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Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions
通过CRISPR-Cas精确诱导双等位基因缺失以对硅藻进行基因组编辑   

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Abstract

Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.

Keywords: CRISPR-Cas(CRISPR-Cas), Diatom(硅藻), Thalassiosira pseudonana(假微型海链藻), Golden Gate(Golden Gate), U6 promoter(U6启动子), Band shift assay(带偏移实验法), Restriction site loss(限制性位点丢失), Phaeodactylum tricornutum(三角褐指藻)

Background

CRISPR-Cas is fast becoming a key method for molecular research. Based on a viral defence mechanism found in bacteria and archaea, CRISPR-Cas induces double-strand breaks (DSBs) at precise locations in the genome. It involves the use of a Cas9 nuclease which forms a complex with a chimeric single guide RNA (sgRNA) formed from CRISPR RNA (crRNA) and trans-activating crRNA (trRNA). Specificity is provided by a 20 nt sequence in the crRNA which corresponds with the target in the genome and guides Cas9 to the correct site by base complementarity. This means that the system is easily programmable and can be applied to new targets simply by changing the 20 nt sequence, provided that a protospacer adjacent motif (PAM) is present in the genome directly following the 20 nt target sequence. For the commonly used Cas9 isolated from Streptococcus pyogenes the PAM sequence is NGG. Gene editing is then achieved either by introducing mutations following imperfect repair by non-homologous end joining (NHEJ), cutting two sites and introducing a precise deletion or through homologous recombination. Since its application in the first eukaryotic systems (Cong et al., 2013; Mali et al., 2013), CRISPR-Cas has been used for genome editing in a wide range of organisms including two diatom species (Hopes et al., 2016; Nymark et al., 2016). Nymark et al. (2016) introduced mutations into the genome of Pheodactylum tricornutum using individual sgRNAs–the protocol for which can be found within Bio-protocol (Nymark et al., 2017). The protocol published in this paper focuses on gene editing in Thalassiosira pseudonana using two sgRNAs to introduce a precise deletion and allow simple screening using the band-shift assay previously described for identifying mutants in higher plants (Brooks et al., 2014). In addition, this method uses Golden Gate cloning (Weber et al., 2011; Engler et al., 2014)–a flexible modular system which allows sequences and cassettes to be easily interchanged and multiple modules to be assembled at once. Whilst this protocol describes targeting of two sites in the same gene to introduce a deletion, the construct can easily be altered to target different genes or greater numbers of genes as previously shown by Sakuma et al. (2014), who demonstrated knock-out of 7 genes using the Golden Gate cloning system.

Materials and Reagents

  1. 0.2 ml PCR tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: AB0620 )
  2. 10 µl filter pipette tips (STARLAB INTERNATIONAL, catalog number: S1121-3810 )
  3. 10 µl pipette tips (STARLAB INTERNATIONAL, catalog number: S1111-3810 )
  4. 200 µl pipette tips (STARLAB INTERNATIONAL, catalog number: S1111-0810 )
  5. 200 µl filter pipette tips (STARLAB INTERNATIONAL, catalog number: S1120-8810 )
  6. 1,000 µl pipette tips (STARLAB INTERNATIONAL, catalog number: S1111-6810 )
  7. 47 mm diameter 1.2 µm isopore filters (Merck, catalog number: RTTP04700 ) (optional)
  8. 1.5 ml Microcentrifuge tubes (Fisher Scientific, catalog number: 11926955 )
  9. 90 mm diameter Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 263991 )
  10. 0.7 µm (M10) tungsten particles (Microcarriers) (Bio-Rad Laboratories, catalog number: 1652266 )
  11. Culture tubes (Fisher Scientific, catalog number: 11317824 )
  12. Macrocarriers (Bio-Rad Laboratories, catalog number: 1652335 )
  13. Macrocarrier holders (Bio-Rad Laboratories, catalog number: 1652322 )
  14. Stopping screens (Bio-Rad Laboratories, catalog number: 1652336 )
  15. 1,350 psi rupture discs (Bio-Rad Laboratories, catalog number: 1652330 )
  16. Thalassiosira pseudonana strain CCMP 1335 (Bigelow) https://ncma.bigelow.org/ccmp1335
  17. pCRTM8/GW/TOPOTM TA Cloning Kit with One ShotTM TOP10 E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: K250020 )
  18. High efficiency competent E. coli (e.g., NEB 5-alpha Competent E. coli (High Efficiency)) (New England Biolabs, catalog number: C2987H )
  19. BsaI (New England Biolabs, catalog number: R0535S )
  20. BpiI (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER1011 )
  21. Taq DNA polymerase (e.g., GoTaq Flexi DNA polymerase) (Promega, catalog number: M8291 )
  22. pICH47732:FCP:NAT (Addgene, catalog number: 85984 ) or pICH47732 (Addgene, catalog number: 48000 )
  23. pICH47742:FCP:Cas9YFP (Addgene, catalog number: 85986 ) or pICH47742 (Addgene, catalog number: 48001 )
  24. pICH47751 (Addgene, catalog number: 48002 )
  25. pICH47761 (Addgene, catalog number: 48003 )
  26. pICH41780 (Addgene, catalog number: 48019 )
  27. pAGM4723 (Addgene, catalog number: 48015 )
  28. pICH86966::AtU6p::sgRNA_PDS (Addgene, catalog number: 46966 )
  29. pCR8/GW:U6 (Addgene, catalog number: 85981 )
  30. Q5 Site-Directed Mutagenesis Kit (New England Biolabs, catalog number: E0554S )
  31. High fidelity DNA polymerase (e.g., Phusion High-Fidelity DNA Polymerase) (New England Biolabs, catalog number: M0530 )
  32. PCR clean up kit (e.g., Illustra GFX PCR DNA and Gel Band Purification Kit) (GE Healthcare, catalog number: 28-9034-70 )
  33. Plasmid mini prep kit (e.g., PureYield Plasmid Miniprep System) (Promega, catalog number: A1223 )
  34. Absolute ethanol (VWR, catalog number: 20821.330 )
  35. Agarose (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17850 )
  36. Ethidium bromide for gel electrophoresis (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15585011 )
  37. TAE buffer for gel electrophoresis (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15558026 )
  38. T4 DNA ligase (Promega, catalog number: M1794 )
  39. 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal) (Sigma-Aldrich, catalog number: B4252-50MG )
  40. IPTG (Sigma-Aldrich, catalog number: I6758-1G )
  41. Half salinity Aquil media (Price et al., 1989, full recipe at: https://ncma.bigelow.org/algal-recipes)
  42. Nourseothricin clonNAT (Werner BioAgents)
  43. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670-100G )
  44. Spermidine (Sigma-Aldrich, catalog number: S0266-1G )
  45. Isopropanol (VWR, catalog number: BDH1133-1LP )
  46. Compressed Helium supply
  47. Mix2seq kit (Eurofins)
  48. Tryptone (Sigma-Aldrich, catalog number: T7293 )
  49. Yeast extract (ForMedium, catalog number: YEA01 )
  50. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
  51. Agar (ForMedium, catalog number: AGA02 )
  52. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166-5G )
  53. Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S4014-5G )
  54. Carbenicillin disodium salt (Sigma-Aldrich, catalog number: C3416-250MG )
  55. Kanamycin sulphate (Sigma-Aldrich, catalog number: 60615-5G )
  56. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  57. Tris-HCl pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15568025 )
  58. EDTA (Sigma-Aldrich, catalog number: EDS )
  59. LB media/LB agar (see Recipes)
  60. Lysis buffer (see Recipes)

Equipment

  1. Pipettes (Thermo Fisher Scientific, model: Finnpipette , volumes: 2 µl, 10 µl, 200 µl and 1,000 µl)
  2. NanoDrop ND-1000 (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 1000 )
  3. Laminar flow hood (Walker safety cabinets, model: Class II 1290 Recirc Gen 6 )
  4. Benchtop microfuge (Eppendorf, model: 5418 R )
  5. Centrifuge (Hettich Lab Technology, model: ROTINA 380 R )
  6. Autoclave (Prestige Medical, catalog number: 210004 )
  7. Tweezers
  8. Vortex (Mo Bio Laboratories, model: Vortex Genie® 2 )
  9. Shaking incubator (37 °C) (IKA, model: KS 4000 i control )
  10. Light incubator at 20 °C (Sanyo versatile Environmental Test chamber)
  11. PCR thermocycler (Bio-Rad Laboratories, model: T100TM Thermal cycler )
  12. Light Microscope and Neubauer chamber (VWR, catalog number: 631-0696 ) or Coulter Counter (Beckman, model: Multisizer 3 ) for counting cells
  13. PDS-1000/He biolistic microparticle delivery system (particle gun) (Bio-Rad Laboratories, catalog number: 1652257 )
  14. Vacuum pump for cell filtration (Welch Vacuum, model: 2534C-02 ) (optional)
  15. Nalgene filtration funnel (Thermo Fisher Scientific, Thermo ScientificTM, model: DS0320-5045 ) (optional)
  16. High Vacuum pump for microparticle delivery system (Uniweld Products, model: HUMM•VACTM HVP6 )
  17. Gel electrophoresis tank (Fisher Scientific, FisherbrandTM, model: Midi Plus Horizontal Gel System )
  18. Electrophoresis power supply (Consort, model: EV243 )

Procedure

  1. Designing sgRNAs
    sgRNAs are designed to facilitate screening once mutations have been introduced. This protocol describes two screening methods: the band shift assay and restriction site loss. We have found the band shift assay to be the most effective–this method is based on introducing a deletion using two sgRNAs which can be screened by amplifying the target and looking for a shorter band on an agarose gel. It has the added benefit of introducing very precise and large deletions. This can be used as a stand-alone method without the need for restriction site loss. Restriction site loss is slightly more complicated and limits the sgRNA design as DSBs need to coincide with a restriction recognition site (preferably at least a 5 nt site cutter). When mutations are introduced through CRISPR-Cas, the restriction site is no longer active and the restriction enzyme cannot cut. This method has been included as it can be a useful way to enrich mutated targets or screen if only one sgRNA is functional.
    1. This protocol uses the chimeric sgRNA for use with the S. pyogenes Cas9 with the following sequence:

      NNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT

      1. The underlined sequence shows the programmable region which corresponds to the 20 nt target sequence in the genome.
      2. In the genome, this sequence is immediately followed by the NGG PAM. Therefore targets need to be identified in the genome with the sequence N20NGG.
        Note: The target sequence of the sgRNA itself should only contain the N20 sequence–i.e., it should not contain the PAM.
      3. In addition, a U6 promoter which recruits RNA polymerase III (pol III) is used to drive expression of the small non-coding sgRNAs. This means that the sgRNA needs to start with a ‘G’ to activate transcription. This can either be incorporated into the target design meaning that targets with the sequence GN19NGG need to be identified OR an additional G can be added to the 5’ end of the sgRNA.
      4. The string of 6 Ts in bold acts as a terminator for transcription when expressing the sgRNA using pol III.  
    2. Two tools are used to design sgRNAs targets:
      1. RGEN Cas-Designer: http://www.rgenome.net/
        1. Includes an off-target finder for several algal and diatom genomes including T. pseudonana. This allows the researcher to reject targets if similar sequences occur elsewhere in the genome which may also be cut.
        2. Allows specified sequences to be omitted such as introns.
        3. Can search for targets with several different PAMs if the researcher wishes to change the Cas9.
      2. Broad Institute sgRNA designer: https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design
        This is used to give on-target scores. The algorithm used by this program is based on empirically tested sgRNAs and predicts their cutting efficiency. Try to choose targets which give a cutting efficiency as close to 1 as possible.
    3. Design two sgRNAs with cut sites about 50-100 bp apart
      1. A deletion of 50-100 bp gives a clear difference between edited targets and the WT (Belhaj et al., 2013; Brooks et al., 2014).
        Note: A larger deletion can be chosen if necessary–research suggests that mutations from larger deletions occur less frequently, however, deletions up to 10 kb are still efficient (Zheng et al., 2014).
      2. Either target the active site of the gene or the beginning of the gene if a frame shift is expected.
        Note: Whole genes can also be removed by targeting the ends of the genes or the end of the promoter/start of the terminator. This can be useful if the active site is unknown or there are problems with designing sgRNAs, for example, when a large number of repeat sequences are present.
    4. If the restriction site loss method is being used for screening, targets need to be designed so that Cas9 cut sites sit over a restriction recognition site or within the deletion site.
      Targets that position cut sites over restriction sites can be determined using software such as PhytoCRISPRex (Rastogi et al., 2016) or by running the target gene through a restriction finder such as the Emboss restriction tool (http://emboss.bioinformatics.nl) and cross-referencing location of the restriction sites with the position of the Cas9 cuts sites/deletions.
      Note: It may not always be possible to design sgRNAs exactly within these specifications, for example, sgRNAs with lower on-target scores may need to be used due to positioning constraints (we have successfully used sgRNAs with a score of 0.4), or a slightly smaller deletion may be necessary. Bear in mind that even if it is possible, targets still need to be empirically tested as other factors such as accessibility may affect their activity. As a result, it is recommended to design at least two sets of sgRNAs for each target gene/sequence.

  2. Making the construct
    The construct is assembled using the Golden Gate system which builds the plasmid in modules using pre-set backbones and pre-set overhangs between sequences (Weber et al., 2011). The basis behind Golden Gate cloning is the use of restriction enzymes BsaI and BpiI, which cut outside of the restriction recognition sites, allowing specific 4 nt overhangs to be created. This allows fragments to be joined together in a specific order and within a single reaction for each level. As the restriction recognition site and cut site are separate, restriction digest and ligation of the insert leads to complete removal of the site, meaning that once the correct assembly is achieved the construct can no longer be cut. This is carried out over three levels, L0, L1 and L2.
    L0 modules contain sequences such as promoters, coding regions and terminators. These are assembled together to form cassettes in L1 backbones. L1 modules are then assembled into the final L2 backbone to create a construct with all the necessary sequences required for CRISPR-Cas. This section describes the construct assembly for CRISPR-Cas in T. pseudonana for targeting two sites in the genome, however, it can easily be adapted for use with other organisms, different modules (e.g., different Cas9, promoters or selective markers) or to target more than two sites. The latter makes this cloning approach ideal for targeting multiple genes.
    L1 backbones contain BsaI sites to allow cloning of L0 modules, as well as BpiI sites to allow L1 modules to be cloned into the L2 backbone. L0 modules are created by amplifying the desired fragments with Taq DNA polymerase and cloning into a pCRTM8/GW/TOPO vector. In order to clone L0 modules into L1 backbones, BsaI sites need to be included through the forward and reverse primers when amplifying the desired fragment. In addition, the overhangs which are created when the BsaI sites are digested need to be the same between adjacent modules. All L1 backbones used within this protocol give standardised overhangs of GGAG and GCTT when digested with BsaI. As a result, the first L0 module in the sequence to be cloned in must contain a GGAG overhang at the 5’ end and the last module must contain a GCTT overhang at the 3’ end. Internal overhangs between L0 modules are decided by the researcher and can be designed to give a seamless join. Below is an example of an amplicon for cloning into L0 showing the start and end of the sequence:

    TggtctcaggagAGCTTGCGCTTTTTCCGAG…CTGATTTACCAAACCAATACCAAAatgtgagacct

    Bold letters denote the BsaI site, underlined regions show the 4 nt overhang generated by restriction with BsaI and the italicised region shows the fucoxanthin chlorophyll a/c-binding protein (FCP) promoter sequence. The first overhang corresponds with the overhang for the end of the L1 backbone. The second overhang corresponds with the overhang from the adjacent Cas9 module. This has been designed so that the overhang contains the ATG at the start of the Cas9, giving a seamless join.
    Figure 1 gives a general overview of the assembly process used in this protocol.


    Figure 1. Overview of the Golden Gate cloning system for assembly of the CRISPR-Cas construct. For T. pseudonana, plasmids for the L0 U6 promoter, L1 FCP:NAT cassette and L1 FCP:Cas9:YFP cassette are already available from Addgene (see the link below). For additional L0 T. pseudonana modules please contact the authors.

    1. The protocol describes creation and assembly of all modules so that researchers can modify the overall system if desired, however, several of the L0 and L1 modules described are already available from Addgene at https://www.addgene.org/Thomas_Mock/. In addition, modules for P. tricornutum will soon be available from Addgene on the same website.
    2. Remove BsaI and BpiI sites from inserts
      1. The original pTpFCP:NAT (Poulsen et al., 2006) cassette for nourseothricin resistance contains a BsaI site in the FCP promoter and a BpiI site in the NAT gene. These sites need to be removed by site-directed mutagenesis to prevent unwanted restriction digest during Golden Gate cloning.
      2. Using pTpFCP/NAT (Poulsen et al., 2006) as a template, remove the BsaI site using forward primer: TCCGCGGCAGaTCTCTGTCG, reverse primer: AGAAGTACCGTGTTGTTGCAGTG, and a Q5 site-directed mutagenesis (SDM) kit (NEB). Repeat SDM on the BpiI site in the NAT gene using the resulting pTpFCP/NAT plasmid (containing the domesticated FCP promoter) as a template, forward (F) primer: CGACACCGTaTTCCGCGTCAC and reverse (R) primer: GTGGTGAAGGACCCATCCAG.
        Note: If using the TpFCP/NAT cassette there is no need to domesticate this sequence as the L1 pICH47732:FCP:NAT module is already available from Addgene.
      3. If using different sequences than those described in this protocol, remove BpiI and BsaI sites in a similar manner as described above. Software for designing SDM primers is available from NEB alongside the kit (http://nebasechanger.neb.com/).
    3. Using U6 promoters in diatoms
      1. All known U6 promoters for diatoms are shown in Table 1. The U6 promoter for P. tricornutum was first described by Nymark et al. (2016) and activity of the T. pseudonana (Hopes et al., 2016) and Fragilariopsis cylindrus promoters (unpublished) have been determined in our lab. In addition, the exact end of the U6 promoters for T. pseudonana and F. cylindrus have been determined empirically using the 5’ TSO RACE (Pinto and Lindblad, 2010) method.

        Table 1. U6 promoters in diatoms and the overhangs required for scar less assembly with the sgRNA. The TATA box is highlighted in yellow and the overhang (last 4 nt of the promoter) in green.


      2. When cloning the U6:sgRNA cassette through Golden Gate, it is important to ensure a scarless junction between the end of the U6 promoter and the start of the sgRNA so that extra nucleotides are not transcribed at the 5’ end of the sgRNA. This is achieved by designing the 4 nt overhang between the U6 promoter and sgRNA as the last 4 nt of the U6 promoter. The overhang necessary to create a scarless junction between the U6 promoter and sgRNA varies for each U6 promoter and requires the overhang to be built into the reverse primer for amplifying the U6 promoter and the forward primer for amplifying the sgRNA. These primers can be found in Table 2 below:

        Table 2. Primers used to introduce the overhang between diatom U6 promoters and sgRNAs. BsaI sites are underlined and overhangs highlighted in green. Ns denote the target site. Uppercase letters A, C, G or T show the section of the primer that will anneal with the initial template.


    4. Creating L0 modules
      1. A full list of primers, annealing temperatures and extension times can be found for all Golden Gate PCR reactions in Table 3.

        Table 3. PCR primers for Golden Gate cloning. BsaI sites are underlined. Overhangs are shown in bold. F denotes the forward primer and R the reverse primer. The string of Ns in the forward sgRNA primer needs to be replaced by the 20 nt target sequence designed for each gene/target. Uppercase letters A, C, G or T show the section of the primer that will anneal with the initial template.


      2. L0 modules contain a Sm/SpR gene to allow selection by spectinomycin in E. coli.
      3. Amplify the FCP promoter from the domesticated pTpFCP/NAT cassette using F primer: tggtctcaggagAGCTTGCGCTTTTTCCGAG and R primer aggtctcacatTTTGGTATTGGT TTGGTAAATCAG (Table 3, numbers 1 and 2) using Taq DNA polymerase. Clone the PCR product directly into a pCRTM8/GW/TOPO vector.
      4. Amplify Cas9:YFP using F primer: aggtctcaaATGGACAAGAAGTACTCCATTGG and R primer: aggtctcaaagcTCACTTGTACAGCTCGTCCATG (Table 3, numbers 3 and 4) using high fidelity (HF) Phusion polymerase. Clean the reaction with a PCR purification kit and incubate with Taq polymerase (Table 4) for 20 min at 72 °C before cloning into the pCRTM8/GW/TOPO vector.
        Note: Amplification of the Cas9 is carried out with HF polymerase to reduce the chance of errors during amplification since Cas9:YFP is a large sequence of 4.8 kbp. As HF polymerases do not add a 3’ ‘A’ overhang, a separate incubation with Taq is required.

        Table 4. Reagents for Cas9:YFP PCR incubation with Taq to add ‘A’ overhangs


      5. Amplify the FCP terminator from domesticated pTpFCP/NAT using F primer: aggtctcagcttATACTGGATTGGTGAATCAATG and R primer: tggtctcaagcgGAGAA CTGGAGCAGCTAC (Table 3, numbers 5 and 6) using Taq DNA polymerase. Clone the PCR product directly into a pCRTM8/GW/TOPO vector.
      6. Amplify the U6 promoter from T. pseudonana genomic DNA using the F primer: cggtctcaggagCTTCATCAAGAGAGCAACCA and R primer: aggtctcaACAATTTCGG CAAAACGT (Table 3, numbers 7 and 8) using Taq DNA polymerase. Clone the PCR product directly into a pCRTM8/GW/TOPO vector. See section 3 for using U6 promoters in diatoms. This module is already available from Addgene.
      7. Due to the guide sequence changing between targets, sgRNAs are not cloned into L0 vectors, instead they are directly assembled into the L1 vector as a PCR product. Target sequences are introduced through the F primer when amplifying the scaffold. Amplify the scaffold from pICH86966::AtU6p::sgRNA_PDS using F primer: aggtctcattgtNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAG and R primer: tggtctcaagcgTAATGCCAACTTTGTACAAG (Table 3, numbers 11 and 12). It is fine to use either Taq or HF DNA polymerase. The run of Ns in the forward primer represents the target sequence. Following amplification, PCR products are purified using a PCR clean-up kit.
    5. Screening L0 modules
      1. Screen all L0 modules before assembly into L1 vectors. Perform colony PCR using the same primers used to amplify fragments for cloning into L0 modules (Table 3) or digest the L0 module to check for size. Sequence L0 modules that contain inserts of the correct size using primers provided in the pCRTM8/GW/TOPO TA kit.
      2. Colony PCR:
        1. Prepare Master Mix for the number of colonies to be tested according to Table 5 and aliquot 20 µl into PCR tubes.

          Table 5. Reagents for colony PCR


        2. Lightly touch a 10 µl pipette tip to each colony (label the colony so that it can be picked following screening), mix using the pipette and briefly spin down.
        3. Run the following PCR cycle.
          1) Initial denaturation: 95 °C for 2 min
          2) 35 cycles:
              Denature at 95 °C for 30 sec
              Anneal for 30 sec (see Table 3 for annealing temperatures)
              Extend at 72 °C (see Table 3 for annealing times)
          3) Carry out the final extension at 72 °C for 10 min
      3. Restriction digest:
        Extract plasmids using a plasmid mini-prep kit, measure the DNA concentration and digest with BsaI to check the size of the insert.
        Note: A NanoDrop spectrophotometer is used to measure DNA concentration.
    6. L1 assembly
      1. Four L1 modules are assembled
        pICH47732:FCP:NAT (available from Addgene)
        pICH47742:FCP:Cas9YFP (available from Addgene)
        pICH47751:U6:sgRNA1
        pICH47761:U6:sgRNA2
      2. Golden Gate assembly is carried out in a single reaction for each L1 assembly.
        1. Set up the reaction as shown in Table 6.

          Table 6. Reagents for L1 Golden Gate assembly


        2. Incubate under the following conditions
          37 °C for 5 h
          50 °C for 5 min
          80 °C for 10 min
        3. Transform 5 µl of the reaction into 50 µl of competent E. coli.
        4. L1 modules contain the cassette for carbenicillin resistance for selection in E. coli.
          Note: Insertion of the correct insert should remove the LacZ gene for blue to white colony screening when incubated with X-gal and IPTG.
      3. Amplify the FCP:NAT cassette from domesticated pTpFCP:NAT with forward primer: tggtctcaggagCTCGAGGTCGACGGTATC and reverse primer aggtctcaagcgCGCAATTA ACCCTCACTAAAGG (Table 3, numbers 9 and 10) using HF Phusion DNA polymerase. Clone into L1 backbone pICH47732. This module is already available from Addgene.
      4. Clone the L0 FCP promoter, L0 Cas9:YFP and L0 FCP terminator into L1 backbone pICH47742. This module is already available from Addgene.
      5. Clone the L0 U6 promoter and sgRNA1 PCR product into L1 backbone pICH47751
      6. Clone the L0 U6 promoter and sgRNA2 PCR product into L1 backbone pICH47761
    7. Screen L1 assemblies
      Screen L1 assemblies either by colony PCR as described or restriction digest with XbaI or EcoRV. Sequence inserts using primers: CCCACTCTGTGAAGACAA and GCCAATATATCCTGTCAAACAC which anneal just upstream and downstream of the insert sites respectively.
    8. L2 assembly
      1. Assemble L1 modules into the Level 2 backbone along with the linker pICH41780 which joins the 4th module to the vector.
        Note: If a different number of modules are to be assembled, the appropriate linker needs to be used to ensure the overhang between the last module and the linker is correct. Details of further Golden Gate modules can be found in Engler et al. (2014).
      2. Set up the reaction as shown in Table 7.

        Table 7. Reagents for L1 Golden Gate assembly


      3. Incubate under the following conditions
        37 °C for 5 h
        50 °C for 5 min
        80 °C for 10 min
      4. Transform 5 µl of the reaction into 50 µl of competent E. coli and prepare plasmids using a plasmid mini-prep kit.
      5. L2 modules contain the cassette for kanamycin resistance for selection in E. coli.
        Note: Insertion of the correct inserts should result in removal of a gene for canthaxanthin synthesis resulting in a colour change from orange/pink to white.
    9. Screen L2 assembly
      1. Check the plasmid is the correct size by digesting with XbaI or EcoRV and running on an agarose gel.
        Note: Restriction sites will vary depending on the construct–choose enzymes that will linearize the plasmid or give a banding pattern that is distinct from the original L2 backbone.
      2. Sequence using the primers shown in Table 8.

        Table 8. Primers for sequencing L2 constructs


      3. Once plasmids have been successfully screened, perform a plasmid maxi-prep kit to produce enough plasmid for transformation.
      4. Ethanol precipitate plasmids to remove any traces of salt and dilute to 1 µg/µl.
    10. Transform the construct into T. pseudonana
      1. Transformation of the CRISPR-Cas construct into T. pseudonana follows the T. pseudonana protocol set out by Poulsen et al. (2006) and the P. tricornutum transformation protocol from Kroth (2007). For ease of use, a combination of both protocols is included in this paper.
      2. Where possible all steps should be prepared aseptically under a laminar flow hood.
      3. Grow T. pseudonana strain CCMP 1335 to exponential phase (approximately 1 x 106 cells/ml) in 24 h light (100-140 μE) at 20 °C in half salinity media (all salt concentrations are halved compared to the full salinity Aquil media recipe, but nutrients, vitamin and trace metal concentrations remain the same). Algal media recipes can be found in full on the Bigalow website (https://ncma.bigelow.org/algal-recipes).
        Note: Full salinity media such as Aquil (Price et al., 1989) can be used to culture T. pseudonana but cells grow just as well in half salinity and the salt concentration needs to be reduced for effective nourseothricin activity.
      4. Prepare tungsten particles
        1. The following preparation uses 60 mg of tungsten particles but can be scaled down.
        2. Weigh 60 mg of 0.7 µm tungsten particles.
        3. Wash by adding 1 ml of 100% ethanol and vortex for 3 min.
        4. Centrifuge at 13,000 x g for 1 min and remove supernatant.
        5. Add 1 ml of sterile, nuclease-free water and mix. Re-centrifuge and remove supernatant.
        6. Repeat the above step two additional times.
        7. Resuspend the particles in 1 ml of water and aliquot 50 µl into tubes.
        8. Store at -20 °C for up to two weeks or keep on ice until needed.
      5. Prepare plates for shooting and selection
        1. Plates for shooting are made with 1.5% agar and half salinity media.
        2. Plates for selection are made with 0.8% agar, half salinity media and 100 µg/ml nourseothricin.
        3. When making plates, in order to avoid precipitation, separately make up and autoclave 2x concentrated agar solution and 2x concentrated media (or in this case full salinity, as the final media needs to be half salinity), without nutrients, vitamins, trace metals or antibiotics. Once solutions have cooled to 50 °C, mix and add nutrients, vitamins, trace metals and, in the case of the selective plates, antibiotics. Plates can then be poured.
      6. Prepare cells
        1. 5 x 107 cells are used per shot and shots are performed in triplicate. A positive control and negative control are also carried out. As a result transformation of one construct plus controls requires enough culture for 9 shots = 4.5 x 108 cells.
        2. Either, gently spin cells down in a centrifuge at 3,000 x g for 10 min and discard all the supernatant. Resuspend in 100 µl of media per shot. 100 µl of suspension is then spread in a 5 cm diameter circle in the centre of each 1.5% agar plate, and allowed to dry at room temperature.
        3. Or, filter 5 x 107 cells onto 47 mm diameter, 1.2 µm isopore filters using gentle vacuum filtration. Place the filter onto a 1.5% agar plate.
      7. Prepare particles
        1. Add the following in order to a 1.5 ml microcentrifuge tube:
          1) 50 µl of prepared tungsten particles.
          2) 5 µg of the plasmid in 5-10 µl of water (for the negative control just use water).
          3) 50 µl of 2.5 M CaCl2 (store solution at -20 °C).
          4) 20 µl of 0.1 M spermidine (store solution at -20 °C for up to one month).
        2. Vortex the tube for 1 min.
        3. Briefly spin down the particles in a centrifuge and remove the supernatant.
        4. Add 250 µl of 100% ethanol and vortex until homogenous.
        5. Briefly spin down the particles in a centrifuge and remove the supernatant.
        6. Resuspend in 50 µl of 100% ethanol. Store on ice. Particles need to be used within 1 h of preparation.
      8. Microparticle bombardment
        1. Set up the particle gun according to manufacturer’s instructions.
        2. Clean the particle gun by wiping with 70% ethanol.
        3. Autoclave the rupture disc retaining cap, microcarrier launch assembly, macrocarrier holders, stopping screes and tweezers.
        4. Sterilise 1,350 psi rupture discs by dipping in isopropanol and allow to dry.
        5. Sterilise macrocarriers by dipping in 70% ethanol and allowing to dry.
        6. Before shooting cells, the helium lines need to be cleared. This can be carried out by performing the following steps required for shooting but without using cells or particles.
        7. Place the macrocarriers inside the macrocarrier holders.
        8. Vortex the coated tungsten particles, and keeping the particles mixed, pipette 10 µl onto the centre of the macrocarrier. Temporarily turn off the laminar flow hood whilst drying particles to prevent loss.
        9. Place a 1,350 psi rupture disc into the retaining cap, and screw the cap tightly into the assembly using the provided tool.
        10. Once the particles have dried, load the macrocarrier and stopping screen into the launch assembly. The launch assembly is then placed in the biolistic chamber.
        11. Place a plate with cells at a flight distance of 7 cm in the biolistic chamber.
        12. Close and turn on the vacuum pump until a vacuum of 25 Hg is reached.
        13. Fire particles into the cells.
        14. Following particle bombardment rinse cells from each plate into 25 ml of media without antibiotics and incubate for 24 h under standard growth conditions.
        15. Following the 24 h incubation, count cells and plate 5 lots of 5 x 106 cells from each transformation onto selective plates.
        16. Using the cells from the negative control, also spread cells onto plates without antibiotics as a positive growth control.
          Note: T. pseudonana requires relatively small concentrations of cells to be plated out in order for nourseothricin to be effective compared to other diatom transformation systems such as P. tricornutum. As a result, there will be a large quantity of transformed cells left in media after plating. If desired, additional plates can be spread, or liquid cultures can be maintained until colonies start to appear on plates in case further plates are needed.
    11. Screen transformants by PCR/Band shift assay
      1. Once colonies appear (after approximately 10 days), transfer into selective liquid media.
      2. Resuspend a colony in 20 µl of media and transfer 10 µl to a PCR tube for colony PCR. The remaining 10 µl can be used to grow up individual cell lines.
        Place the remaining 10 µl in 1 ml of selective liquid media. Once cells reach around 1 x 106 cells/ml, this can then be used to inoculate larger volumes.
      3. Spin the 10 µl of cells, set aside for colony PCR, down in a centrifuge for 1-2 min at full speed and remove the supernatant.
      4. Resuspend cells in lysis buffer (see Recipes).
      5. Incubate on ice for 15 min.
      6. Incubate at 95 °C for 10 min.
      7. Use 1 µl as template in a 20 µl PCR reaction using Taq polymerase.
      8. Amplify the target region. For the urease gene described in Hopes et al. (2016) the forward primer: AAACAGACCACCTTCACCTC and reverse primer: CTCCACCTGTACGTCTCG were used. Run PCR products on a 1% w/v agarose gel at 4-5 V/cm (80-100 V with the specified gel system). In the Hopes et al. (2016) paper, as the deletion was only 37 nt, instead of the 1% w/v gel, a 2% w/v agarose gel was run at 3 V/cm (60 V with the specified gel system) to visualise the shift in band size compared to the WT. See Figure 2 for gels demonstrating deletion induced band shifts.
      9. Amplify a section of the Cas9 gene using forward primer: CCGAGACAAGCAGAGTGGAAAG and reverse primer: AGAGCCGATTGATGTCCAGTTC. Run PCR products on a 1% w/v agarose gel at 4-5 V/cm (80-100 V with the specified gel system) to check for the presence of Cas9.
        Note: We found that around 11% of primary clones contained Cas9 but 100% of clones with Cas9 contained a deletion. Screen at least 30-40 colonies to isolate several primary clones with deletions.
      10. Include WT samples and negative controls using water instead of template in all PCR reactions.
      11. Due to the nature of CRISPR-Cas, primary colonies can be mosaic with a mixture of WT and mutant cells (Figure 2). In order to procure a clean cell-line with one clone, cultures from primary clones, with evidence of mutations, need to be re-spread onto additional selective plates. Streak 100 µl of cells from the primary cultures at exponential phase onto new selective plates and incubate until colonies appear. Sub-clones can then be screened as shown above.


        Figure 2. Screening of the CRISPR-Cas induced genome editing in T. pseudonana. Screening by PCR and sequencing. Expected sgRNA cut indicated by ↓. Red text shows the sgRNA target sequence and bold text the PAM motif. Primary clones: Several primary clones contain sequences showing both CRISPR-induced mutations and the wildtype (WT) sequence, as seen by the presence of two bands following PCR, these are indicated by (+WT). Remaining samples represent different cell lines. M1 shows a 4 nt deletion from the second sgRNA whilst mutants M2-M4 show a 37 nt deletion between the two CRISPR-Cas cut sites. Sub-clones: The gel shows examples of a selection of sub-clones derived from the primary clones. Sub-clones are labelled according to the primary clone and sub-clone number. With the exception of M1_9, which gives a WT sequence and 4 nt deletion as seen in the primary clone, all sub-clones chosen for sequencing are bi-allelic. Two-thirds of sequenced bi-allelic sub-clones show a single sequence with a 37 nt deletion suggesting that both alleles carry the same mutation. In sub-clones where mutations differ between alleles both sequences are shown.

    12. Screen by restriction digest (optional)
      1. Amplify the target region.
      2. Digest the PCR product with the restriction enzyme that coincides with the sgRNA cut site/resides within the expected deletion site.
      3. Run fragments on an agarose gel. If CRISPR-Cas has induced a mutation, then the restriction site will no longer be functional and fragments will remain uncut. Take care to amplify fragments or use restriction enzymes that give a unique restriction site. Otherwise several small fragments, which are difficult to visualise may occur, regardless of mutation.
      4. If enriching for edited sequences, extract genomic DNA and perform the restriction digest. PCR amplify the target. Only WT sequences should be cut, allowing preferential amplification of the mutant fragment.
    13. Screen by sequencing
      1. Sequence PCR products to verify deletions (Figure 2) using a service such as the Eurofins Mix2seq kit.
      2. It can also be worth sequencing products which appear to have a WT sequence based on size. This can identify mutants which arise from activity of one sgRNA, leading to smaller indels at only one site. An example of this can be seen with M1 (Figure 2).

Data analysis

  1. Screening is carried out using the band shift assay and/or restriction site loss as described above. This allows assay products to be run on a gel to easily determine genome editing either by deletion (band shift assay) or mutation of the target site (restriction site loss). Products are then sequenced to confirm deletions/mutations.
  2. For primary clones around 30-40 colonies are tested for both Cas9 and deletions.
    Note: We sequenced PCR products from all primary colonies, irrespective of the presence of Cas9 or deletions, and found that 11% of clones contained Cas9. 100% of clones with Cas9 contained a mutation–75% of the mutant clones contained deletions but the remaining 25% showed a mutation at one target site with a band similar to that of the WT. As a result, it can be worth screening for Cas9 as an indicator for potential mutations, as editing may not always result in a deletion. Cas9 clones can then be sub-cloned as described and subjected to further analysis including restriction site loss and sequencing.
  3. Around 30 sub-clones are typically tested by band shift assay to determine the bi-allelic deletion frequency. This is calculated as the number of sub-clones with only the lower band (indicative of a deletion in both alleles), compared to the total number of sub-clones tested. Other colonies may give only the higher WT band or a mixture of both.
  4. Further analysis is dependent on which gene/sequence is being targeted. For example, when targeting the urease gene (which allows cells to use urea as a nitrogen source) in T. pseudonana, cell-lines were grown in media with either nitrate (positive control) or urea as the sole nitrogen source (Hopes et al., 2016). Cell counts and cell size were measured once a day until stationary phase was reached. Growth curves/cell size was then compared between cell lines (WT and mutant) and growth conditions (nitrate or urea) to determine nitrogen starvation or limitation in edited cell lines.

Recipes

  1. LB media/LB agar


    Combine tryptone, yeast extract and sodium chloride. If making plates also add agar. Make up to 1 L with MilliQ/distilled water and autoclave. If making selective media add the relevant antibiotic once the media has cooled to 50 °C. Plates are also poured once media has reached 50 °C. The recipe can be scaled up or down as needed
  2. Lysis buffer

Acknowledgments

Thanks to The Sainsbury Laboratory for supplying the Golden Gate destination vectors and linkers. This work has been funded by a PhD studentship from the Natural Environment Research Council (NERC) awarded to Amanda Hopes. TM acknowledges partial funding from NERC (NE/K004530/1) and the School of Environmental Sciences at University of East Anglia, Norwich.
Author’s contributions: AH and TM conceived the project. AH designed and developed the protocol with input on Golden Gate cloning and the band shift assay method from Vladimir Nekrasov. AH and NB determined the U6 promoter for F. cylindrus and IG tested its activity in the same species. AH wrote the paper.
This protocol is based on the work presented in Hopes et al. (2016).
The authors declare that they have no competing interests.

References

  1. Belhaj, K., Chaparro-Garcia, A., Kamoun, S. and Nekrasov, V. (2013). Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9(1): 39.
  2. Brooks, C., Nekrasov, V., Lippman, Z. B. and Van Eck, J. (2014). Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166(3): 1292-1297.
  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. Engler, C., Youles, M., Gruetzner, R., Ehnert, T. M., Werner, S., Jones, J. D., Patron, N. J. and Marillonnet, S. (2014). A golden gate modular cloning toolbox for plants. ACS Synth Biol 3(11): 839-843.
  5. Hopes, A., Nekrasov, V., Kamoun, S. and Mock, T. (2016). Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. Plant Methods 12: 49.
  6. Kroth, P. G. (2007). Genetic transformation: a tool to study protein targeting in diatoms. Methods Mol Biol 390: 257-267.
  7. 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.
  8. Nymark, M., Sharma, A. K., Hafskjold, M. C., Sparstad, T., Bones, A. M. and Winge, P. (2017). CRISPR/Cas9 gene editing in the marine diatom Phaeodactylum tricornutum. Bio Protoc e2442.
  9. Nymark, M., Sharma, A. K., Sparstad, T., Bones, A. M. and Winge, P. (2016). A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 6: 24951.
  10. Pinto, F. L. and Lindblad, P. (2010). A guide for in-house design of template-switch-based 5' rapid amplification of cDNA ends systems. Anal Biochem 397(2): 227-232.
  11. Poulsen, N., Chesley, P. M. and Kröger, N. (2006). Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). J Phycolo 42(5):1059-1065.
  12. Price, N. M., Harrison, G. I., Hering, J. G., Nirel, P. M., Palenik, B. and Morel, F. (1989). Preparation and chemistry of the artificial algal culture medium aquil. Biological Oceanography 6: 443-461.
  13. Rastogi, A., Murik, O., Bowler, C. and Tirichine, L. (2016). PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing. BMC Bioinformatics 17(1): 261.
  14. Sakuma, T., Nishikawa, A., Kume, S., Chayama, K. and Yamamoto, T. (2014). Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep 4: 5400.
  15. Weber, E., Engler, C., Gruetzner, R., Werner, S. and Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS One 6(2): e16765.
  16. Zheng, Q., Cai, X., Tan, M. H., Schaffert, S., Arnold, C. P., Gong, X., Chen, C. Z. and Huang, S. (2014). Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques 57(3): 115-124.

简介

最近为三角褐指藻(Phaeodactylum tricornutum)和海绵假丝酵母(Thalassiosira pseudonana)建立了硅藻基因组编辑。 目前的协议,虽然开发的 T。 pseudonana ,可以修改编辑任何硅藻基因组,因为我们利用灵活,模块化的金门克隆系统。 主要步骤包括如何设计构建使用金门克隆靶向两个网站,允许一个精确的删除被引入目标基因。 解释转化方案,以及使用带移位测定和/或限制性位点丢失进行筛选的方法。

【背景】CRISPR-Cas正在迅速成为分子研究的一个关键方法。基于在细菌和古细菌中发现的病毒防御机制,CRISPR-Cas诱导基因组中精确位置的双链断裂(DSBs)。它涉及使用与CRISPR RNA(crRNA)和反式激活crRNA(trRNA)形成的嵌合单引导RNA(sgRNA)形成复合物的Cas9核酸酶。特异性由crRNA中的20nt序列提供,其与基因组中的靶标相对应,并通过碱基互补性将Cas9引导至正确的位点。这意味着该系统很容易编程,只要改变20个核苷酸序列即可将其应用于新的目标,前提是在20nt目标序列之后,基因组中存在原型间隔区相邻基序(PAM)。对于从酿脓链球菌分离的常用Cas9,PAM序列是NGG。然后通过在通过非同源末端连接(NHEJ)进行不完全修复后引入突变,切割两个位点并引入精确缺失或通过同源重组来实现基因编辑。由于其在第一个真核系统中的应用(Cong等人,2013; Mali等人,2013),CRISPR-Cas已被用于广泛的基因组编辑包括两个硅藻种类的生物体的范围(Hopes等人,2016; Nymark等人,2016)。 Nymark等(2016)使用单个sgRNA将突变引入到三角褐指藻的基因组中 - 在Bio-protocol中可以找到该方案(Nymark等,et al。 ,2017)。本文中发表的方案着重于使用两种sgRNA引入精确缺失并使用之前描述的用于鉴定高等植物中的突变体的带移测定进行简单筛选的 Thalassiosira pseudonana 中的基因编辑(Brooks 等人,,2014)。另外,该方法使用Golden Gate克隆(Weber等人,2011; Engler等人,2014) - 灵活的模块化系统,其允许序列和盒子是易于互换,多个模块一次装配。虽然该协议描述了靶向相同基因中的两个位点以引入缺失,但是可以容易地改变构建体以靶向不同基因或更多数量的基因,如以前由Sakuma等人(2014)所示的。他们利用金门克隆系统证明了7个基因的敲除。

关键字:CRISPR-Cas, 硅藻, 假微型海链藻, Golden Gate, U6启动子, 带偏移实验法, 限制性位点丢失, 三角褐指藻

材料和试剂

  1. 0.2ml PCR管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:AB0620)
  2. 10μl过滤吸头(STARLAB INTERNATIONAL,目录号:S1121-3810)
  3. 10μl移液枪头(STARLAB INTERNATIONAL,目录号:S1111-3810)
  4. 200μl移液枪头(STARLAB INTERNATIONAL,目录号:S1111-0810)
  5. 200μl过滤吸头(STARLAB INTERNATIONAL,目录号:S1120-8810)
  6. 1,000μl移液枪头(STARLAB INTERNATIONAL,目录号:S1111-6810)
  7. 直径为47毫米的1.2微米等孔过滤器(默克,目录号:RTTP04700)(可选)
  8. 1.5ml微量离心管(Fisher Scientific,目录号:11926955)
  9. 直径90mm的培养皿(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:263991)
  10. 0.7微米(M10)钨颗粒(微载体)(Bio-Rad Laboratories,目录号:1652266)
  11. 培养管(Fisher Scientific,目录号:11317824)
  12. 大载体(Bio-Rad Laboratories,目录号:1652335)
  13. Macrocarrier holder(Bio-Rad Laboratories,产品目录号:1652322)
  14. 停止屏幕(Bio-Rad Laboratories,产品目录号:1652336)

  15. 1350psi爆破片(Bio-Rad Laboratories,目录号:1652330)
  16. Thalassiosira pseudonana CCMP 1335(Bigelow) https://ncma.bigelow.org/ccmp1335
  17. 具有One Shot TM TOP10 E的pCR TM TM / GW / TOPO TM TA克隆试剂盒。 (Thermo Fisher Scientific,Invitrogen TM,目录号:K250020)。
  18. 高效率的主管E。 (New England Biolabs,产品目录编号:C2987H)(例如,NEB 5-alpha Competent E.coli(高效率))。
  19. Bsa(新英格兰生物实验室,目录号:R0535S)
  20. (Thermo Fisher Scientific,Thermo Scientific TM,目录号:ER1011)
  21. Taq DNA聚合酶(例如,GoTaq Flexi DNA聚合酶)(Promega,目录号:M8291)
  22. pICH47732:FCP:NAT(Addgene,目录号:85984)或pICH47732(Addgene,目录号:48000)
  23. pICH47742:FCP:Cas9YFP(Addgene,目录号:85986)或pICH47742(Addgene,目录号:48001)
  24. pICH47751(Addgene,目录号:48002)
  25. pICH47761(Addgene,目录号:48003)
  26. pICH41780(Addgene,目录号:48019)
  27. pAGM4723(Addgene,目录号:48015)
  28. pICH86966 :: AtU6p :: sgRNA_PDS(Addgene,目录号:46966)
  29. pCR8 / GW:U6(Addgene,目录号:85981)
  30. Q5定点突变试剂盒(新英格兰生物实验室,目录号:E0554S)
  31. 高保真度DNA聚合酶(例如Phusion High-Fidelity DNA Polymerase)(New England Biolabs,目录号:M0530)
  32. PCR清洁试剂盒(例如,Illustra GFX PCR DNA和Gel Band纯化试剂盒)(GE Healthcare,目录号:28-9034-70)
  33. 质粒小量制备试剂盒(,例如,PureYield质粒小量制备系统)(Promega,目录号:A1223)
  34. 无水乙醇(VWR,目录号:20821.330)
  35. 琼脂糖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17850)
  36. 用于凝胶电泳的溴化乙锭(Thermo Fisher Scientific,Invitrogen TM,目录号:15585011)
  37. 用于凝胶电泳的TAE缓冲液(Thermo Fisher Scientific,Invitrogen TM,产品目录号:15558026)
  38. T4 DNA连接酶(Promega,目录号:M1794)
  39. 5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷(X-Gal)(Sigma-Aldrich,目录号:B4252-50MG)
  40. IPTG(Sigma-Aldrich,目录号:I6758-1G)
  41. 半盐度Aquil媒体(价格 ,1989年,完整的食谱在:
  42. Nourseothricin clonNAT(Werner BioAgents)
  43. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670-100G)
  44. 亚精胺(Sigma-Aldrich,目录号:S0266-1G)
  45. 异丙醇(VWR,目录号:BDH1133-1LP)
  46. 压缩氦气供应
  47. Mix2seq套件(Eurofins)
  48. 胰蛋白胨(Sigma-Aldrich,目录号:T7293)
  49. 酵母提取物(ForMedium,目录号:YEA01)
  50. 氯化钠(Sigma-Aldrich,目录号:S7653)
  51. 琼脂(ForMedium,目录号:AGA02)
  52. 氨苄青霉素钠盐(Sigma-Aldrich,目录号:A0166-5G)
  53. 大观霉素二盐酸盐五水合物(Sigma-Aldrich,目录号:S4014-5G)
  54. 羧苄青霉素二钠盐(Sigma-Aldrich,目录号:C3416-250MG)
  55. 卡那霉素硫酸盐(Sigma-Aldrich,目录号:60615-5G)
  56. Triton X-100(Sigma-Aldrich,目录号:T8787)
  57. Tris-HCl pH 8.0(Thermo Fisher Scientific,Invitrogen TM,产品目录号:15568025)
  58. EDTA(Sigma-Aldrich,目录号:EDS)
  59. LB培养基/ LB琼脂(见食谱)
  60. 裂解缓冲液(见食谱)

设备

  1. 移液器(Thermo Fisher Scientific,型号:Finnpipette,体积:2μl,10μl,200μl和1,000μl)
  2. NanoDrop ND-1000(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 1000)
  3. 层流罩(Walker安全柜,型号:Class II 1290 Recirc Gen 6)
  4. 台式微型离心机(Eppendorf,型号:5418 R)
  5. 离心机(海蒂诗实验室技术,型号:ROTINA 380 R)

  6. 高压灭菌器(Prestige Medical,目录号:210004)
  7. 镊子
  8. 涡(Mo Bio Laboratories,型号:Vortex Genie 2)
  9. 摇动培养箱(37°C)(IKA,型号:KS 4000 i control)
  10. 在20°C的光照培养箱(三洋多功能环境测试室)
  11. PCR热循环仪(Bio-Rad Laboratories,型号:T100 TM热循环仪)
  12. 用于计数细胞的光学显微镜和Neubauer室(VWR,目录号:631-0696)或Coulter Counter(Beckman,型号:Multisizer 3)
  13. PDS-1000 / He生物射弹微粒输送系统(粒子枪)(Bio-Rad Laboratories,目录号:1652257)
  14. 用于细胞过滤的真空泵(Welch Vacuum,型号:2534C-02)(可选)
  15. Nalgene过滤漏斗(Thermo Fisher Scientific,Thermo Scientific TM,型号:DS0320-5045)(可选)
  16. 用于微粒输送系统的高真空泵(Uniweld Products,型号:HUMM•VAC HVP6)
  17. 凝胶电泳槽(Fisher Scientific,Fisherbrand TM,型号:Midi Plus水平凝胶系统)
  18. 电泳电源(配套,型号:EV243)

程序

  1. 设计sgRNAs
    sgRNA被设计成一旦引入突变便于筛选。该协议描述了两种筛选方法:带移位测定和限制性位点丢失。我们发现带移位测定法是最有效的 - 这种方法是基于使用两种sgRNA引入缺失,所述sgRNA可以通过扩增靶并在琼脂糖凝胶上寻找更短的带来进行筛选。它具有引入非常精确和大量删除的附加好处。这可以作为一个独立的方法,而不需要限制站点的丢失。限制性位点丢失稍微复杂一些,并且限制了sgRNA的设计,因为DSB需要与限制性识别位点(优选至少5nt位点切割位点)重合。当通过CRISPR-Cas引入突变时,限制性位点不再有活性,限制酶不能切割。这个方法已经包含了,因为如果只有一个sgRNA有功能的话,它可以是富集突变目标或筛选的有用方法。
    1. 该协议使用嵌合sgRNA与S一起使用。 pyogenes Cas9的序列如下:

      NNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC的 TTTTTT
      1. 带下划线的序列显示了与基因组中20nt靶序列相对应的可编程区域。
      2. 在基因组中,这个序列紧接着是NGG PAM。因此需要在基因组中用序列N 20 NGG来鉴定靶标。
        注意:sgRNA本身的目标序列应该只包含序列 - 即它不应包含PAM。
      3. 另外,使用募集RNA聚合酶III(pol III)的U6启动子来驱动小的非编码sgRNA的表达。这意味着sgRNA需要以“G”开始以激活转录。这可以被结合到目标设计中,这意味着需要识别具有序列GN19 NGG的目标,或者可以将额外的G添加到sgRNA的5'端。
      4. 使用pol III表达sgRNA时,粗体字6的字符串作为转录的终止子。  
    2. 使用两种工具来设计sgRNA靶:
      1. RGEN Cas-Designer: http://www.rgenome.net/
        1. 包括几个藻类和硅藻基因组的非目标查找器,包括T。 pseudonana 。这使得研究人员可以拒绝目标,如果类似的序列发生在基因组的其他地方,也可能会被削减。
        2. 允许指定的序列被省略,如内含子。
        3. 如果研究人员希望更改Cas9,可以搜索具有多个不同PAM的目标。
      2. Broad Institute sgRNA设计师: https://portals.broadinstitute.org/gpp/公共/分析工具/ sgrna-设计
        这是用来给目标的分数。该程序使用的算法是基于经验测试的sgRNA,并预测其切割效率。尝试选择切割效率尽可能接近1的目标。
    3. 设计两个切割位点相距约50-100 bp的sgRNA
      1. 删除50-100bp使得编辑的目标与WT之间有明显的区别(Belhaj等人,2013; Brooks等人,2014)。 > 注意:如果需要,可以选择更大的缺失 - 研究表明较大缺失的突变发生较少,但是直到10kb的缺失仍然有效(Zheng et al。,2014)。
      2. 如果预期有帧移动,则可以靶向基因的活性位点或基因的起点。
        注意:整个基因也可以通过靶向基因的末端或终止子/启动子的末端来去除。如果活性位点是未知的,或者在设计sgRNA时存在问题,例如当存在大量的重复序列时,这可能是有用的。
    4. 如果使用限制性丢失的方法进行筛选,则需要设计靶标,使得Cas9切割位点位于限制识别位点或位于删除位点内。
      可以使用诸如Phyto CRISPRex(Rastogi等人,2016)之类的软件或通过限制发现者(诸如浮雕限制工具())运行目标基因来确定将位点限制在限制性位点上的目标和限制性位点的交叉引用位置与Cas9切割位置的交叉参考位置网站/删除。
      注意:设计完全符合这些规范的sgRNA可能并不总是可能的,例如,由于定位限制,可能需要使用具有较低目标分数的sgRNA(我们已成功使用了评分为0.4的sgRNA) ,或者稍小的删除可能是必要的。请记住,即使有可能,目标仍然需要经验测试,因为其他因素,如无障碍可能会影响他们的活动。因此,建议为每个目标基因/序列设计至少两组sgRNA。

  2. 构建
    使用Golden Gate系统组装构建体,该系统使用预先设定的主链和序列之间的预设突出端来建立模块中的质粒(Weber等人,2011)。金门克隆背后的基础是使用在限制性识别位点外切割的限制性酶BsaI和BpiI,从而产生特定的4nt突出端。这允许片段以特定顺序连接在一起,并且在每个级别的单个反应内。由于限制识别位点和切割位点是分开的,插入物的限制性消化和连接导致完全移除位点,这意味着一旦实现正确的组装,构建体就不能再被切割。这是在三个级别上进行的,L0,L1和L2。
    L0模块含有序列,如启动子,编码区和终止子。这些组装在一起形成L1骨干中的盒子。然后将L1模块组装到最终的L2骨架中以创建具有CRISPR-Cas所需的所有必需序列的构建体。本节将介绍 T中用于CRISPR-Cas的构建组件。 pseudonana 用于靶向基因组中的两个位点,然而,它可以很容易地适用于其他生物体,不同的模块(例如,不同的Cas9,启动子或选择性标记)比两个网站。后者使这种克隆方法适用于多个基因的目标。
    L1骨干包含BsaI站点以允许克隆L0模块以及BpiI站点以允许将L1模块克隆到L2骨干中。通过用Taq DNA聚合酶扩增所需片段并克隆入pCR TM 8 / GW / TOPO载体来产生L0模块。为了将L0模块克隆到L1骨架中,当扩增所需片段时,需要通过正向和反向引物来包含<!> Bsa I位点。另外,消解 Bsa I站点时创建的突出部分需要在相邻模块之间相同。在这个协议中使用的所有L1主链在用 Bsa I消化时给出GGAG和GCTT的标准化突出端。结果,要克隆的序列中的第一个L0模块必须在5'端包含GGAG突出端,并且最后一个模块在3'端必须包含GCTT突出端。 L0模块之间的内部悬垂由研究人员决定,可设计为无缝连接。下面是一个克隆到L0的扩增子的例子,显示序列的开始和结束:

    Ť的 GGTCTC 一个 GGAG AGCTTGCGCTTTTTCCGAG ... CTGATTTACCAA ACCAATACCAA AATG 吨的 gagacc < / strong> t

    粗体字母表示BsaI位点,下划线区域显示由限制性酶切位点产生的4nt突出端,斜体区域显示岩藻黄素叶绿素a / c结合蛋白( FCP)启动子序列。第一个突出部分与L1主干结束处的突出部分相对应。第二个突出部分与相邻的Cas9模块的突出部分相对应。这样做的目的是为了在Cas9的开始部分包含ATG,从而实现无缝连接。
    图1给出了在这个协议中使用的汇编过程的一般概述。


    图1.用于装配CRISPR-Cas构建体的Golden Gate克隆系统的概述。 对于 T。 pseudonana,L0 U6启动子,L1 FCP:NAT盒和L1 FCP:Cas9:YFP盒的质粒已经可以从Addgene获得(参见以下链接)。额外的L0 em。 pseudonana 模块请联系作者。

    1. 该协议描述了所有模块的创建和组装,以便研究人员可以根据需要修改整个系统,但是,所描述的一些L0和L1模块已经可以从Addgene获得 https://www.addgene.org/Thomas_Mock/ 。另外,在同一个网站上很快就可以从Addgene获得 P. tricornutum 的模块。
    2. 从插入中删除 Bsa I和 Bpi I网站
      1. 原始的pTpFCP:NAT(Poulsen等人,2006)用于耐磺丝菌素抗性的盒含有在FCP启动子中的BsaI位点和BipI /我在NAT基因网站。这些网站需要通过定点突变,以防止金门克隆过程中不必要的限制消化。
      2. 使用pTpFCP / NAT(Poulsen等人,2006)作为模板,使用正向引物:TCCGCGGCAGaTCTCTGTCG,反向引物:AGAAGTACCGTGTTGTTGCAGTG和Q5位点去除BsaI位点 - 定向诱变(SDM)试剂盒(NEB)。用所得到的pTpFCP / NAT质粒(包含驯化的FCP启动子)作为模板,在正向(F)引物:CGACACCGTaTTCCGCGTCAC和反向(R)引物上重复在NAT基因中的BpiI位点上的SDM: GTGGTGAAGGACCCATCCAG。
        注意:如果使用TpFCP / NAT磁带盒,则不需要将此序列归属,因为已经可以从Addgene获得L1 pICH47732:FCP:NAT模块。
      3. 如果使用的协议序列不同于本协议中描述的序列,则按照上述类似的方式移除BMI I和BSS I站点。 NEB提供了用于设计SDM引物的软件( http://nebasechanger.neb.com/ ) 。
    3. 在硅藻中使用U6启动子
      1. 所有已知的用于硅藻的U6启动子显示在表1中。三角茄(T. tricornutum)首先由Nymark等人(2016)描述,而T. pseudonana(Hopes等人, ), 2016)和我们的实验室已经确定了Fragilariopsis cylindr us启动子(未发表)。另外,对于T. pseudonana 和 F的U6启动子的确切末端。 cylindrus 已经使用5'TSO RACE(Pinto和Lindblad,2010)方法凭经验确定。

        表1.在硅藻中的U6启动子和缺少与sgRNA组装的瘢痕所需的突出端。 TATA框以黄色突出显示,突出部分(启动子的最后4个字符)为绿色。


      2. 当通过Golden Gate克隆U6:sgRNA盒时,确保U6启动子的末端和sgRNA的起始之间的无疤连接是重要的,以使得额外的核苷酸不在sgRNA的5'末端转录。这是通过设计U6启动子和sgRNA之间的4nt突出端作为U6启动子的最后4nt而实现的。在U6启动子和sgRNA之间产生无瘢痕连接所必需的突出部分对于每个U6启动子而言是不同的,并且需要将突出端构建到用于扩增U6启动子的反向引物和用于扩增sgRNA的正向引物中。这些引物可以在下面的表2中找到:

        表2.用于引入硅藻U6启动子和sgRNA之间的突出端的引物。 Bsa I位点以下划线标出,突出部分以绿色突出显示。 N表示目标站点。大写字母A,C,G或T显示将与初始模板退火的引物部分。


    4. 创建L0模块
      1. 表3中的所有金门PCR反应都可以找到完整的引物列表,退火温度和延伸时间。

        表3.用于Golden Gate克隆的PCR引物。 Bsa 我的网站有下划线。突出显示为粗体。 F表示正向引物,R表示反向引物。正向sgRNA引物中的Ns序列需要被为每个基因/靶标设计的20nt靶序列取代。大写字母A,C,G或T显示将与初始模板退火的引物部分。


      2. L0模块含有一个Sm / SpR基因,可以通过E中的壮观霉素进行选择。大肠杆菌。
      3. 使用Taq DNA聚合酶,使用F引物:tggtctcaggagAGCTTGCGCTTTTTCCGAG和R引物aggtctcacatTTTGGTATTGGT TTGGTAAATCAG(表3,编号1和2)从驯化的pTpFCP / NAT盒中扩增FCP启动子。将PCR产物直接克隆到pCR TM 8 / GW / TOPO载体中。
      4. 使用高保真(HF)Phusion聚合酶,使用F引物:aggtctcaaATGGACAAGAAGTACTCCATTGG和R引物:aggtctcaaagcTCACTTGTACAGCTCGTCCATG(表3,编号3和4)扩增Cas9:YFP。用PCR纯化试剂盒清洁反应,并在72℃下与Taq聚合酶(表4)温育20分钟,然后克隆到pCR TM 8 / GW / TOPO载体中。
        注意:用HF聚合酶进行Cas9的扩增以减少扩增期间的错误机会,因为Cas9:YFP是4.8kbp的大序列。由于HF聚合酶不添加3''A突出端,需要与Taq单独孵育。

        表4. Cas9的试剂:YFP PCR与Taq孵育以增加“A”突出物


      5. 使用Taq DNA聚合酶,使用F引物:aggtctcagcttATACTGGATTGGTGAATCAATG和R引物:tggtctcaagcgGAGAA CTGGAGCAGCTAC(表3,编号5和6)从家驯的pTpFCP / NAT扩增FCP终止子。将PCR产物直接克隆到pCR TM 8 / GW / TOPO载体中。
      6. 扩增来自 T的U6启动子。假基因组DNA使用F引物:cggtctcaggagCTTCATCAAGAGAGCAACCA和R引物:aggtctcaACAATTTCGG CAAAACGT(表3,编号7和8),使用Taq DNA聚合酶。将PCR产物直接克隆到pCR TM 8 / GW / TOPO载体中。关于在硅藻中使用U6启动子,请参阅第3节。该模块已经可以从Addgene获得。
      7. 由于目标序列之间的指导序列发生改变,sgRNA不能被克隆到L0载体上,而是直接组装成L1载体作为PCR产物。当扩增支架时通过F引物引入靶序列。使用F引物:aggtctcattgtNNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGCAAG和R引物:tggtctcaagcgTAATGCCAACTTTGTACAAG(表3,编号11和12)扩增来自pICH86966 :: AtU6p :: sgRNA_PDS的支架。使用Taq或HF DNA聚合酶是很好的。 Ns在正向引物中的运行表示靶序列。扩增后,使用PCR清洁试剂盒纯化PCR产物。
    5. 筛选L0模块
      1. 在将所有L0模块组装到L1矢量中之前,屏幕上。使用与用于扩增片段的相同引物进行菌落PCR以克隆到L0模块(表3)或消化L0模块以检查大小。序列L0模块,其使用在pCR TM 8 / GW / TOPO TA试剂盒中提供的引物包含正确大小的插入片段。
      2. 菌落PCR:
        1. 根据表5准备主混合物的待测菌落数,并将20μl等分到PCR管中。

          表5.用于菌落PCR的试剂


        2. 轻轻地向每个菌落轻轻接触10μl移液枪头(标记菌落,以便在筛选后可以将其挑出),使用移液管混匀并短暂旋转。
        3. 运行以下PCR循环。
          1)初始变性:95℃2分钟
          2)35个周期:
          &NBSP;&NBSP;&NBSP; 95°C变性30秒
          &NBSP;&NBSP;&NBSP;退火30秒(退火温度见表3)
          &NBSP;&NBSP;&NBSP;在72℃延伸(退火时间见表3)
          3)在72℃下进行10分钟的最后延伸
      3. 限制摘要:
        用质粒mini-prep试剂盒提取质粒,测量DNA浓度并用Bsa I消化以检测插入物的大小。
        注:使用NanoDrop分光光度计测量DNA浓度。
    6. L1装配
      1. 四个L1模块组装在一起
        pich47732:FCP:NAT(可从Addgene获得)
        pICH47742:FCP:Cas9YFP(可从Addgene获得)
        pICH47751:U6:sgRNA1
        pICH47761:U6:sgRNA2
      2. 对于每个L1组件,金门组件在一个反应中进行。
        1. 建立反应如表6所示。

          表6. L1金门装配试剂


        2. 在以下条件下孵育
          37°C 5小时
          50°C 5分钟
          80°C 10分钟
        3. 将5μl反应物转化成50μl感受态E.大肠杆菌。
        4. L1模块含有用于在大肠杆菌中选择的耐羧苄西林的盒。
          注意:插入正确的插入片段后,应当移除LacZ基因,以便与X-gal和IPTG一起温育时进行蓝色至白色的菌落筛选。
      3. 使用HF Phusion DNA聚合酶,用正向引物:tggtctcaggagCTCGAGGTCGACGGTATC和反向引物aggtctcaagcgCGCAATTA ACCCTCACTAAAGG(表3,编号9和10)从家驯pTpFCP:NAT扩增FCP:NAT盒。克隆入L1骨干pICH47732。该模块已经可以从Addgene获得。
      4. 将L0 FCP启动子L0Cas9:YFP和L0FCP终止子克隆到L1骨架pICH47742中。该模块已经可以从Addgene获得。
      5. 将L0 U6启动子和sgRNA1 PCR产物克隆到L1骨架pICH47751中
      6. 将L0 U6启动子和sgRNA2 PCR产物克隆到L1骨架pICH47761中
    7. 屏幕L1组件
      筛选L1装配,如所描述的通过菌落PCR或用Xba I或Eco Eco RV限制消化。使用引物的序列插入片段:CCCACTCTGTGAAGACAA和GCCAATATATCCTGTCAAACAC,分别在插入位点的上游和下游退火。
    8. L2装配
      1. 将L1模块与链接器pICH41780一起组装到Level 2骨干网中,该链接器将第4个模块连接到矢量上。
        注:如果要组装不同数量的模块,则需要使用相应的链接器来确保最后一个模块和链接器之间的悬空是正确的。进一步的金门模块的详细信息可以在Engler et al。 (2014)。
      2. 建立如表7所示的反应。

        表7. L1金门装配试剂


      3. 在以下条件下孵育
        37°C 5小时
        50°C 5分钟
        80°C 10分钟
      4. 将5μl反应物转化成50μl感受态E.使用质粒小型制备试剂盒制备质粒。
      5. L2模块含有卡那霉素抗性的盒子,用于在E中选择。大肠杆菌。
        注意:插入正确的插入片段会导致角黄素合成基因的去除,导致颜色从橙色/粉红色变为白色。
    9. 屏幕L2组装
      1. 通过用Xba I或Eco Eco RV消化并在琼脂糖凝胶上进行消化来检查质粒的大小是否正确。
        注意:限制性位点将根据构建体选择的酶而变化,这些酶将使质粒线性化,或者提供与原始L2骨架不同的条带模式。
      2. 使用表8所示的引物进行测序。

        表8.用于测序L2构建体的引物


      3. 一旦质粒成功筛选,使用质粒Maxi-prep试剂盒生成足够的质粒用于转化。
      4. 乙醇沉淀质粒去除任何痕量的盐,并稀释到1微克/微升。
    10. 将构造转换为 T。 pseudonana
      1. 将CRISPR-Cas构建体转化到T细胞中。 pseudonana 跟在 T之后。由Poulsen等人提出的pseudonana 协议。 (2006)和 P。来自Kroth(2007)的三角酵母转化方案。为了便于使用,本文包含两种协议的组合。
      2. 在可能的情况下,所有的步骤应该在层流罩下进行无菌处理。
      3. 在20°C,24小时光照(100-140μE)内,将T.pseudonana菌株菌株CCMP1335增殖至指数期(约1×10 6个细胞/ ml)盐度介质(所有的盐浓度都比全盐度Aquil介质配方减半,但营养物质,维生素和微量金属浓度保持不变)。藻类媒体食谱可以在Bigalow网站上找到( https://ncma.bigelow.org/algal-食谱)。
        注意:Aquil(Price等人,1989)等完全盐度培养基可用于培养T.pseudonana,但细胞在半盐度下也能生长,并且需要降低盐浓度以有效地抑制nothothricin活性。
      4. 准备钨颗粒
        1. 以下制备使用60毫克的钨颗粒,但可以缩小。
        2. 称量60毫克0.7微米的钨颗粒。
        3. 加入1毫升100%乙醇并涡旋3分钟,洗净。

        4. 13,000×g离心1分钟,去除上清液。
        5. 加入1毫升无菌,无核酸的水并混合。重新离心并除去上清液。
        6. 重复上述步骤两次。
        7. 重悬在1毫升水中的颗粒,并等分50μL管。

        8. 在-20°C储存长达两个星期或冰上直到需要
      5. 准备用于拍摄和选择的盘子

        1. 用1.5%琼脂和一半盐度介质制作拍摄用的板
        2. 用0.8%琼脂,半盐度培养基和100μg/ ml Nourseothricin进行选择。
        3. 制作平板时,为了避免沉淀,分别补充和高压灭菌2次浓缩琼脂溶液和2次浓缩培养基(或在这种情况下,全盐度,因为最终培养基需要一半的盐度),没有营养素,维生素,微量金属或抗生素。一旦溶液冷却到50°C,混合和添加营养物质,维生素,微量金属和在选择性板材的情况下抗生素。然后可以浇注板。
      6. 准备细胞
        1. 每个镜头使用5×10 7个细胞并且一式三份进行照射。阳性对照和阴性对照也被执行。结果一个构建体加上对照的转化需要足够的培养9次= 4.5×10 8个细胞。
        2. 轻轻地将细胞在离心机中以3000gxg离心10分钟并弃去所有的上清液。重悬在100μl的媒体每枪。然后将100μl悬浮液铺在每个1.5%琼脂板中心的5cm直径的圆圈中,并在室温下干燥。
        3. 使用轻柔的真空过滤将5×10 7个细胞过滤到47mm直径,1.2μm等孔过滤器上。将过滤器放在1.5%的琼脂平板上。
      7. 准备颗粒
        1. 添加以下为了一个1.5毫升的离心管:
          1)50μL准备好的钨颗粒。
          2)5微克在5-10微升水中的质粒(阴性对照只使用水)。
          3)50μl的2.5M CaCl 2(在-20℃储存溶液)。
          4)20μl0.1M亚精胺(在-20℃储存一个月)。
        2. 漩涡管1分钟。
        3. 简要地在离心机中旋转颗粒并除去上清液。
        4. 加入250μl的100%乙醇并涡旋至均质。
        5. 简要地在离心机中旋转颗粒并除去上清液。
        6. 在50μl的100%乙醇中重悬。存放在冰上。粒子需要在准备1小时内使用。
      8. 微粒轰击
        1. 根据制造商的说明安装粒子枪。

        2. 用70%的乙醇清洗颗粒枪
        3. 高压灭菌器的爆破片保持帽,微载体发射组件,宏载载体,停止screes和镊子。

        4. 通过浸入异丙醇消毒1,350 psi的爆破片

        5. 用70%乙醇浸泡消毒微生物,使其干燥。
        6. 在拍摄细胞之前,需要清除氦气线。这可以通过执行拍摄所需的以下步骤来执行,但不使用细胞或颗粒。
        7. 将宏载体放在微载体内。
        8. 涡旋涂覆的钨颗粒,保持颗粒混合,吸取10微升到宏载体的中心。暂时关闭层流罩,同时干燥颗粒,以防止损失。
        9. 将一个1350 psi的防爆片放入固定帽中,用提供的工具将帽盖紧紧地拧入组件。
        10. 一旦颗粒干燥,加载宏载体并停止屏幕进入发射组件。然后发射组件被放置在biolistic室。

        11. 在生物弹道室中,在距离7厘米处放置一个细胞板
        12. 关闭并打开真空泵,直至达到25Hg的真空度。
        13. 火颗粒进入细胞。
        14. 粒子轰击后,将每个平板上的细胞冲洗到25ml不含抗生素的培养基中,并在标准生长条件下孵育24小时。
        15. 培养24小时后,对细胞进行计数并从每个转化中将5个5×10 6个细胞铺在选择平板上。
        16. 使用来自阴性对照的细胞,也将细胞扩散到没有抗生素的平板上作为阳性生长对照。
          注意:与其他硅藻转化系统如三角褐指藻相比,假丝酵母菌需要相对较小浓度的细胞才能被有效地利用。结果,电镀后将会有大量的转化细胞留在培养基中。如果需要的话,可以铺平另外的平板,或者可以保持液体培养物,直到菌落开始出现在平板上,以防需要更多的平板。
    11. 通过PCR /带位移测定筛选转化体
      1. 一旦出现菌落(约10天后),转移到选择性液体培养基中。
      2. 在20μl培养基中重悬菌落,并将10μl转移至PCR管进行菌落PCR。剩余的10μl可用于培养单个细胞系。
        将剩余的10μl置于1ml选择性液体培养基中。一旦细胞达到约1×10 6个细胞/毫升,这可以用来接种更大的体积。
      3. 旋转10μL的细胞,留出菌落PCR,在离心机下全速1-2分钟,去除上清液。
      4. 在裂解缓冲液中重悬细胞(参见食谱)。
      5. 在冰上孵育15分钟。

      6. 在95°C孵育10分钟
      7. 使用1μl作为模板在使用Taq聚合酶的20μlPCR反应中。
      8. 放大目标区域。对于Hopes等人(2016)中描述的脲酶基因,使用正向引物:AAACAGACCACCTTCACCTC和反向引物:CTCCACCTGTACGTCTCG。在1%w / v琼脂糖凝胶上以4-5 V / cm(80-100 V,使用指定的凝胶系统)运行PCR产物。在Hopes等人(2016)的论文中,由于缺失只有37nt,而不是1%w / v凝胶,所以2%w / v琼脂糖凝胶以3V /厘米(具有指定凝胶系统的60V)以显现与WT相比带区大小的偏移。见图2的凝胶显示删除引起的带移。
      9. 使用正向引物:CCGAGACAAGCAGAGTGGAAAG和反向引物:AGAGCCGATTGATGTCCAGTTC扩增一段Cas9基因。在1%w / v琼脂糖凝胶上以4-5 V / cm(80-100 V,使用指定的凝胶系统)进行PCR产物检测是否存在Cas9。
        注:我们发现大约11%的主克隆含有Cas9,但是含有Cas9的克隆中有100%含有一个缺失。筛选至少30-40个菌落,以分离几个具有缺失的主要克隆。

      10. 在所有PCR反应中,使用水而不是模板包括WT样本和阴性对照。
      11. 由于CRISPR-Cas的性质,初级菌落可以与WT和突变细胞的混合物进行镶嵌(图2)。为了获得具有一个克隆的干净的细胞系,来自具有突变证据的原始克隆的培养物需要重新铺展到另外的选择性平板上。将来自指数期的原代培养物的100μl细胞连接到新的选择性平板上并温育直至出现菌落。然后可以如上所示筛选亚克隆。


        图2.筛选CRISPR-Cas在 T中诱导的基因组编辑。 pseudonana 。通过PCR和测序进行筛选。期望的sgRNA减少由↓表示。红色文字显示sgRNA靶序列和粗体文本的PAM图案。初级克隆:几个初级克隆包含显示CRISPR诱导的突变和野生型(WT)序列的序列,如在PCR之后存在两条带所见,这些由(+ WT)表示。剩余的样品代表不同的细胞系。 M1显示来自第二个sgRNA的4nt缺失,而突变体M2-M4显示两个CRISPR-Cas切割位点之间的37nt缺失。亚克隆:凝胶显示了来自主克隆的亚克隆的选择实例。根据主克隆和亚克隆号标记亚克隆。除了在主克隆中产生WT序列和4nt缺失的M1_9以外,选择用于测序的所有亚克隆都是双等位基因的。三分之二的测序双等位基因亚克隆显示具有37nt缺失的单一序列,表明两个等位基因携带相同的突变。在等位基因突变不同的亚克隆中,显示了两个序列。

    12. 按限制摘要筛选(可选)
      1. 放大目标区域。
      2. 用与sgRNA切割位点重合的限制性酶消化PCR产物/位于预期的缺失位点内。
      3. 在琼脂糖凝胶上运行片段。如果CRISPR-Cas已经诱导了突变,那么限制性位点将不再起作用,并且片段将保持未切割。注意扩增片段或使用限制性内切酶酶切位点。否则,不管突变如何,都可能出现几个难以显现的小碎片。
      4. 如果编辑序列富集,提取基因组DNA并进行限制性消化。 PCR扩增目标。只有野生型序列应该被切断,允许优先扩增突变片段。
    13. 通过排序屏幕
      1. 使用Eurofins Mix2seq试剂盒等服务对PCR产物进行序列验证(图2)。
      2. 对于似乎有基于大小的WT序列的产品也是值得的。这可以识别由一个sgRNA的活性引起的突变体,导致仅在一个位点处的较小的indel。一个例子可以看到M1(图2)。

数据分析

  1. 使用如上所述的带位移测定和/或限制性位点丢失进行筛选。这使得测定产物能够在凝胶上运行以通过缺失(带移测定)或靶位点突变(限制性位点缺失)容易地确定基因组编辑。然后对产物进行测序以确认缺失/突变。
  2. 对于30-40个菌落的主要克隆进行Cas9和缺失的检测。
    注:我们对来自所有原代菌落的PCR产物进行了测序,无论是否存在Cas9或缺失,发现11%的克隆含有Cas9。具有Cas9的100%的克隆含有突变-75%的突变克隆含有缺失,但是其余的25%在一个靶位点处显示具有与WT类似的带的突变。因此,可以将Cas9作为潜在突变的指标进行筛选,因为编辑可能并不总是导致删除。然后可以如所描述的那样对Cas9克隆进行亚克隆,并进行进一步分析,包括限制性位点丢失和测序。
  3. 大约30个亚克隆通常通过带移测定来测定以确定双等位基因缺失的频率。这被计算为与只有较低的带(指示两个等位基因中的缺失)的亚克隆的数目相比,所测试的亚克隆的总数。其他菌落可能只给出较高的WT带或两者的混合物。
  4. 进一步的分析取决于哪个基因/序列正在被靶向。例如,当靶向尿素酶基因(其允许细胞使用尿素作为氮源)时。假单胞菌(pseudonana),使细胞系在硝酸盐(阳性对照)或尿素作为唯一氮源的培养基中生长(Hopes等人,2016)。每天测量一次细胞计数和细胞大小,直到达到稳定期。然后比较细胞系(WT和突变体)和生长条件(硝酸盐或尿素)之间的生长曲线/细胞大小,以确定编辑的细胞系中的氮饥饿或限制。

食谱

  1. LB培养基/ LB琼脂


    将胰蛋白胨,酵母提取物和氯化钠组合。如果制作平板也加琼脂。用MilliQ /蒸馏水和高压灭菌器补足1L。一旦介质冷却到50°C,如果选择性培养基添加相关的抗生素。一旦介质已经达到50°C,板也被浇注。配方可根据需要放大或缩小
  2. 裂解缓冲液

致谢

感谢塞恩斯伯里实验室提供的金门目标载体和连接器。这项工作是由自然环境研究委员会(NERC)授予Amanda Hopes的博士生资助的。 TM承认NERC(NE / K004530 / 1)和诺里奇东安格利亚大学环境科学学院的部分资金。
作者贡献:AH和TM构思了这个项目。 AH设计并开发了Golden Gate克隆和Vladimir Nekrasov带移测定法的输入方案。 AH和NB确定了F. cylindrus的U6启动子,IG在同一物种中测试了它的活性。 AH写了这篇文章。
该协议基于Hopes等人(2016年)提出的工作。
作者声明他们没有竞争利益。

参考

  1. Belhaj,K.,Chaparro-Garcia,A.,Kamoun,S.和Nekrasov,V.(2013)。 使植物基因组编辑变得简单:使用CRISPR / Cas系统在模式和作物植物中进行定向诱变< / a> Plant Methods 9(1):39.
  2. Brooks,C.,Nekrasov,V.,Lippman,Z.B。和Van Eck,J。(2014)。 第一代番茄的高效基因编辑使用聚类规则间隔短回文重复序列/ CRISPR相关9系统。植物生理学 166(3):1292-1297。
  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系统的多重基因组工程。 Scienc e 339(6121):819-823。
  4. Engler,C.,Youles,M.,Gruetzner,R.,Ehnert,T.M。,Werner,S.,Jones,J.D。,Patron,N.J。和Marillonnet,S。(2014)。 用于植物的金门模块化克隆工具箱。 ACS Synth Biol 3(11):839-843。
  5. Hopes,A.,Nekrasov,V.,Kamoun,S。和Mock,T。(2016)。 通过硅藻中的CRISPR-Cas编辑尿素酶基因海绵假丝酵母 。植物方法 12:49。
  6. Kroth,P.G。(2007)。 遗传转化:研究硅藻中蛋白质靶向的工具方法Mol Biol 390:257-267。
  7. Mali,P.,Yang,L.,Esvelt,K. M.,Aach,J.,Guell,M.,DiCarlo,J. E.,Norville,J.E。和Church,G.M。(2013)。 通过Cas9进行RNA指导的人类基因组工程。 科学 339(6121):823-826。
  8. Nymark,M.,Sharma,A. K.,Hafskjold,M. C.,Sparstad,T.,Bones,A. M.和Winge,P.(2017)。 在海洋硅藻Phaeodactylum tricornutum中进行CRISPR / Cas9基因编辑。 > Bio Protoc e2442。
  9. Nymark,M.,Sharma,A.K.,Sparstad,T.,Bones,A.M。和Winge,P。(2016)。 适用于海洋藻类基因编辑的CRISPR / Cas9系统。 Sci Rep 6:24951.
  10. Pinto,F.L。和Lindblad,P。(2010)。 内部设计基于模板切换的5'cDNA末端系统快速扩增指南。 Anal Biochem 397(2):227-232。
  11. Poulsen,N.,Chesley,P.M。和Kröger,N。(2006)。 硅藻的分子遗传操作假单胞菌Thalassiosira pseudonana (Bacillariophyceae)。
  12. Price,N.M.,Harrison,G.I.,Hering,J.G.,Nirel,P.M。,Palenik,B。和Morel,F。(1989)。 人工藻类培养基aquil的制备和化学。 生物海洋学 6:443-461。
  13. Rastogi,A.,Murik,O。,Bowler,C。和Tirichine,L.(2016)。 PhytoCRISP-Ex:一个基于网络的独立应用程序,用于查找CRISPR / CAS编辑。 BMC Bioinformatics 17(1):261.
  14. Sakuma,T.,Nishikawa,A.,Kume,S.,Chayama,K.和Yamamoto,T。(2014)。 使用一体化CRISPR / Cas9载体系统在人类细胞中进行多重基因组工程。 Sci Rep 4:5400。
  15. Weber,E.,Engler,C.,Gruetzner,R.,Werner,S.和Marillonnet,S.(2011)。 用于多基因构建体标准化组装的模块化克隆系统 PLoS One < / em> 6(2):e16765。
  16. Zheng,Q.,Cai,X.,Tan,M.H.,Schaffert,S.,Arnold,C.P.,Gong,X.,Chen,C.Z.and Huang,S.(2014)。 使用人类细胞中的CRISPR / Cas9系统进行精确的基因缺失和替换 生物技术 57(3):115-124。
  • English
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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: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hopes, A., Nekrasov, V., Belshaw, N., Grouneva, I., Kamoun, S. and Mock, T. (2017). Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions. Bio-protocol 7(23): e2625. DOI: 10.21769/BioProtoc.2625.
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