DNA purification kit (MG Med, catalog number: MD008 )
    6. TAP medium (Harris, 1989 or Thermo Fisher Scientific, GibcoTM, catalog number: A1379801 )
    7. TAP sucrose solution (see Recipes)
    8. TAP agar plates (see Recipes)
    9. Top agar (see Recipes)

Equipment

  1. Cas9 protein purification
    1. Centrifuge: swing rotor (LaboGene, model: 1580R )
    2. Sonicators (Qsonica, model: Q125 )
    3. Pipettes
    4. Shaking incubator (JS Research, model: JSSI-300C )

  2. In vitro transcription and library PCR
    1. Incubator (JS Research, model: JSGI-050T )
    2. Thermal cycler (Bio-Rad Laboratories, model: C1000 TouchTM Thermal Cycler )
    3. Micro centrifuge (LaboGene, model: 1730R )
    4. Spectrophotometer

  3. Transfection
    1. 100 ml flask
    2. Spectrophotometer (GE Healthcare, Amersham Biosciences, model: Ultrospec 2100 pro )
    3. Hemocytometer (Marienfeld-Superior, catalog number : 0650030 )
    4. Microscope (Olympus, model: CH30 )
    5. Micro high speed centrifuge (Hanil, model: MICRO 17TR )
    6. Orbital shaker (N-biotek, model: NB-101M )
    7. Clean bench (BioFree, model: BF-150BSC )
    8. Electroporation device (Bio-Rad Laboratories, model: Gene Pulser XcellTM Electroporation Systems )
    9. Electroporation cuvettes (Bio-Rad Laboratories, 0.4 cm gap)

Procedure

  1. CRISPR target selection for creating gene knock-outs of interest
    1. Obtain the coding sequence (CDS) of the gene of interest from an appropriate database. We use Phytozome from JGI (https://phytozome.jgi.doe.gov/pz/portal.html#) (Figure 1).


      Figure 1. Main page of Phytozome: The plant genome database. The coding sequence information of each gene can be obtained from Phytozome database.

    2. For target design, we use CRISPR RGEN tools (http://www.rgenome.net/), a web-based set of tools for CRISPR target design and next-generation sequencing (NGS) data analysis (Figure 2).


      Figure 2. Main page of CRISPR RGEN Tools. A web-based set of tools which provides all required tools from CRISPR target design to NGS data analysis.

    3. In Cas-Designer, select the type of CRISPR endonuclease, such as SpCas9 from Streptococcus pyogenes, and the target genome, in this case that of the model organism Chlamydomonas reinhardtii. Cas-Designer now contain the versions 4 and 5 of it at ‘Plants’ organism type on our website. Then insert the CDS of the gene of interest in the ‘Target Sequence’ box. We recommend choosing target sites within the first half of the full CDS to increase the probability of generating complete knock-out mutations (Figure 3).


      Figure 3. Overview of Cas-Designer. Users can select various types of CRISPR endonucleases and the target genome including Chlamydomonas reinhardtii (versions 4 or 5).

    4. Choose several targets among the candidates from Cas-Designer. You should avoid targets that are associated with potential off-target sites bearing 1-2 mismatches compared to the on-target. Targets with GC content ranging from 30 to 70% and higher out-of-frame scores are recommended (Figure 4).


      Figure 4. An example data of an output table from Cas-Designer. Cas-Designer shows possible CRISPR target sites from input sequences along with useful information such as GC content, out-of-frame score, and off-target information.
      Note: N/A means ‘not applicable’ due to the lack of flanking DNA nucleotides.

  2. Purification of recombinant Cas9 protein
    1. Transform 50 μl competent BL21-Pro cells with a pET28 vector containing 6xHis-SpCas9.
    2. Spread the cells on an agar plate containing kanamycin. Incubate the plate overnight at 37 °C.
    3. Inoculate 10 ml LB medium containing 50 μg/ml kanamycin in a conical tube with a single colony from the plate. Grow to saturation overnight at 37 °C with shaking at 200 rpm.
    4. The next morning, inoculate 200 ml of LB medium containing kanamycin in a flask with 2 ml saturated overnight culture.
    5. Grow the cells at 37 °C with shaking at 200 rpm to mid-log phase (to an OD600 of approximately 0.4-0.5).
    6. Add 160 μl of 1 M IPTG (0.8 mM final concentration) and incubate the cells overnight at 18 °C with shaking at 200 rpm.
    7. Transfer the culture to a 50 ml conical centrifuge tube and pellet the cells by centrifuging for 20 min at 3,000 x g and 4 °C. Discard the supernatant and repeat the harvest in the same tube.
    8. Re-suspend the cell pellets in 10 ml lysis buffer (see Recipes).
    9. Add 20 mg lysozyme and 100 μl of 100 mM PMSF (1 mM final concentration).
    10. Incubate 1 h on ice.
    11. Sonicate the cells in a conical tube on ice (amplitude 25%, 1 sec ON/1 sec OFF, pulse 30 times). Repeat the sonication process 5 times.
    12. Transfer the sonicated cells to a 1.5 ml microtube using micropipette and pellet debris by centrifuging at 4 °C, 11,000 x g for 30 min.
    13. Filter the supernatant through a 0.45-μm syringe filter and collect it in a 50 ml conical tube.
    14. Add 2 ml Ni-NTA agarose to the lysate. Swirl agarose beads before adding. Incubate for 1 h with rotation at 4 °C.
    15. Transfer the mixture to a chromatography column with pipette. Collect the flow-through in a conical tube.
    16. Wash the column with 10 ml wash buffer (see Recipes). Collect this first batch of wash buffer in a conical tube.
    17. Repeat the wash. Collect the second batch of wash buffer in a conical tube.
    18. Elute the bound protein with 10 ml elution buffer (see Recipes). Collect the eluate in 1 ml sequential aliquots in 10 microtubes.
    19. Using a Bradford assay, determine the protein concentration in each fraction.
    20. Apply the eluate that contains the fusion protein to a protein concentrator (100K MWCO).
    21. Centrifuge at 4 °C, 3,000 x g until the sample volume is reduced by 90%.
    22. Apply 4 ml protein storage buffer (see Recipes) and centrifuge at 4 °C, 3,000 x g, until the volume is reduced by 90%.
    23. Elute the protein on the filter with 300 μl protein storage buffer.
    24. Measure the protein concentration through Bradford assay with standard BSA in the spectrophotometer (the final concentration of SpCas9 should be approximately ~10 mg/ml).
    Notes:
    1. Identify which samples from each step contain the fusion protein by SDS-PAGE.
    2. You should verify the activity of Cas9 protein using an in vitro cleavage assay.

  3. In vitro transcription of sgRNA
    1. Template extension


      After template extension, purify DNA using a PCR purification kit.
      Note: Purified DNA template can be stored at -20 °C.

      Table 3. Oligos for in vitro transcription
      Oligo F
      (target specific)
      5’-GAAATTAATACGACTCACTATAG (target 20 nt - 5’ to 3’ direction, without PAM) GTTTTAGAGCTAGAAATAGCAAG-3’
      Oligo R
      (common )
      5’-AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGAC
      TAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3’

    2. In vitro transcription reaction


      4 h-overnight reaction, 37 °C water bath or incubator.
    3. Removal of DNA template
      1. Add 11 μl DNase I buffer
      2. Add 1 μl (2 U/μl) DNase I in 100 μl reaction
      3. Incubate at 37 °C, 30 min
    4. RNA purification
      1. Use an RNA cleanup kit following the manufacturer’s manual.
      2. Elute RNA in 10-25 μl DEPC treated water or RNase-free water. The final RNA concentration should be 5-15 μg/μl.
      Note: RNA should be stored at -80 °C until the Chlami cells ready.

  4. Transfection of ribonucleoproteins (RNPs) to Chlamydomonas
    1. Preparation of cells and RNP complex for transformation
      1. Cultivate the Chlamydomonas cells mixotrophically (in the light with acetate) in 50 ml liquid TAP medium at 25 °C under continuous light (50-70 μmol photons m-2 sec-1) in a 100 ml flask with shaking at 90 rpm until the cell density reaches an OD750 of 0.3-0.5 as measured by a spectrophotometer.
      2. Estimate the cell density with a hemocytometer and microscope.
      3. Harvest 5 x 105 cells by centrifugation in the 1.5 ml Eppendorf tubes (2,000 x g, 3 min, room temperature), discard the supernatant, and resuspend the cells in 250 μl of TAP sucrose solution (see Recipes) by gently pipetting (prepare one aliquot of cells per transformation reaction).
      4. To prepare the RNP complex, premix purified Cas9 protein (200 μg) with in vitro transcribed sgRNA (140 μg) in the 1.5 ml Eppendorf tubes and incubate for 10 min at room temperature (prepare one aliquot of RNP complexes per transformation reaction).
      5. Add the RNP complex to the resuspened cells and gently tap the tube.
      6. Transfer 250 μl (+ volume of the RNP complex) of the transformation mixture to an electroporation cuvette.
      7. Incubate at room temperature for 5 min.
    2. Electroporation of RNP complex
      1. Set parameters in the electroporation device (Bio-Rad Laboratories) (voltage: 600 V, capacity: 50 μF, resistance: infinity).
      2. Gently tap the cuvette to mix the contents and place the cuvette in the cuvette chamber.
      3. Electroporate the cells and then resuspend them by gently pipetting in 750 μl of TAP sucrose solution (total volume, 1 ml).
      4. Transfer all of the transformation mixture into a 15 ml conical tube.
      5. To obtain single mutant colonies, immediately transfer 2-4 x 103 cells (4-6 μl) from the transformation mixture to a new 15 ml conical tube and then plate them on a 1.5% TAP agar plate (see Recipes) using top agar (see Recipes). After transformation, 400-600 colonies generally appear (Figure 5).


        Figure 5. Visual coloration examination to investigate CpFTSY gene knockout. Red circles indicate the putative CpFTSY knockout mutant lines which have pale green colors grown on TAP agar medium.

      6. Incubate the rest of the cells for 12 h in dim light (5-10 μmol photons m-2 sec-1), after which they should be harvested for genomic DNA extraction for use in targeted deep-sequencing analysis.

  5. Preparation of NGS library using PCR
    1. Deep sequencing library preparation by PCR amplification
      1. (Optional) Target gene (~cleavage site ± 250 bp) amplification
        For successful library construction, we recommend amplifying the target gene, which should include the cleavage site ± 250 bp, first. We use Phusion DNA polymerase (Thermo).
      2. Pre-indexing PCR (~25 cycles)


        For paired-end sequencing, the DNA library must have two adapters that include the i5 and i7 indices. Amplify the target DNA using Phusion polymerase with the pre-index tailed primers.
        Note: We recommend that the length of the target DNA be ~250 bp for Miseq 300 cycles or ~450 bp for Miseq 500 cycles for optimal merging of the paired-end reads.
      3. Indexing PCR (~25 cycles)


        After the pre-indexed amplification, amplify the PCR products with universal index primers. Purify the amplicons using a PCR DNA purification kit and measure the concentration of library using a spectrophotometer.
    2. Targeted deep sequencing
      We perform targeted deep sequencing using an Illumina Miseq Reagent Kit (v2).

Data analysis

  1. Analysis of targeted deep-sequencing data
    1. We use Cas-Analyzer in RGEN tools (http://www.rgenome.net/cas-analyzer/) (Figure 6)


      Figure 6. Overview of Cas-Analyzer. Users analyze their targeted deep-sequencing data using Cas-Analyzer without uploading any data to the server or local tool installation.

    1. Upload the NGS data files. Single-end reads, paired-end reads, and already merged sequencing data are allowed.
    2. Input the reference sequence and target DNA sequence without the PAM (protospacer adjacent motif) sequences that are the recognition site of CRISPR endonucleases.
    3. Submit the data.
    4. The results contain information about mutation counts and frequencies in brief. Statistical data and detailed sequence alignment data are displayed further down on the page.

  2. Analysis of off-target mutations in an isolated mutant
    1. Find more detailed information about potential off-targets associated with each selected target in the Chlamydomonas genome using Cas-OFFinder. Select a mismatch number (typically 4) and DNA/RNA bulge size (typically 1) (Figure 7).


      Figure 7. Overview of Cas-OFFinder. Cas-OFFinder (http://www.rgenome.net/cas-offinder/) has a broad range of searching options on the number of mismatches (10 bp) and DNA or RNA bulges (2 bp).

    2. Genomic DNA preparation from wild-type cells and isolated mutants.
    3. For predicted off-targets, perform NGS library preparation, targeted deep sequencing (Procedure E), and NGS data analysis.

Recipes

  1. Cas9 protein purification
    1. LB medium (for 1 L)
      10 g NaCl
      10 g tryptone
      5 g yeast extract
      Distilled water
    2. 1 M IPTG stock
      2.383 g of IPTG
      10 ml distilled water
      Filtered by syringe filter
    3. Lysis buffer, pH 7.4 (10 ml for 200 ml E.coli culture)
      50 mM NaH2PO4
      300 mM NaCl
      10 mM imidazole
      Distilled water
      Note: Adjust pH to 7.4 using NaOH, filtered.
    4. Wash buffer, pH 7.4 (20 ml for 200 ml E.coli culture)
      50 mM NaH2PO4
      300 mM NaCl
      20 mM imidazole
      Distilled water
      Note: Adjust pH to 7.4 using NaOH, filtered.
    5. Elution buffer, pH 7.4 (10 ml for 200 ml E.coli culture)
      50 mM NaH2PO4
      300 mM NaCl
      250 mM imidazole
      Distilled water
      Note: Adjust pH to 7.4 using NaOH, filtered.
    6. Cas9 storage buffer, pH 7.5 (10 ml for 200 ml E.coli culture)
      150 mM NaCl
      20 mM HEPES
      0.1 mM EDTA
      1 mM DTT
      2% sucrose
      20% glycerol
      Distilled water
      Note: Adjust pH to 7.5 using NaOH, filtered.
  2. Transfection
    1. TAP sucrose solution
      TAP medium with 40 mM sucrose
    2. TAP agar plates
      TAP medium with 1.5% agar
    3. Top agar
      TAP medium with 0.5% agar

Acknowledgments

This protocol was originally published as part of Baek et al. (2016). The authors specially thank to Dr. Duk Hyoung Kim for developing the method. This work was supported by grants from the Korea CCS R&D Center (KCRC) (NRF-2014M1A8A1049273) to E.J. and the Plant Molecular Breeding Center of Next Generation BioGreen 21 Program (PJ01119201) to S.B.

References

  1. Baek, K., Kim, D. H., Jeong, J., Sim, S. J., Melis, A., Kim, J. S., Jin, E. and Bae, S. (2016). DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep 6: 30620.
  2. Bae, S., Kweon, J., Kim, H. S. and Kim, J. S. (2014a). Microhomology-based choice of Cas9 nuclease target sites. Nat Methods 11(7): 705-706.
  3. Bae, S., Park, J. and Kim, J. S. (2014b). Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30(10): 1473-1475.
  4. Harris, E. H. (2001). Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 52: 363-406.
  5. Kim, S., Kim, D., Cho, S. W., Kim, J. and Kim, J. S. (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24(6): 1012-1019.
  6. Park, J., Bae, S. and Kim, J. S. (2015). Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31(24): 4014-4016.
  7. Park, J., Lim, K., Kim, J. S. and Bae, S. (2017). Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33(2): 286-288.

简介

我们通过使用无DNA的CRISPR成功地引入了目标克隆在赖氨酸衣藻中的目标基因敲除。在该协议中,整个工作流程的详细过程涵盖从初始目标选择CRISPR到使用下一代测序(NGS)技术的突变体分析。此外,我们介绍一种基于Web的工具集,名为CRISPR RGEN工具( http:// www.rgenome.net/ ),其中提供了CRISPR目标设计到NGS数据分析的所有必需工具。

背景 我们最近报道(Baek等人,2016),使用预先组装的Cas9蛋白质指导RNA核糖核蛋白,模型绿色微藻(Chlamydomonas reinhardtii)(Harris,2001)进行了一步改造核蛋白(RNP)。无DNA的CRISPR-Cas9递送的方式具有几个优点,例如不需要密码子优化和不同种类的微藻中的特异性启动子。此外,它减少了脱靶效应,并且在细胞中也可能具有较少的细胞毒性,因为Cas9蛋白是瞬时活性的,然后被细胞中的内源性蛋白酶降解(Kim等人,2014)。此外,由于不存在外来DNA序列,得到的基因编辑的微藻可以免于转基因生物(GMO)的规定。在该协议中,整个工作流程的详细过程包含从CRISPR的初始目标选择到使用NGS技术的突变分析(Bae等人,2014a和2014b; Park等人,2015和2017)。

关键字:基因组编辑,  CRISPR-Cas9,  微藻,  核糖核蛋白,  莱茵衣藻, DNA-free转化

材料和试剂

  1. Cas9蛋白纯化
    1. 1.5 ml Eppendorf管
    2. 无菌50ml锥形管
    3. 10ml注射器
    4. 0.45μm注射器过滤器
    5. 蛋白质浓缩器-100K MWCO(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88533)
    6. Poly-Prep色谱柱(Bio-Rad Laboratories,目录号:7311550)
    7. BL21-Pro细胞
    8. 含有6xHis-SpCas9(Addgene)的pET28质粒
    9. 卡那霉素
    10. 溶菌酶
    11. PMSF
    12. Ni-NTA琼脂糖珠(QIAGEN,目录号:30210)
    13. Bradford试剂(Bio-Rad Laboratories,目录号:5000205)
    14. 牛血清白蛋白(BSA)
    15. 氯化钠(NaCl)
    16. 胰蛋白胨
    17. 酵母提取物
    18. IPTG
    19. 磷酸二氢钠(NaH 2 PO 4)
    20. 咪唑
    21. 氢氧化钠(NaOH)
    22. HEPES
    23. 乙二胺四乙酸(EDTA)
    24. DL-二硫苏糖醇(DTT)
    25. 蔗糖
    26. 甘油
    27. LB培养基(参见食谱)
    28. 1 M IPTG库存(见配方)
    29. 裂解缓冲液(见配方)
    30. 洗涤缓冲液(见配方)
    31. 洗脱缓冲液(见配方)
    32. Cas9存储缓冲区(见配方)

  2. 体外转录和文库PCR
    1. 1.5 ml Eppendorf管
    2. 正向和反向寡核苷酸(参见表1和2)

      表1.预索引引物
    3. dNTP mix
    4. Phusion DNA聚合酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:F530L)
    5. PCR纯化试剂盒
    6. ATP,CTP,GTP,UTP,100mM MgCl 2,DEPC处理水
    7. T7 RNA聚合酶(New England Biolabs,目录号:M0251L)
    8. DNase I(不含RNase)(New England Biolabs,目录号:M0303L)
    9. RNase抑制剂鼠(New England Biolabs,目录号:M0314L)
    10. RNeasy MinElute清理套件(QIAGEN,目录号:74204)
    11. Illumina Miseq试剂盒(v2) 

    12. 转染
      1. 1.5 ml Eppendorf管
      2. 无菌15毫升和50ml圆锥管
      3. 纯化的Cas9蛋白和sgRNA
      4. 衣原体衣原体CC-4349 cw15 mt-(衣藻资源中心)
      5. PCR DNA纯化试剂盒(MG Med,目录号:MD008)
      6. TAP培养基(Harris,1989或Thermo Fisher Scientific,Gibco TM,目录号:A1379801)
      7. TAP蔗糖溶液(见食谱)
      8. TAP琼脂平板(参见食谱)
      9. 顶级琼脂(见食谱)
    13. 设备

      1. Cas9蛋白纯化
        1. 离心机:摆动转子(LaboGene,型号:1580R)
        2. 超声波发生器(Qsonica,型号:Q125)
        3. 移液器
        4. 振动孵化器(JS研究,型号:JSSI-300C)

      2. 体外转录和文库PCR
        1. 孵化器(JS研究,型号:JSGI-050T)
        2. 热循环仪(Bio-Rad Laboratories,型号:C1000 Touch TM 热循环仪)
        3. 微型离心机(LaboGene,型号:1730R)
        4. 分光光度计

      3. 转染
        1. 100ml烧瓶
        2. 分光光度计(GE Healthcare,Amersham Biosciences,型号:Ultrospec 2100 pro)
        3. 血细胞计数器(Marienfeld-Superior,目录号:0650030)
        4. 显微镜(Olympus,型号:CH30)
        5. 微型高速离心机(Hanil,型号:MICRO 17TR)
        6. 轨道摇床(N-biotek,型号:NB-101M)
        7. 洁净台(BioFree,型号:BF-150BSC)
        8. 电穿孔装置(Bio-Rad Laboratories,型号:Gene Pulser Xcell TM 电穿孔系统)
        9. 电穿孔试管(Bio-Rad Laboratories,0.4cm间隙)

      程序

      1. CRISPR目标选择用于创建感兴趣的基因敲除
        1. 从适当的数据库获取感兴趣的基因的编码序列(CDS)。我们使用JGI的植物志( https://phytozome.jgi .doe.gov / pz / portal.html#)(图1)。


          图1.植物志的主页:植物基因组数据库。每个基因的编码序列信息可以从Phytozome数据库获得。

        2. 对于目标设计,我们使用CRISPR RGEN工具( http://www.rgenome.net/ < / a>),一种用于CRISPR目标设计和下一代排序(NGS)数据分析的基于Web的工具集(图2)。


          图2. CRISPR RGEN工具的主页一种基于Web的工具集,可从CRISPR目标设计到NGS数据分析提供所有必需的工具。

        3. 在Cas-Designer中,选择CRISPR内切核酸酶的类型,例如来自化脓链球菌(Streptococcus pyogenes)的SpCas9,以及目标基因组,在这种情况下是模型生物体衣原体衣原体的靶基因组。 Cas-Designer现在包含我们网站上“植物”类型的版本4和5。然后将感兴趣的基因的CDS插入“目标序列”框中。我们建议在整个CDS的前半部分内选择目标网站,以增加产生完全敲除突变的可能性(图3)。


          图3. Cas-Designer的概述 用户可以选择各种类型的CRISPR内切核酸酶和目标基因组,包括重组衣原体(<4>或5)。

        4. 从Cas-Designer中选出候选人中的几个目标。与目标对象相比,您应避免与潜在的脱靶网站相关联的目标与1-2个不匹配。推荐使用GC含量范围在30到70%和更高分辨率的目标(图4)

          图4.来自Cas-Designer的输出表的示例数据。 Cas-Designer从输入序列中显示可能的CRISPR目标站点,以及有用的信息,如GC内容,帧外得分和非目标信息。
          注意:由于缺乏侧翼DNA核苷酸,N / A意味着“不适用”。

      2. 重组Cas9蛋白的纯化
        1. 用含有6xHis-SpCas9的pET28载体转化50μl有效的BL21-Pro细胞。
        2. 将细胞扩散到含有卡那霉素的琼脂平板上。在37℃孵育板过夜。
        3. 将含有50μg/ ml卡那霉素的10ml LB培养基接种在具有来自板的单个菌落的锥形管中。在37℃下以200转/分钟摇动,使其饱和过夜
        4. 第二天早上,将含有卡那霉素的200ml LB培养基接种在具有2ml饱和过夜培养物的烧瓶中
        5. 在37℃下以200rpm振荡培养细胞至对数期(OD 600)约为0.4-0.5。
        6. 加入160μl1M IPTG(终浓度为0.8mM),并在18℃下以200rpm摇动孵育细胞一夜。
        7. 将培养物转移到50ml圆锥形离心管中,并以3,000xg和4℃离心20分钟来沉淀细胞。丢弃上清液并在同一管中重复收获。
        8. 将细胞沉淀重新悬浮于10ml裂解缓冲液中(参见食谱)。
        9. 加入20 mg溶菌酶和100μl100 mM PMSF(1 mM终浓度)
        10. 在冰上孵育1小时。
        11. 在冰上的锥形管中振荡细胞(振幅25%,1秒ON / 1秒OFF,脉冲30次)。重复超音波处理5次。
        12. 将超声处理的细胞转移到1.5ml微量管中,使用微量移液管和颗粒碎片通过在4℃,11,000×g离心30分钟。
        13. 通过0.45μm注射器过滤器过滤上清液,并将其收集在50ml锥形管中
        14. 向裂解液中加入2 ml Ni-NTA琼脂糖。加入前旋入琼脂糖珠。孵育1 h,旋转4°C。
        15. 用移液管将混合物转移到色谱柱上。收集锥形管中的流通。
        16. 用10ml洗涤缓冲液洗涤柱子(参见食谱)。在锥形管中收集第一批洗涤缓冲液。
        17. 重复洗涤。在锥形管中收集第二批洗涤缓冲液。
        18. 用10ml洗脱缓冲液洗脱结合的蛋白质(参见食谱)。收集10 ml微量管中1 ml顺序等分试样的洗脱液
        19. 使用Bradford测定法,确定每个级分中的蛋白质浓度
        20. 将含有融合蛋白的洗脱液应用于蛋白质浓缩器(100K MWCO)
        21. 在4℃,3,000×g离心,直到样品体积减少90%。
        22. 应用4 ml蛋白质储存缓冲液(见食谱),并在4℃,3,000 x g下离心,直到体积减少90%。
        23. 用300μl蛋白质储存缓冲液洗涤过滤器上的蛋白质。
        24. 在分光光度计中用标准BSA测定Bradford测定蛋白质浓度(SpCas9的最终浓度应为约10mg / ml)。
        注意:
        1. 通过SDS-PAGE识别来自每个步骤的哪些样品含有融合蛋白。
        2. 您应该使用体外切割测定来验证Cas9蛋白的活性。

      3. sgRNA的体外转录
        1. 模板扩展


          扩增模板后,使用PCR纯化试剂盒纯化DNA 注意:纯化的DNA模板可以储存在-20°C。

          表3.体外转录
          的Oligos
        2. 转录反应


          4小时过夜反应,37℃水浴或培养箱
        3. 去除DNA模板
          1. 加入11μlDNase I缓冲液
          2. 加入1μl(2 U /μl)DNase I,在100μl反应物中
          3. 在37℃,30分钟内孵育
        4. RNA纯化
          1. 按照制造商手册使用RNA清洁套件。
          2. 在10-25μlDEPC处理过的水或无RNase的水中洗脱RNA。最终RNA浓度应为5-15μg/μl。
          注意:RNA应保存在-80°C,直到Chlami细胞准备好。

        5. 将核糖核蛋白(RNP)转染至衣藻类
          1. 细胞和RNP复合物的制备用于转化
            1. 在50℃的液体TAP培养基中,在连续的光(50-70μmol光子,约 sec)下,在25℃下,在50ml的液体TAP培养基中混合(在乙酸根的光中)培养“衣藻”细胞< sup> -1 )在100ml烧瓶中以90rpm振荡直至细胞密度达到通过分光光度计测得的0.3-0.5的OD 750以下。
            2. 用血细胞计数器和显微镜估算细胞密度
            3. 通过在1.5ml Eppendorf管(2,000×g×,3分钟,室温)中离心收获5×10 5个细胞,弃去上清液,并将细胞重悬于250μl的TAP蔗糖溶液(参见食谱)通过轻轻移液(每转化反应制备一份细胞)。
            4. 为了制备RNP复合物,将预混合纯化的Cas9蛋白(200μg)在体外转录于1.5ml Eppendorf管中的sgRNA(140μg),并在室温下孵育10分钟(准备一份RNP复合物每转化反应)
            5. 将RNP复合物加入重悬细胞,轻轻敲打管
            6. 将转化混合物的250μl(+体积的RNP复合物)转移到电穿孔试管中
            7. 在室温下孵育5分钟。
          2. 电穿孔RNP复合体
            1. 在电穿孔设备(Bio-Rad Laboratories)(电压:600 V,容量:50μF,电阻:无穷大)中设置参数。
            2. 轻轻点击比色杯混合内容物,并将比色皿放入比色皿室。
            3. 将细胞电穿孔,然后通过在750μlTAP蔗糖溶液(总体积,1ml)中轻轻移液来重悬。
            4. 将所有转化混合物转移到15ml锥形管中
            5. 为了获得单个突变菌落,立即将2-4×10 3个细胞(4-6μl)从转化混合物中转移到新的15ml锥形管中,然后将其置于1.5%TAP琼脂平板(参见食谱)使用顶部琼脂(见食谱)。转化后,通常出现400-600个菌落(图5)

              图5.用于调查CpFTSY基因敲除的视觉着色检查。红色圆圈表示在TAP琼脂培养基上生长的具有淡绿色的推定的CpFTSY敲除突变体系。

            6. 将其余细胞在暗淡的光(5-10μmol光子,sup sec -1)中孵育12小时,然后将其收获用于基因组DNA提取用于靶向深测序分析。

        6. 使用PCR制备NGS文库
          1. 通过PCR扩增深度测序文库制备
            1. (可选)靶基因(〜切割位点±250 bp)扩增 为了成功构建图书馆,我们建议首先扩增目标基因,其中应包括切割位点±250 bp。我们使用Phusion DNA聚合酶(Thermo)。
            2. 预索引PCR(〜25个周期)


              对于配对末端测序,DNA文库必须具有两个包含i5和i7索引的适配器。扩增目标DNA使用Phusion聚合酶与前索引尾引物 注意:对于Miseq 300循环,我们建议目标DNA的长度为〜250 bp,Miseq 500循环的长度为450 bp,以便最佳合并成对的读数。
            3. 索引PCR(〜25个周期)


              在预索引扩增后,用通用引物引物扩增PCR产物。使用PCR DNA纯化试剂盒纯化扩增子,并使用分光光度计测量文库的浓度
          2. 目标深度测序
            我们使用Illumina Miseq Reagent Kit(v2)进行定向深度测序。
        7. 数据分析

          1. 目标深度测序数据分析
            1. 我们在RGEN工具中使用Cas-Analyzer( http://www.rgenome。 net / cas-analyzer / )(图6)


              图6. Cas-Analyzer概述。用户使用Cas-Analyzer分析其目标深度排序数据,而无需将任何数据上传到服务器或本地工具安装。

            1. 上传NGS数据文件。允许单端读取,配对读取和已合并的排序数据。
            2. 输入参考序列和目标DNA序列,而不使用作为CRISPR核酸内切酶识别位点的PAM(原始相邻基序)序列。
            3. 提交数据。
            4. 结果包含关于突变计数和频率的信息。统计数据和详细的序列比对数据将在页面上进一步显示。

          2. 分离突变体中非靶突变的分析
            1. 使用Cas-OFFinder查找有关使用“衣藻”基因组中每个选定目标相关的潜在脱靶的更多详细信息。选择不匹配的数字(通常为4)和DNA / RNA的隆起大小(通常为1)(图7)

              图7. Cas-OFFinder概述 Cas-OFFinder( http://www.rgenome.net/cas-offinder/ )对不匹配数(10 bp)和DNA或RNA突起(2 bp)有广泛的搜索选项。 />
            2. 野生型细胞和分离突变体的基因组DNA制备
            3. 对于预测的脱靶,执行NGS库准备,靶向深度测序(方法E)和NGS数据分析。

          食谱

          1. Cas9蛋白纯化
            1. LB培养基(1升)
              10克NaCl
              10克胰蛋白胨
              5克酵母提取物
              蒸馏水
            2. 1 M IPTG股票
              2.383克IPTG
              10ml蒸馏水
              通过注射器过滤器过滤器
            3. 裂解缓冲液,pH 7.4(200ml用于200ml大肠杆菌培养物)
              50mM NaH 2 PO 4
              300 mM NaCl
              10 mM咪唑
              蒸馏水
              注意:使用NaOH将pH调节至7.4,过滤。
            4. 洗涤缓冲液,pH 7.4(200ml用于200ml大肠杆菌培养物)
              50mM NaH 2 PO 4
              300 mM NaCl
              20mM咪唑
              蒸馏水
              注意:使用NaOH将pH调节至7.4,过滤。
            5. 洗脱缓冲液,pH 7.4(对于200ml大肠杆菌培养物为10ml)
              50mM NaH 2 PO 4
              300 mM NaCl
              250毫克咪唑
              蒸馏水
              注意:使用NaOH将pH调节至7.4,过滤。
            6. Cas9储存缓冲液,pH7.5(200ml用于200ml大肠杆菌培养物)×/ 150 mM NaCl
              20 mM HEPES
              0.1 mM EDTA
              1 mM DTT
              2%蔗糖
              20%甘油
              蒸馏水
              注意:使用NaOH将pH调节至7.5,过滤。
          2. 转染
            1. TAP蔗糖溶液
              具有40mM蔗糖的TAP培养基
            2. TAP琼脂板
              具有1.5%琼脂的TAP培养基
            3. 顶级琼脂
              具有0.5%琼脂的TAP培养基

          致谢

          该协议最初是作为Baek等人的一部分发布的。 (2016)。作者特别感谢杜金亨博士研制的方法。这项工作得到韩国CCS研发中心(NCR-2014M1A8A1049273)授予E.J.的资助。和下一代BioGreen 21程序植物分子育种中心(PJ01119201)。

          参考

          1. Baek,K.,Kim,DH,Jeong,J.,Sim,SJ,Melis,A.,Kim,JS,Jin,E。和Bae,S。(2016)。通过CRISPR-Cas9核糖核酸蛋白在衣藻衣原体中的无DNA双基因敲除 Sci Rep 6:30620.
          2. Bae,S.,Kweon,J.,Kim,HS和Kim,JS(2014a)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed / 24972169“target =”_ blank“> Cas9核酸酶靶位点的基于微生物学的选择。 Nat方法 11(7):705-706。
          3. Bae,S.,Park,J.和Kim,JS(2014b)。&nbsp; Cas-OFFinder:一种快速而通用的算法,用于搜索Cas9 RNA引导内切核酸酶潜在的目标外位点。生物信息学30(10):1473-1475。
          4. Harris,EH(2001)。&nbsp; 衣藻/ em>作为模型生物体。 Annu Rev Plant Physiol Plant Mol Biol 52:363-406。
          5. Kim,S.,Kim,D.,Cho,SW,Kim,J.and Kim,JS(2014)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm。 nih.gov/pubmed/24696461“target =”_ blank“>通过递送纯化的Cas9核糖核蛋白,在人类细胞中高效率的RNA指导基因组编辑。基因组研究24(6):1012 -1019。
          6. Park,J.,Bae,S.and Kim,JS(2015)。&nbsp; Cas-Designer:用于选择CRISPR-Cas9目标网站的基于网络的工具。生物信息学 31(24):4014-4016。
          7. Park,J.,Lim,K.,Kim,JS和Bae,S。(2017)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/ pubmed / 27559154“target =”_ blank“> Cas-analyzer:用于使用NGS数据评估基因组编辑结果的在线工具。生物信息学 33(2):286-288。 >
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yu, J., Baek, K., Jin, E. and Bae, S. (2017). DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis. Bio-protocol 7(11): e2352. DOI: 10.21769/BioProtoc.2352.
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