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Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
酿酒酵母中使用人工靶DNA序列进行多重CRISPR/ Cas9的方法   

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Abstract

Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed ‘multiplexing’) is achieved by the expression of multiple sgRNAs within the same nucleus. However, an ongoing concern of the CRISPR field has been the accidental targeting of Cas9 to alternative (‘off-target’) DNA locations within a genome. We describe the use (dubbed Multiplexing of Cas9 at Artificial Loci) of installed artificial Cas9 target sequences into the yeast genome that allow for (i) multiplexing with a single sgRNA; (ii) a reduction/elimination in possible off-target effects, and (iii) precise control of the placement of the intended target sequence(s).

Keywords: CRISPR/Cas9(CRISPR/Cas9), Budding yeast(出芽酵母), Multiplexing(复制), Genome DNA editing(基因组DNA编辑), sgRNA(sgRNA)

Background

The CRISPR (Clustered Regularly Interspaced Palindromic Repeats) mechanism has evolved in prokaryotes as a primitive adaptive immune system with the capability to edit any genome with great precision (Jinek et al., 2012; Sorek et al., 2013). This biotechnology requires the use of an endonuclease (Cas9) from S. pyogenes (or othologous species), a single RNA ‘guide’ sequence, and exogenous donor DNA (if needed). In only a few years, CRISPR/Cas9 has been utilized in numerous research laboratories in both academic and industry settings to target DNA sequences within any genome (Doudna and Charpentier, 2014). A variety of research areas including basic research, biofuels, agriculture, genetic disorders, and human pathogens/disease have begun harnessing this technology to address important scientific questions (Estrela and Cate, 2016; Demirci et al., 2017; Men et al., 2017). Recent work in S. cerevisiae has piloted the development of novel CRISPR-based applications including automated genomic engineering (Si et al., 2017), chromosome splitting (Sasano et al., 2016), and the use of nuclease-dead Cas9 (dCas9) to modulate gene expression (Jensen et al., 2017). While this editing system has proved extremely useful, a number of concerns are still being actively addressed. These include off-target effects–the propensity of Cas9 to accidentally target additional genomic positions (Cho et al., 2014; Zhang et al., 2015), the required cloning step(s) needed to generate multiple sgRNAs for Cas9 multiplexing (Ryan and Cate, 2014), and the safety and application of Cas9-based ‘gene drives’ (DiCarlo et al., 2015). Our methodology addresses some of these issues by engineering artificial Cas9 target site(s) within the yeast genome. We describe (i) the selection of the artificial sequences used to multiplex Cas9; (ii) the cloning strategies used to construct plasmids harboring the unique target sites flanking several genes including Cas9 itself; (iii) integration of these constructs into a single yeast genome in successive steps, and (iv) editing using expressed Cas9, sgRNA, and donor DNA to demonstrate proof of concept. This system allows for seamless, marker-less, multi-loci genomic editing with only a single sgRNA. We envision this method could be useful for synthetic genome construction, yeast library generation, and simultaneous manipulation of related genes within a common genetic or signaling pathway.

Materials and Reagents

  1. Pipette tips (LTS tips 1,000 μl, 250 μl, 20 μl, Mettler-Toledo, Rainin, catalog numbers: GPS-L1000 , GPS-L250 , and GPS-L10 )
  2. Tubes (Axygen Microtubes 1.5 ml clear, homo-polymer, boil-proof) (Corning, Axygen®, catalog number: MCT-150-C )
  3. Disposable sterile plastic 15 ml (Corning, Falcon®, catalog number: 352099 ) and 50 ml (Corning, Falcon®, catalog number: 352098 ) conical centrifuge tubes
  4. Disposable glass test tubes (20 x 150 mm) (Fisher Scientific, catalog number: 14-958K )
  5. Kimble Kim-Kap test tube closures (Fisher Scientific, catalog number: 14-957-91C)
    Manufacturer: DWK Life Sciences, Kimble®, catalog number: 7366020 .
  6. Plastic Petri dish (100 x 15 mm size) (VWR, catalog number: 25384-088 )
  7. 0.5 mm glass beads (Bio Spec Products, catalog number: 11079105 )
  8. Yeast strains
    1. SF838-1Dα (MATα ura3-52 leu2-3,122 his4-519 ade6 pep4-3 gal2) (Univ. of Oregon; Rothman and Stevens, 1986)
    2. THS4218 (SF838-1Dα; HIS4 his3Δ::HygR) used for in vivo plasmid assembly and recovery (Univ. of California, Berkeley; Finnigan and Thorner, 2015)
    3. BY4741 (MATα his3∆1 ura3∆0 leu2∆0 met15∆0) (Univ. of California, Berkeley; Brachmann et al., 1998) for construction of all yeast strains tested
  9. One Shot® TOP10 chemically competent E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404003 )
    Note: See (Finnigan and Thorner, 2015) for propagation and preparation of TOP10 seed cultures.
  10. Plasmid containing prGalL-Cas9-CYC1(t) used as template DNA for construction of Cas9-containing cassettes (Addgene, catalog number: 43804) from (DiCarlo et al., 2013)
  11. Plasmid containing prSNR52-sgRNA-SUP4(t) (Addgene, catalog number: 43803 ; synthesized de novo by Genscript) (DiCarlo et al., 2013)
  12. ssDNA: deoxyribonucleic acid sodium salt from salmon testes (boiled for 10 min and cooled on ice prior to each use of a 10 mg/ml stock solution in water) (Sigma-Aldrich, catalog number: D1626 )
  13. Zymolyase® 100T from Arthrobacter leuteus (25 mg/ml) (Amsbio, catalog number: 120493-1 ) in 50% glycerol stock (certified ACS grade) (Fisher Scientific, catalog number: G33 )
  14. Appropriate restriction enzyme(s) for digestion
    1. NotI-HF (New England Biolabs, catalog number: R3189 )
    2. DpnI (New England Biolabs, catalog number: R0176 )
    3. BamHI-HF (New England Biolabs, catalog number: R3136 )
    4. XhoI (New England Biolabs, catalog number: R0146 )
  15. QIAquick gel extraction kit (QIAGEN, catalog number: 28706 )
  16. T4 DNA ligase (New England Biolabs, catalog number: M0202 )
  17. GeneJET plasmid miniprep kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K0503 )
  18. KOD hot start DNA polymerase (EMD Millipore, catalog number: 71086-3 , distributed by VWR, catalog number: 80511-384)
  19. Custom DNA oligonucleotide primers (25-100 nmol concentration; Integrated DNA Technologies)
  20. GeneJet PCR purification kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K0701 )
  21. Ethidium bromide (Sigma-Aldrich, catalog number: E8751 )
  22. Agarose powder (U.S. Biotech Sources, catalog number: G01PD-500 )
  23. Hygromycin, used at 300 μg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 10687010 )
  24. G418 sulfate (Geneticin), used at 200 μg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 11811031 )
  25. D-(+)-Raffinose pentahydrate (20% stock in water, filter sterilized, not autoclaved) (Sigma-Aldrich, catalog number: R7630 ). Filtered using disposable cellulose nitrate filter (0.2 μm filter size) (Corning, catalog number: 430186 )
  26. Sucrose (20% stock in water, filter sterilized, not autoclaved) (Fisher Scientific, catalog number: S3 )
  27. 1 M lithium acetate dihydrate (CH3COOLi·2H2O, reagent grade) (Sigma-Aldrich, catalog number: L6883 )
  28. 50% PEG: Poly (ethylene glycol), BioXtra avg. molecular weight 3,350 (Sigma-Aldrich, catalog number: P4338 )
  29. Ampicillin (final concentration of 100 μg/ml) (RPI, catalog number: A40040-100.0 )
  30. Kanamycin (final concentration of 50 μg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 11815024 )
  31. Yeast extract (BD, BactoTM, catalog number: 212750 )
  32. Peptone (BD, BactoTM, catalog number: 211677 )
  33. Dextrose (20% stock in water) (Thermo Fisher Scientific, catalog number: D16 )
  34. D-(+)-Galactose (20% stock in water, filter sterilized, not autoclaved) (Sigma-Aldrich, catalog number: G0750 )
  35. Tryptone (BD, BactoTM, catalog number: 211705 )
  36. Sodium chloride (NaCl, certified ACS grade ≥ 99.0%) (Fisher Scientific, catalog number: S271 )
  37. Potassium chloride (KCl, BioXtra ≥ 99.0%) (Sigma-Aldrich, catalog number: P9333 )
  38. Magnesium chloride hexahydrate (MgCl2·6H2O, BioXtra ≥ 99.0%) (Sigma-Aldrich, catalog number: M2670 )
  39. Magnesium sulfate solution (MgSO4, molecular biology grade) (Sigma-Aldrich, catalog number: M3409 )
  40. SuperPure agar, bacteriological grade (US Biotech Sources, catalog number: A01PD-500 )
  41. Yeast nitrogen base minus amino acids and minus ammonium sulfate (Sigma-Aldrich, catalog number: Y1251 )
  42. Ammonium sulfate ((NH4)2SO4 certified ACS grade ≥ 99.0%) (Fisher Scientific, catalog number: A702 )
  43. ‘Almost complete’ amino acid mixture
    Adenine HCl (Sigma-Aldrich, catalog number: A9795 ), 20 mg/L
    Arginine (Sigma-Aldrich, catalog number: A5131 ), 20 mg/L
    Tyrosine (Sigma-Aldrich, catalog number: T3754 ), 30 mg/L
    Isoleucine (Sigma-Aldrich, catalog number: I2752 ), 30 mg/L
    Phenylalanine (Sigma-Aldrich, catalog number: P2126 ), 50 mg/L
    Glutamic acid (Sigma-Aldrich, catalog number: G1251 ), 100 mg/L
    Aspartic acid (Sigma-Aldrich, catalog number: A9256 ), 100 mg/L
    Threonine (Sigma-Aldrich, catalog number: T8625 ), 200 mg/L
    Serine (Sigma-Aldrich, catalog number: S4500 ), 400 mg/L
    Valine (Sigma-Aldrich, catalog number: V0500 ), 150 mg/L
  44. Methionine (Sigma-Aldrich, catalog number: M9625 )
  45. Lysine (Sigma-Aldrich, catalog number: L5626 )
  46. Histidine (Sigma-Aldrich, catalog number: H8125 )
  47. Leucine (Sigma-Aldrich, catalog number: L8000 )
  48. Uracil (Sigma-Aldrich, catalog number: U0750 )
  49. 5-Fluoroorotic acid (5-FOA) (Oakwood Products, catalog number: 003234 )
  50. Sodium hydroxide (NaOH certified ACS grade ≥ 97.0%) (Fisher Scientific, catalog number: S318 )
  51. Tris base, molecular biology grade ≥ 99.8% (Fisher Scientific, catalog number: BP152 )
  52. Glacial acetic acid (certified ACS grade) (Fisher Scientific, catalog number: A38 )
  53. Ethylenediaminetetraacetic acid (EDTA 99%-101%) (Fisher Scientific, catalog number: S311 )
  54. Ultrapure sterile water (Millipore Sigma, Milli-Q water purification system)
  55. YPD liquid media (see Recipes)
  56. YPGal (see Recipes)
  57. SOC medium (see Recipes)
  58. YPD plates (with appropriate drugs optional) (see Recipes)
  59. Synthetic drop-out plates/media (see Recipes)
  60. 5-FOA plates (see Recipes)
  61. LB plates (with appropriate drug included) (see Recipes)
  62. TAE buffer (see Recipes)

Equipment

  1. Pipettes (Rainin, Pipet-Lite LTS, 1,000 μl, 250 μl, 20 μl, and 2 μl sizes)
  2. PCR machine (MJ Research PTC-200 Peltier Thermo Cycler, dual 30-well alpha blocks) (MJ Research, model: PTC-200 )
  3. Centrifuge (Eppendorf microcentrifuge) (Eppendorf, model: 5415 D , catalog number: 022621408)
  4. Eppendorf rotor (for 24 x 1.5/2 ml) (Eppendorf, model: F-45-24-11 , catalog number: 022636502)
  5. Vortexing adaptor (Microtube foam insert for Fisher Vortex Genie 2 mixer) (Scientific Industries, model: Vortex Genie 2 , catalog number: 504-0234-00)
  6. Ice maker (Hoshizaki American)
  7. Water bath (Thermomix circulating water bath, Model B, type 852 013/5)
  8. DNA gel electrophoresis apparatus (HE 33 Mini Submarine Unit) (GE Healthcare, catalog number: 80-6052-45 )
  9. ChemiDoc UV transilluminator (Bio-Rad Laboratories, model: ChemiDocTM XRS+, catalog number: 1708265 )
  10. Incubator rotator (Labquake shaker) (Labindustries, model: T-415-110 )
  11. Incubators (VWR, model: Model 1535 )
  12. Autoclave (Univ. of California, Barker Hall)

Procedure

Here we describe the strategy used to design, construct, integrate, and edit with Cas9 artificial target sequences within the yeast genome. We have focused heavily on the cloning methodologies for how to precisely install the programmed target sites (other methods may be substituted at certain steps and we highlight these) and multiplex with Cas9 in vivo. We provide a detailed description of our published (Finnigan and Thorner, 2016) proof of concept describing this methodology using both an essential and non-essential gene as well as the Cas9 gene itself to illustrate the utility and power of this approach.

  1. Selection of artificial site(s)
    1. To begin, a unique target sequence (designated ‘[u1]’) of a minimum of 23 bp (20 target + 3 Protospacer Adjacent Motif [PAM]) must be selected that has a maximum mismatch to the organism of choice (budding yeast genome) and all other exogenous components of the system (added vectors, GFP, drug cassettes, tags, etc.). The chosen sequence may be from another organism (in our case, two human gene sequences were selected), or may be generated de novo.
      Note: Other studies have found a variety of limitations to the target sequence (and PAM). It is recommended (in yeast) to not have a poly-T sequence (five or more) as this represents a termination signal for RNA Pol III (DiCarlo et al., 2013). Additionally, sequences that are extremely AT or GC rich are not recommended.
    2. Putative artificial 23 bp sequences should be checked against the yeast genome (and other exogenous sequences) using the NCBI Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). An initial screening of the 3’ most 15 bp (including the PAM) should first be searched. This represents the ‘seed’ region used by the Cas9/sgRNA complex to first associate with a putative matching genomic target sequence (Jinek et al., 2012; DiCarlo et al., 2013; Jiang et al., 2013). A maximum mismatch between these bases should be identified with particular emphasis on mismatches within the GG bases of the PAM sequence. Once putative target(s) are found, a full search of the entire 23 bp sequence can be interrogated against the yeast genome with the same preference for a high level of mismatch.
      Note: If the artificial target is to be included within the open reading frame (and translated as an appended N- and/or C-terminal tag), then an extra base (23 + 1) should be included to keep the sequence in-frame. Moreover, the occurrence of any in-frame stop codon within the sequence should be avoided.
    3. Design of this multiplexing system using a minimum number of sgRNAs should be carefully considered prior to the construction of any assembled plasmids and/or genome integration. For instance, the total number of unique site(s) and their placement across gene(s) may be difficult to undo once the initial investment of strain construction has been made. Our system utilizes a [u1] target site flanking multiple loci to be manipulated whereas the Cas9 gene itself is flanked by either the [u1] or [u2] sites providing the option for either simultaneous or sequential seamless excision of the nuclease.

  2. Construction of tagged genes, Cas9, and sgRNA plasmids
    1. Following selection of the unique [u1, u2, u3…] artificial sites (Procedure A), as well as the intended strategy to flanking the loci of interest with said sites (Figure 1, bottom), in vivo plasmid assembly (Finnigan and Thorner, 2015) can be used to install the sites at their precise position(s).
      Note: We have previously described the entire process for plasmid ‘in vivo ligation’ followed by recovery of the constructed vectors from yeast (Finnigan and Thorner, 2015). We will include additional comments/steps pertaining to pertinent modifications from the original protocol.


      Figure 1. Design, construction, and use of artificial DNA sequences for Cas9 multiplex gene editing in budding yeast. Top. Workflow overview. This protocol begins with selection of the artificial DNA Cas9 target sites followed by construction of the desired plasmid(s) including Cas9 and sgRNA expression cassettes. Next, successive integration events into the yeast genome place the target sites at multiple loci including Cas9. Transformation of the sgRNA plasmid and amplified donor DNA fragment(s) in the presence of Cas9 allows for in vivo editing. Finally, verification of the genome by diagnostic PCR and DNA sequencing confirms the intended modifications. Bottom. An example genome containing six identical [u1] (unique engineered sequence) sites at three loci (one of which includes Cas9). The 24 bp sequence (20 target + 3 PAM + 1 extra base) is appended as an 8-residue tag at both the N- and C-termini of Cdc11 (single red asterisk). In the case of the deleted GeneY (Shs1), the added [u1] sites (23 bp) are part of the UTR (two red asterisks). Use of only one sgRNA allows for double stranded break (DSB) formation at all six genomic positions. The presence of corresponding amplified donor DNA (only one is illustrated for clarity) with flanking homology to the UTR allows for simultaneous multi-gene marker-less integration events via homologous recombination. Depending on the identity of the artificial site [u1 or u2] flanking the Cas9 gene, concomitant or sequential excision of Cas9 can be achieved depending on the need for future editing events in the given strain.

    2. Design of oligonucleotides should include the intended 23 (or 24) bp artificial sequence and place either in-frame as part of the gene to be tagged, or within the flanking UTR by a two-step PCR amplification. As shown in Figure 2, we describe the construction process for two identical [u1] tags at the N- and C-termini of a given gene. However, we have chosen to separate inclusion of each tag into two intermediate plasmids to be built in parallel. If a single plasmid assembly process was used, then there would be competition between PCR fragments containing the intended [u1] sequences; it is possible that rather than include the WT gene between the two artificial sites, exclusion of the central fragment could result and create only a single [u1] site. To eliminate this possibility, we constructed each [u1] sequence separately. This may be useful in other applications where a gene might purposely be flanked by two different artificial sites.


      Figure 2. Plasmid assembly of sample gene containing flanking unique sequence sites [u1]. 1. Using in vivo plasmid assembly in yeast (Finnigan and Thorner, 2015) one artificial site [u1] is inserted between the generic promoter (prGeneX) and open reading frame of a sample gene (GeneX). This strategy can also be applied for a deletion of a gene using the MX cassette (Goldstein and McCusker, 1999) or any developed genetic allele. The inserted [u1] sequences are included within two overlapping oligonucleotides that allow for the inclusion of the desired target de novo within the assembled plasmid. The example shown includes use of the S. pombe HIS5 MX cassette; selection for recircularization of the plasmid is also sufficient. 2. A similar plasmid is also constructed in parallel using in vivo ligation which inserts the [u1] site after the last codon of the gene (as shown) and upstream of the native terminator sequence. For selection purposes, the ADH1-MX cassette was also included. 3. Each created plasmid serves as the template for two PCRs which contain both identical [u1] sites at the proper position; the overlapping homology created between the fragments spans the entire open reading frame of the sample gene (as shown). 4. The newly designed cassette can be amplified by PCR and contains the gene of interest flanked by the two [u1] sites as well as 5’ and 3’ UTR sequences (also referred to as prGeneX and GeneX(t), respectively) for integration (see Figure 4).

    3. Following successful construction of the two separate [u1] tagged sequences, a second round of assembly should be performed using amplified products from each of the original vectors. In this case, while the [u1] could technically provide 23 (or 24) bp of overlapping homology, the entirety of the full gene provides > 1,200 bp of homology to greatly bias the cross-over to occur within this sequence and shift the propensity for assembly to include both [u1] sites as well as the central gene. Both the CDC11 (WT gene) and SHS1 (deletion cassette) were constructed with flanking [u1] sites.
      Note: This process could also be achieved using a deletion cassette rather than the WT coding sequence of a gene. Should the tagged cassette include a selectable marker, then construction may be done in a single step since active selection would ensure the presence of both [u1] sites as well as the deletion marker.
    4. For the construction of the Cas9-expression cassette (Figure 3), we demonstrate the use of an additional intermediate plasmid that was built due to the complexity of the final intended product (nine separate DNA sequences to be assembled). Following the individual plasmid assemblies to insert the [u1] (shown) or [u2] target sites, the first intermediate created a vector with a unique restriction site (NotI) between the inducible promoter and generic terminator sequences. Another round of in vivo assembly allowed for the inclusion of the Cas9 gene (in two amplified fragments; amplified from Addgene plasmid #43804) and a C-terminal NLS sequence.
      Note: This intermediate integrating vector (Figure 3, #3) would allow for the rapid inclusion of any Cas9 ortholog into the [u1]- or [u2]-containing HIS3 locus in yeast.


      Figure 3. Plasmid assembly of the Cas9 expression cassette. 1. Similar to assembly of the flanking [u1] sites surrounding the sample gene (Figure 2), construction of the [u1] or [u2] flanked expression cassette is done in a stepwise fashion (red asterisks). S. pyogenes Cas9 is under control of the GAL1/10 promoter and is placed downstream of the unique site and the 5’ UTR of the yeast HIS3 gene. A unique restriction site (RS, NotI) was included after the start codon and before the ADH1 terminator sequence. 2. The [u1] or [u2] site was placed after the entire MX terminator sequence but upstream of the yeast HIS3 3’ UTR. 3. Following construction of each separate vector, both fragments were PCR amplified and combined into a larger vector using in vivo ligation to create an intermediate expression vector. 4. Digestion of the unique NotI restriction site allows for a final step of plasmid assembly to insert Cas9 and a NLS (nuclear localization signal), both amplified in two overlapping PCR fragments in-frame with the start codon. 5. Following verification of the entire Cas9 expressing cassette, PCR amplification allows for integration into the yeast genome at the HIS3 locus (see Figure 4).


      Figure 4. Integration of [u1] flanked gene(s) and Cas9 expression cassette into the yeast genome. As a proof of concept, we chose to pilot this methodology in yeast using two native yeast genes (CDC11 and SHS1) as well as integrate Cas9 at a safe-harbor (HIS3) locus. CDC11 is an essential gene, and our strategy illustrates the additional steps taken to allow for modification and Cas9-based editing of both essential and non-essential genes. 1. WT BY4741 yeast were transformed with a URA3-based covering vector expressing a WT copy of GeneX (CDC11) lacking 3’ UTR sequence. 2. The endogenous GeneX copy was deleted using the standard MX-based deletion cassette and selected on G418-containing medium. 3. The entire [u1]-flanked GeneX construct (Figure 2) was amplified and transformed into the GeneX∆ strain and selected on media containing 5-FOA to counter-select for the URA3-based covering vector. The only viable yeast remaining would have integrated the [u1]-flanked GeneX (WT) copy in place of the KanR cassette resulting in a marker-less modification. Yeast integration events were all confirmed by diagnostic PCR and DNA sequencing following each round of successive modifications. 4. At a separate GeneY locus (SHS1), a deletion cassette was constructed and flanked by [u1] Cas9 sites (similar strategy as GeneX in Figure 2) and transformed into yeast to delete the endogenous GeneY copy. 5. Finally, the entire Cas9-expression cassette (Figure 3) was amplified, transformed into yeast, and integrated at the HIS3 locus (his3∆1), providing the strain G418 resistance. As shown, the final strain contains six identical [u1] target sites. However, an alternative construct was also tested using a [u2] sequence distinct from the [u1] site that flanked the Cas9 cassette allowing for separate and sequential editing at the HIS3 locus.

    5. Following construction and verification of the desired plasmids (Figures 2, 3 bottom), a final PCR amplification allows for the entire designed cassettes to be integrated into the yeast genome (see Procedure C).
    6. Donor PCRs may be amplified from plasmid or chromosomal preparations with at least 30 bp of flanking homology on either side of the DSB break(s). The PCR fragments (CDC11, SHS1, and HIS3) used as donor DNA for our proof of concept were PCR amplified from an isolated chromosomal DNA prep of WT yeast with several hundred bases of flanking UTR sequence. For amplification of WT HIS3, SF838-1Dα genomic DNA was used.
    7. The sgRNA-expression plasmid may be constructed using a variety of protocols. The construction of our sgRNA included the following:
      1. The sgRNA cassette sequence was modeled after (DiCarlo et al., 2013) with the SNR52 promoter, the SUP4 terminator, and included both the [u1] 20 bp target sequence and the required structural RNA sequence (79 bp) for S. pyogenes Cas9. This entire cassette was synthesized de novo from Genscript (Piscataway, NJ).
        Note: Numerous methodologies have been developed that allow for construction of the sgRNA plasmid including restriction digest and ligation, in vivo assembly, or Gibson cloning (Laughery et al., 2015; Ryan et al., 2016).
      2. Flanking restriction sites should be included in the synthesized sgRNA cassette (Figure 1C of Finnigan and Thorner, 2016). Our synthesized gene (Genscript) was cloned into pUC57 and contained flanking BamHI and XhoI sites. The high-copy pRS425 yeast plasmid and the [u1] sgRNA plasmid should be digested with both BamHI and XhoI overnight at 37 °C (incubator), linearized fragments separated on a 1% agarose gel, and extracted using a Qiaquick gel extraction kit (QIAGEN).
      3. Purified DNA should be ligated together at 16 °C for 16 h using T4 DNA ligase (NEB); the reaction halted by incubating at 65 °C for 10 min (performed in PCR block).
      4. 100 μl of TOP10 competent E. coli should be transformed with 10 μl of the ligation reaction, incubated on ice for 15 min, heat-shocked at 42 °C for 45 sec (water bath), recovered on ice for 2 min, gently mixed with 500 μl of SOC medium (see Recipes), and incubated at 37 °C for 1 h (incubator with slow rotation).
      5. Cells should be plated onto LB + AMP plates (see Recipes) and incubated overnight at 37 °C. Clonal isolates are to be cultured in liquid LB + AMP medium (overnight in a 37 °C water bath for 14-16 h) and plasmids are extracted using a bacterial plasmid extraction kit (GeneJet). Diagnostic digests followed by DNA sequencing of sgRNA plasmids can be used to confirm proper subcloning from pUC57 to pRS425.
      6. In order to create the [u2] targeting sgRNA plasmid (which is identical except for the 20 bp target sequence), successive rounds of a modified quick-change PCR (Zheng et al., 2004) can be used to mutate the [u1] sequence into [u2]. (This could be achieved on either the smaller pUC57 vector followed by the aforementioned subcloning steps, or the final pRS425 vector). Briefly, 60 bp oligonucleotides (a forward and reverse primer) should be designed that are complementary and contain a central 1-4 base substitution (the changes do not have to be successive but should be within a relatively close region).
      7. The entire vector should be PCR amplified using the high-fidelity KOD hot-start polymerase (EMD Millipore) with the recommended conditions, an annealing temperature of 50 °C, and an extension time of 40 sec/Kb. Briefly, a 50 μl reaction (total volume) containing the KOD components (Mg2+, 1x buffer solution, and 1x dNTPs, all provided), template DNA (100-500 ng), oligonucleotides (forward and reverse, final concentration 1 μM each primer), and 1 μl of KOD enzyme should be prepared. An initial hot-start cycle (95 °C, 2 min) followed by cycles consisting of (i) 95 °C, 15 sec, (ii) 50 °C, 30 sec, and (iii) 72 °C, X sec (where X is the total size of the template plasmid divided by 40 sec/Kb). Following 16 total cycles, a final 10 min extension time at 72 °C should be included.
      8. Following confirmation on a DNA agarose gel, 7.5 μl of the PCR reaction should be digested with 1 μl of DpnI overnight at 37 °C (incubator) with a final reaction volume of 50 μl. Digestion with DpnI is used to destroy the circular (methylated) template yeast plasmid DNA within the PCR reaction in order to remove false positives clones that may propagate in yeast due to template DNA rather than the intended plasmid assembly.
      9. 2 μl of the DpnI-treated digestion reaction should be transformed into E. coli as previously described.
      10. Clonal isolates should be confirmed via DNA sequencing.

  3. Integration of constructs into the yeast genome
    1. Our strategy for placement of the [u1]/[u2] flanked DNA sequences into the yeast genome illustrated the ability of both essential (CDC11) and non-essential (SHS1) genes to be manipulated with this methodology (Figure 4). Variations of our step-wise integration protocol could be achieved using different gene deletions or other methods including (i) integration of separate tagged genes into opposite mating types in parallel and subsequent mating, diploid formation, and sporulation (given the manipulated genes include a selectable marker of some sort); (ii) integration vectors such as the pRS300-series (Sikorski and Hieter, 1989), and/or (iii) CRISPR/Cas9 itself to create double stranded breaks and allow for insertion of the [u1]-flanked fragments as donor DNA. Our procedure illustrates one possible strategy to construct the intended yeast strain.
    2. WT BY4741 yeast should be transformed with a protective ‘covering vector’ (URA3-marked) using a modified lithium acetate protocol (Finnigan and Thorner, 2015) and selected on SD-URA plates.
    3. A knock-out deletion cassette (Finnigan et al., 2015) for the essential gene (e.g., CDC11) should be PCR amplified (with sufficient 5’ and 3’ UTR flanking sequences–several hundred bps), digested overnight at 37 °C with DpnI, transformed into yeast, and selected on medium containing G418. Additionally, colonies should also be sensitive on plates containing 5-FOA (to counter-select for the URA3-marked covering plasmid) since loss of an essential gene (e.g., cdc11∆) render yeast inviable.
      Note: The URA3-marked covering vector must not include any native 3’ UTR, otherwise the deletion cassette may delete the plasmid-borne copy rather than the endogenous gene.
    4. For clarity/simplicity, we will refer to our proof of concept with regards to the CDC11 essential gene, the SHS1 non-essential gene, and Cas9 itself. The [u1]-CDC11-[u1] (essential gene) PCR fragment with flanking UTR (> 300 bp) (Figure 2) should be transformed into cdc11∆ yeast following treatment with DpnI. Since no selectable marker is present on the integrating fragment, yeast should be plated onto medium containing 5-FOA (to select for the restoration of the CDC11 locus and cell viability). All generated yeast strains should be verified by diagnostic PCR and DNA sequencing.
    5. Second, the SHS1 locus should be deleted using the [u1]-shs1∆::HygR-[u1] with flanking UTR sequence (Figure 2) in the standard fashion, by transforming yeast (from step C4) with the amplified PCR product following DpnI digestion, and selection on medium containing hygromycin.
    6. Third, the HIS3 locus should be modified with the addition of one (or both) versions of the Cas9-expression cassette–flanked by either the [u1] or [u2] sites. Due to the large size of the entire Cas9 cassette (Figure 3) including the HIS3 5’ and 3’ UTR sequences (500 bp), two amplified PCR products may be generated using primers internal to the Cas9 gene itself (generating at least 100 bp of internal overlap for recombination), treated with DpnI, co-transformed into yeast (from step C5), and selected on medium containing G418. Colonies should also be tested for hygromycin resistance; due to the common promoter and terminator sequences between all the MX-based drug cassettes (Goldstein and McCusker, 1999), it is advisable to confirm all marked loci after each round of selection (by growth phenotypes and possibly by diagnostic PCRs) since there is a propensity for marker ‘swapping’ that may compete with integration at the desired site. The integration vector for the Cas9-expression cassette can target any S. cerevisiae strain harboring some 5’ and 3’ UTR sequences (within 1,000 bp on either side of the HIS3 gene). This integration will also be effective in strains containing an intact HIS3 gene, his3∆1, or his3∆0 yeast.
      Note: Confirmation of the final [u1]- and [u2]-flanked strains should include PCR amplification of the three manipulated loci (CDC11, SHS1, and HIS3) with a high-fidelity polymerase, PCR purification, and DNA sequencing.

  4. CRISPR/Cas9 multiplexed editing in budding yeast using artificial target sites
    1. A modified yeast transformation protocol is used for Cas9 editing. First, overnight cultures (synthetic drop out media minus uracil, 2% raffinose, 0.2% sucrose, see Recipes) are prepared for the yeast strain containing all six [u1] sites as well as the URA3-marked covering vector expressing WT CDC11 and incubated at 30 °C.
    2. Yeast should be back-diluted to an OD600 of approximately 0.30 into YPGal (2% galactose) and incubated for 4.5 additional hours at 30 °C to induce Cas9 expression.
    3. Approximately 10 OD600 of cells should be harvested at 7,300 x g, washed with 0.5 ml water, centrifuged once more at the same speed, and the water should be removed with a sterile pipet.
    4. Yeast should be resuspended in 0.5 ml of 100 mM sterile lithium acetate, centrifuged at 7,300 x g, and the supernatant should be removed (leaving the cells in the tube).
    5. A ‘PEG master mix’ should be created (fresh) containing the following: 240 μl of 50% polyethylene glycol (PEG), 36 μl of 1 M lithium acetate, 50 μl of SS DNA (boiled for 10 min and cooled on ice) and a variable amount of sterile water (donor DNA, plasmid DNA, and water should total 34 μl). The mixture (including water) should be vortexed separately and added to the yeast pellet.
    6. DNA for the Cas9 editing should include the following:
      1. The sgRNA plasmid (between 1,000-1,500 ng total DNA).
      2. Donor DNA (for each edited locus) as amplified PCR fragments (1,000-1,500 ng total DNA, varied based on length of PCR product).
    7. Control reactions for Cas9 editing should include (i) empty pRS425 vector with no sgRNA–this serves as an upper bound for the total number of transformants even in the absence of donor DNA of any kind; (ii) cultured yeast in YPDex (2% dextrose) (see Recipes) rather than galactose–however, this may yield a significantly different number of transformed colonies given the difference in metabolic activity and total cell number, and/or (iii) inclusion of the sgRNA plasmid and no donor DNA of any kind (sterile water)–this causes Cas9-induced double stranded breaks but requires any surviving yeast to perform NHEJ at each DSB location (two controls are demonstrated in Figure 5).


      Figure 5. Example results from Cas9-based editing in vivo. A. Schematic of the generated yeast strain with six identical [u1] artificial sites flanking three loci (GeneX, GeneY, and Cas9 at the HIS3 locus) and, after addition of the sgRNA for [u1] and donor DNA for each locus, the expected replacement alleles (WT) for all edited genomic locations. B. As a proof of concept, we provide example data from our Cas9 mCAL editing in vivo. Plate A includes yeast transformed with the sgRNA and all three donor PCR fragments. Plate B includes sgRNA but no donor DNA. Plate C includes an empty plasmid (identical to the guide RNA plasmid backbone) and no donor DNA. Because DSBs are lethal in yeast, Plate B results in no viable colonies whereas inclusion of the donor DNA allows for repair of each manipulated locus and a significant number of colonies. The provided example does not use any selection for integrated donor DNA and merely selects for the presence of a covering vector (URA3) and the single sgRNA plasmid (LEU2).

    8. Following addition of the PEG master mixture to yeast, the appropriate amount of DNA (plasmid and PCRs) should be added, and the tube vortexed at maximum setting for 10 sec to achieve full resuspension.
    9. Yeast should be heat shocked at 42 °C for 45 min, centrifuged at 7,300 x g for 1 min, and the PEG mixture should be removed with a pipet.
    10. Yeast should be resuspended in fresh YPGal liquid and placed at 30 °C overnight (14-16 h) to recover.
    11. Following recovery, cells (and the YPGal liquid) should be plated directly onto SD-URA-LEU plates and incubated at 30 °C for three days prior to imaging. Three example conditions from our proof of concept are demonstrated in Figure 5. Plate A. Yeast were transformed with the sgRNA [u1] plasmid and donor DNA for all three loci. Plate B. Yeast were transformed with the sgRNA [u1] plasmid and no donor DNA. Plate C. Yeast were transformed with an empty pRS425 vector and no donor DNA. The total number of colonies is recorded on each plate (Figure 5 bottom). Cas9-induced DSBs render cells inviable (Figure 5B), but the inclusion of donor DNA for all three loci allows for subsequent repair by homologous recombination, and rescue of cell viability (Figure 5A).
      Note: The proof of concept illustrates use of Cas9 editing with the artificial sites with no selection for markers (lack of uracil selects for the covering vector whereas lack of leucine selects for the presence of the [u1] sgRNA). However, selection/markers can be used if needed.

  5. Verification of editing genome sequences
    1. Confirmation of Cas9 editing of yeast should be performed by first preparing genomic DNA from clonal isolates (Amberg et al., 2006).
    2. Diagnostic PCRs should be performed using genomic DNA as a template to test for both the identity and sizes of the manipulated loci (in our case, CDC11, SHS1, and HIS3) to assess whether donor DNAs were properly integrated into the genome. As an example, removal of the flanking [u1] sites for CDC11 (and replacement with the native gene) displayed a small shift upon loss of the artificial site when separated on a 2% agarose DNA gel. Similarly, the integration of the WT SHS1 gene (in place of the deletion) allowed for PCR combinations that provided evidence of proper integration.
    3. Finally, manipulated loci should be amplified once more, purified, and sent for DNA sequencing.
      Note: As shown, the example strategy illustrates the Cas9-expression cassettes flanked by [u1] sites. We also tested the sequential elimination of Cas9 after editing of additional loci by construction of a [u2]-flanked Cas9 cassette along with the appropriate guide RNA and donor DNA (Finnigan and Thorner, 2016). In this scenario, consecutive transformations would allow for removal of the Cas9 gene at a later time.

Data analysis

For all stages of plasmid and yeast strain construction (Procedures A-E), diagnostic PCRs and final confirmation via DNA sequencing were performed to assess that all assembled DNA fragments (plasmid or genome) were generated as expected. An online DNA alignment program (Biology Workbench, San Diego Supercomputer Center) was used to analyze Sanger sequences. For Figure 5, colonies were counted using a sectoring method; independent experiments were performed in triplicate–only a representative plate for each trial is presented (http://www.g3journal.org/content/6/7/2147.long) (Finnigan and Thorner, 2016).

Notes

Our system includes a number of significant improvements and novel uses for CRISPR/Cas9-based gene editing in budding yeast.

  1. The use of a single sgRNA to target multiple genomic loci (reduces the need for multiple sgRNA constructs).
  2. Construction of an initial yeast strain pre-programmed with all unique sites flanking all genes of a complex or pathway would allow for massively parallel future multiplexing and strain generation via Cas9; all combinations would be possible because WT copies of each unmodified gene could easily be repaired (as we have shown).
  3. Allows for seamless excision of the Cas9 gene itself either in conjunction with modification of other loci or sequentially at a future time.
  4. Programming of the artificial sites on either side (flanking) the locus of interest ensures excision of the entire gene/marker/cassette and helps prevent promiscuous cross-over should a single allele or similar mutant need to be integrated in place of the WT copy.
  5. This process can be used to edit essential genes.
  6. This method could be applied to deletion or modification of entire chromosome segments, groups of genes, or multiple genes.
  7. Cas9-based editing allows for marker-less integration (the plasmid harboring the guide RNA can be easily lost with no selective pressure).
  8. Reduction/elimination off-target effects–since there is absolute control over the sequence that will be targeted within the genome, this should aid in optimizing editing while reducing recruitment of Cas9 to similar or other genomic loci.
  9. As a consequence of having the exact same unique artificial site multiple times within a genome, it would allow for a comprehensive screen/search for local epigenetic or positional effects that might alter Cas9 editing since the sgRNA and the target sequence would be identical across the genome.
  10. Aid in synthetic genome engineering–inclusion of Cas9 target site(s) would allow future editing, chromosome splitting, rearrangements, etc., across the entire genome.

Recipes

  1. YPD liquid media
    1% yeast extract
    2% peptone
    2% dextrose
    Autoclaved
  2. YPGal
    1% yeast extract
    2% peptone
    2% galactose
    YP autoclaved, galactose filter sterilized, mixed
  3. SOC medium
    2% tryptone
    0.5% yeast extract
    10 mM NaCl
    2.5 mM KCl
    1 mM MgCl2
    1 mM MgSO4
    20 mM glucose final concentration
  4. YPD plates (with appropriate drug included if needed)
    1% yeast extract
    2% peptone
    2% dextrose
    20 g/L Bacto agar
    Autoclaved
    Liquid drug stocks added once media cooled < 55 °C
  5. Synthetic drop-out media (e.g., -URA-LEU)
    1.7 g yeast nitrogen base minus ammonium sulfate and minus amino acids
    5.0 g ammonium sulfate
    1.1 g of ‘almost complete’ amino acid mixture
    Remaining amino acid mixture (All from Sigma-Aldrich, methionine 150 mg/L; lysine 180 mg/L; histidine 60 mg/L
    Note: If needed, leucine 260 mg/L and uracil 20 mg/L.
    10 mg/L tryptophan (filter sterilized, not autoclaved)
    2% dextrose
    20 g/L Bacto agar
    Sterile water and agar are mixed and autoclaved separately from remaining component
  6. 5-FOA plates
    Synthetic media components (appropriate amino acid drop-out, if any)
    0.5 g/L 5-FOA
    0.5 g/L uracil
    Heat all components (separate from water/agar mixture) to 75 °C for 30 min, filter sterilize and combine with autoclaved water/agar mixture
  7. LB plates (with appropriate drug included)
    1% tryptone
    0.5% yeast extract
    15 g/L agar
    10 g/L NaCl
    4 mM NaOH
  8. TAE buffer (1x final concentration), pH 8.4
    40 mM Tris (1 M stock solution)
    20 mM glacial acetic acid
    1 mM EDTA (0.5 M stock solution, pH 8.0)

Acknowledgments

This project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418 to G.C.F. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. This work was also supported by an Innovative Research Award (to G.C.F.) from the Johnson Cancer Research Center at Kansas State University. This work was supported by an Undergraduate Research Award (to R.M.G.) from the College of Arts and Sciences at Kansas State University. This protocol was modified and adapted from (Finnigan and Thorner, 2016). We would like to thank Jeremy Thorner (Univ. of California, Berkeley) for useful advice and comments.

References

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  4. Demirci, Y., Zhang, B. and Unver, T. (2017). CRISPR/Cas9: An RNA-guided highly precise synthetic tool for plant genome editing. J Cell Physiol.
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简介

鉴于CRISPR(集群定期间隔短回归重复)编辑技术的出现,基因组操纵变得更加易于使用。 Cas9核酸内切酶将募集复合物的单链(单向导)RNA(sgRNA)片段结合到相应的基因组靶序列,引发双链断裂。真核修复系统允许引入外源DNA,修复现有突变或内源基因产物的缺失。通过在同一核内表达多个sgRNA来实现Cas9对多个基因组位置的定位(称为“多重”)。然而,CRISPR领域的持续关注是将Cas9意外地定位到基因组内的替代(“脱靶”)DNA位置。我们将安装的人造Cas9靶序列的使用(称为人造基因座上的Cas9复制)描述为允许(i)与单个sgRNA复用的酵母基因组中的用途; (ii)减少/消除可能的脱靶效应,以及(iii)精确控制预定目标序列的放置。
【背景】CRISPR(集群定期间隔回归重复)机制已经在原核生物中演变为具有很高精度编辑任何基因组的能力的原始适应性免疫系统(Jinek等,2012; Sorek等,2013)。这种生物技术需要使用来自化脓性链球菌(或othologous物种)的内切核酸酶(Cas9),单个RNA'引导'序列和外源供体DNA(如果需要)。仅在短短几年内,CRISPR / Cas9已被许多研究实验室用于学术和行业环境,以靶向任何基因组中的DNA序列(Doudna和Charpentier,2014)。包括基础研究,生物燃料,农业,遗传疾病和人类病原体/疾病在内的各种研究领域已经开始利用这一技术来解决重要的科学问题(Estrela和Cate,2016; Demirci et al。,2017; Men et al。 2017年)。近来在酿酒酵母中的工作已经开发了基于CRISPR的新型应用,包括自动基因工程(Si et al。,2017),染色体分裂(Sasano et al。,2016),以及使用核酸酶死Cas9(dCas9 )调控基因表达(Jensen等,2017)。虽然这个编辑系统已被证明是非常有用的,但仍然有一些问题正在积极解决。这些包括脱靶效应 - Cas9意外地靶向另外的基因组位置的倾向(Cho等人,2014; Zhang等人,2015),为Cas9复用产生多个sgRNA所需的克隆步骤(Ryan和Cate,2014)以及基于Cas9的“基因驱动”的安全性和应用(DiCarlo et al。,2015)。我们的方法通过在酵母基因组内工程化人造Cas9靶位点来解决其中的一些问题。我们描述(i)用于复用Cas9的人工序列的选择; (ii)用于构建携带包括Cas9本身在内的若干基因侧翼的独特靶位点的质粒的克隆策略; (iii)在连续的步骤中将这些构建体整合到单个酵母基因组中,以及(iv)使用表达的Cas9,sgRNA和供体DNA编辑以证明概念证明。该系统允许仅使用单个sgRNA的无缝,无标记,多位点基因组编辑。我们设想,这种方法可用于合成基因组构建,酵母文库生成以及在共同遗传或信号通路中同时操作相关基因。

关键字:CRISPR/Cas9, 出芽酵母, 复制, 基因组DNA编辑, sgRNA

材料和试剂

  1. 移液器提示(LTS提示1,000μl,250μl,20μl,Mettler-Toledo,Rainin,目录号:GPS-L1000,GPS-L250和GPS-L10)
  2. 管(Axygen Microtubes 1.5ml透明,均聚物,防沸腾)(Corning,Axygen,目录号:MCT-150-C)
  3. 将一次性无菌塑料15ml(Corning,Falcon ,目录号:352099)和50ml(Corning,Falcon,目录号:352098)锥形离心管>
  4. 一次性玻璃试管(20 x 150 mm)(Fisher Scientific,目录号:14-958K)
  5. Kimble Kim-Kap试管封闭件(Fisher Scientific,目录号:14-957-91C)
    制造商:DWK Life Sciences,Kimble ® ,目录号码:7366020。
  6. 塑料培养皿(100 x 15毫米尺寸)(VWR,目录号:25384-088)
  7. 0.5毫米玻璃珠(Bio Spec Products,目录号:11079105)
  8. 酵母菌株
    1. SF838-1Dα(MATLAB ura3-52 leu2-3,122 his4-519 ade6 pep4-3 gal2)(俄勒冈大学; Rothman和Stevens,1986)
    2. 用于体内质粒装配和回收的THS4218(SF838-1Dα; HIS4his3Δ:: Hyg R )(加利福尼亚大学伯克利分校; Finnigan和Thorner,2015)
    3. 用于构建所有酵母的BY4741(MATαhis3Δ1ura3Δ0leu2Δ0met15Δ0)(加利福尼亚大学伯克利分校; Brachmann等人,1998)菌株测试了
  9. 单击® TOP10具有化学能力。 (Thermo Fisher Scientific,Invitrogen TM,目录号:C404003)
    注意:参见(Finnigan和Thorner,2015),用于繁殖和制备TOP10种子培养物。
  10. 含有prGalL-Cas9-CYC1(t)的质粒用作构建含Cas9的盒的模板DNA(Addcene,目录号:43804)(DiCarlo等人,2013)
  11. 含有prSNR52-sgRNA-SUP4(t)的质粒(Addcene,目录号:43803;由Genscript合成)(DiCarlo等人,2013)
  12. ssDNA:来自鲑鱼睾丸的脱氧核糖核酸钠盐(煮沸10分钟,并在每次使用10mg / ml水溶液前在冰上冷却)(Sigma-Aldrich,目录号:D1626)
  13. (25mg / ml)(Amsbio,目录号:120493-1)在50%甘油储备液(经认证的ACS级)(Fisher Scientific,Inc。)中的酵母菌素<100>目录号:G33)
  14. 适当的限制酶用于消化
    1. I-HF(New England Biolabs,目录号:R3189)
    2. I(New England Biolabs,目录号:R0176)
    3. HI-HF(New England Biolabs,目录号:R3136)
    4. Xho I(New England Biolabs,目录号:R0146)
  15. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28706)
  16. T4 DNA连接酶(New England Biolabs,目录号:M0202)
  17. GeneJET质粒微量稀释试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:K0503)
  18. KOD热启动DNA聚合酶(EMD Millipore,目录号:71086-3,由VWR分发,目录号:80511-384)
  19. 定制DNA寡核苷酸引物(25-100nmol浓度; Integrated DNA Technologies)
  20. GeneJet PCR纯化试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:K0701)
  21. 溴化乙锭(Sigma-Aldrich,目录号:E8751)
  22. 琼脂糖粉末(美国生物技术公司,目录号:G01PD-500)
  23. 以300μg/ ml(Thermo Fisher Scientific,Gibco TM,目录号:10687010)使用的潮霉素
  24. 以200μg/ ml(Thermo Fisher Scientific,Gibco TM,目录号:11811031)使用的G418硫酸盐(遗传霉素),
  25. D-(+) - 五水合棉子糖(20%储存于水中,过滤灭菌,不经高压灭菌)(Sigma-Aldrich,目录号:R7630)。使用一次性硝酸纤维素过滤器(0.2μm过滤器尺寸)过滤(Corning,目录号:430186)
  26. 蔗糖(20%储存在水中,过滤灭菌,不经高压灭菌)(Fisher Scientific,目录号:S3)
  27. 1M乙酸锂二水合物(CH 3 CO 2•2H 2 O,试剂级)(Sigma-Aldrich,目录号:L6883)
  28. 50%PEG:聚(乙二醇),BioXtra平均分子量3,350(Sigma-Aldrich,目录号:P4338)
  29. 氨苄青霉素(终浓度为100μg/ ml)(RPI,目录号:A40040-100.0)
  30. 卡那霉素(终浓度为50μg/ ml)(Thermo Fisher Scientific,Gibco TM,目录号:11815024)
  31. 酵母提取物(BD,Bacto TM ,目录号:212750)
  32. 蛋白胨(BD,Bacto TM ,目录号:211677)
  33. 右旋糖(20%储存在水中)(Thermo Fisher Scientific,目录号:D16)
  34. D-(+) - 半乳糖(20%储存在水中,过滤灭菌,不经高压灭菌)(Sigma-Aldrich,目录号:G0750)
  35. Tryptone(BD,Bacto TM ,目录号:211705)
  36. 氯化钠(NaCl,认证ACS等级≥99.0%)(Fisher Scientific,目录号:S271)
  37. 氯化钾(KCl,BioXtra≥99.0%)(Sigma-Aldrich,目录号:P9333)
  38. 氯化镁六水合物(MgCl 2•6H 2 O,BioXtra≥99.0%)(Sigma-Aldrich,目录号:M2670)
  39. 硫酸镁溶液(分子生物学级别的MgSO 4)(Sigma-Aldrich,目录号:M3409)
  40. 超纯琼脂,细菌级(美国生物技术资料公司,目录号:A01PD-500)
  41. 酵母氮碱减去氨基酸和负硫酸铵(Sigma-Aldrich,目录号:Y1251)
  42. 硫酸铵((NH 4)2 SO 4认证的ACS等级≥99.0%)(Fisher Scientific,目录号:A702)
  43. “几乎完全”的氨基酸混合物
    腺嘌呤HCl(Sigma-Aldrich,目录号:A9795),20mg / L
    精氨酸(Sigma-Aldrich,目录号:A5131),20mg / L
    酪氨酸(Sigma-Aldrich,目录号:T3754),30mg / L
    异亮氨酸(Sigma-Aldrich,目录号:I2752),30mg / L
    苯丙氨酸(Sigma-Aldrich,目录号:P2126),50mg / L
    谷氨酸(Sigma-Aldrich,目录号:G1251),100mg / L 天门冬氨酸(Sigma-Aldrich,目录号:A9256),100mg / L 苏氨酸(Sigma-Aldrich,目录号:T8625),200mg / L
    丝氨酸(Sigma-Aldrich,目录号:S4500),400mg / L
    缬氨酸(Sigma-Aldrich,目录号:V0500),150mg / L
  44. 甲硫氨酸(Sigma-Aldrich,目录号:M9625)
  45. 赖氨酸(Sigma-Aldrich,目录号:L5626)
  46. 组氨酸(Sigma-Aldrich,目录号:H8125)
  47. 亮氨酸(Sigma-Aldrich,目录号:L8000)
  48. 尿嘧啶(Sigma-Aldrich,目录号:U0750)
  49. 5-氟尿酸(5-FOA)(Oakwood Products,目录号:003234)
  50. 氢氧化钠(NaOH认证ACS等级≥97.0%)(Fisher Scientific,目录号:S318)
  51. 三碱基,分子生物学等级≥99.8%(Fisher Scientific,目录号:BP152)
  52. 冰醋酸(认证ACS级)(Fisher Scientific,目录号:A38)
  53. 乙二胺四乙酸(EDTA 99%-101%)(Fisher Scientific,目录号:S311)
  54. 超纯无菌水(Millipore Sigma,Milli-Q水净化系统)
  55. YPD液体介质(见配方)
  56. YPGal(见配方)
  57. SOC介质(参见食谱)
  58. YPD板(适当的药物可选)(见配方)
  59. 合成辍学板/媒体(见配方)
  60. 5-FOA板(参见食谱)
  61. LB平板(含适当药物)(见食谱)
  62. TAE缓冲区(见配方)

设备

  1. 移液器(Rainin,Pipet-Lite LTS,1,000μl,250μl,20μl和2μl大小)
  2. PCR机(MJ Research PTC-200Peltier Thermo Cycler,双30孔α嵌段)(MJ Research,型号:PTC-200)
  3. 离心机(Eppendorf微量离心机)(Eppendorf,型号:5415D,目录号:022621408)
  4. Eppendorf转子(24 x 1.5 / 2 ml)(Eppendorf型号:F-45-24-11,目录号:022636502)
  5. 涡旋适配器(Fisher Vortex Genie 2混合器的Microtube泡沫插入件)(Scientific Industries,型号:Vortex Genie 2,目录号:504-0234-00)
  6. 制冰机(Hoshizaki American)
  7. 水浴(Thermomix循环水浴,型号B,852 013/5型)
  8. DNA凝胶电泳仪(HE 33 Mini Submarine Unit)(GE Healthcare,目录号:80-6052-45)
  9. ChemiDoc UV透射仪(Bio-Rad Laboratories,型号:ChemiDoc TM XS +,目录号:1708265)
  10. 孵化器旋转器(Labquake摇床)(Labindustries,型号:T-415-110)
  11. 孵化器(VWR,型号:Model 1535)
  12. 高压灭菌器(加利福尼亚大学巴克厅)

程序

这里我们描述了用于设计,构建,整合和编辑酵母基因组中的Cas9人工靶序列的策略。我们重点关注如何精确安装编程的目标位点的克隆方法(其他方法可能在某些步骤中被替代,我们强调这些),并与Cas9 体内复用。我们详细说明了我们发表的(Finnigan和Thorner,2016)概念证明,描述了使用基本和非必需基因以及Cas9基因本身的方法来说明这种方法的效用和功能。

  1. 人造地点的选择
    1. 首先,必须选择与选择的生物体(发芽酵母基因组)具有最大不匹配的至少23bp(20个靶标+ 3个Protospacer相邻基序[PAM])的唯一靶序列(称为“[u1]”) )和系统的所有其他外生成分(添加的载体,GFP,药物盒,标签,等等)。所选择的序列可以来自另一个生物体(在我们的例子中,选择了两个人基因序列),或者可以从新生成生成。
      注意:其他研究已经发现了靶序列(和PAM)的各种限制。推荐(在酵母中)不具有poly-T序列(五个或更多个),因为它代表RNA Pol III的终止信号(DiCarlo等,2013)。另外,不推荐使用极端AT或GC丰富的序列。
    2. 应使用NCBI Basic Local Alignment Search Tool(BLAST)( https://blast.ncbi.nlm.nih.gov/Blast.cgi )。应首先搜索3'最多15 bp(包括PAM)的初步筛选。这表示Cas9 / sgRNA复合物使用的“种子”区域,其首先与推定的匹配的基因组靶序列相关联(Jinek et al。,2012; DiCarlo等人。 ,2013; Jiang等人,2013)。应确定这些碱基之间的最大不匹配,特别强调PAM序列的GG碱基内的错配。一旦发现了推定的靶标,就可以对酵母基因组询问整个23bp序列的全面搜索,同样偏好高水平的错配。
      注意:如果将人造目标包含在开放阅读框内(并作为附加的N-和/或C-terminal标签转换),则应包括额外的基数(23 + 1)以保持序列在帧内。此外,应避免在序列内发生任何帧内终止密码子。
    3. 在构建任何组装的质粒和/或基因组整合之前,应仔细考虑使用最少数量的sgRNA的该多路复用系统的设计。例如,一旦创建了应变施工的初始投资,就可能难以撤销基因中唯一位点的总数及其位置。我们的系统利用位于多个基因座侧翼的[u1]靶位点进行操作,而Cas9基因本身侧面是[u1]或[u2]位点,提供了同时或连续无缝切割核酸酶的选择。 >
  2. 标记基因,Cas9和sgRNA质粒的构建
    1. 在选择独特的[u1,u2,u3 ...]人造位点(方法A)之后,以及在所述位点(图1,底部),体内质粒组装(Finnigan and Thorner,2015)可用于将位点安装在其精确位置。
      注意:我们以前描述了质粒“体内连接”的整个过程,然后从酵母中回收构建的载体(Finnigan and Thorner,2015)。我们将包括与原始协议有关的相关修改的附加注释/步骤。


      图1.在芽殖酵母中Cas9多态基因编辑的人造DNA序列的设计,构建和使用。 热门。工作流概述。该方案开始于人造DNA Cas9靶位点的选择,随后构建包含Cas9和sgRNA表达盒的所需质粒。接下来,进入酵母基因组的连续整合事件将目标位点放置在包括Cas9在内的多个位点。在Cas9存在下转化sgRNA质粒和扩增的供体DNA片段允许在体内编辑。最后,通过诊断PCR和DNA测序验证基因组,证实了预期的修改。底部。在三个位点(其中一个包括Cas9)含有六个相同[u1](独特工程序列)位点的实例基因组。在Cdc11(单个红色星号)的N-和C-末端附有24bp序列(20个靶标+ 3个PAM + 1个额外碱基)作为8个残基标签。在缺失的GeneY(Shs1)的情况下,添加的[u1]位点(23bp)是UTR的一部分(两个红色星号)。仅使用一种sgRNA可以在所有六个基因组位置上形成双链断裂(DSB)。与UTR具有侧翼同源性的相应的扩增供体DNA的存在(为了清楚而仅示出一个)允许通过同源重组同时进行多基因标记的整合事件。根据Cas9基因侧翼的人工位点[u1或u2]的身份,可以根据给定菌株未来编辑事件的需要,实现Cas9的伴随或连续切除。

    2. 寡核苷酸的设计应包括预期的23(或24)bp人工序列,并将其置于框内作为待标记基因的一部分,或通过两步PCR扩增置于侧翼UTR内。如图2所示,我们描述了给定基因的N-和C-末端两个相同的[u1]标签的构建过程。然而,我们选择将每个标签的包含分成两个并行构建的中间质粒。如果使用单个质粒组装过程,则含有预期的[u1]序列的PCR片段之间将存在竞争;可能的是,不是将WT基因包括在两个人造部位之间,而是可能导致排除中心片段,并且仅产生单个[u1]位点。为了消除这种可能性,我们分别构建了每个[u1]序列。这可能在其他应用中有用,其中基因可能有意地位于两个不同的人造位点的两侧

      图2.含有侧翼独特序列位点的样品基因的质粒装配[u1]。 1。在酵母中使用体内质粒组装(Finnigan and Thorner,2015),将一个人工位点[u1]插入到通用启动子(prGeneX)和样品基因(GeneX)的开放读框之间。该策略也可以应用于使用MX盒(Goldstein和McCusker,1999)或任何开发的遗传等位基因的基因的缺失。插入的[u1]序列包括在两个重叠的寡核苷酸之内,这些寡核苷酸允许在组装的质粒中包含所需的靶向靶标。所示的示例包括使用 S。 pombe HIS5 MX盒式磁带;质粒再循环的选择也是足够的。也使用体内连接构建类似的质粒,其在基因的最后一个密码子(如图所示)和天然终止子序列的上游插入[u1]位点。为了选择目的,还包括了ADH1 -MX磁带。每个产生的质粒用作两个PCR的模板,其中两个相同的[u1]位点在适当的位置;片段之间产生的重叠同源性跨越了样本基因的整个开放阅读框架(如图所示)。 4.新设计的盒可以通过PCR扩增,并包含两个[u1]位点以及5'和3'UTR序列(也分别称为prGeneX和GeneX(t))侧翼的基因)整合(见图4)
    3. 在成功构建两个单独的[u1]标记序列之后,应使用来自每个原始载体的扩增产物进行第二轮装配。在这种情况下,虽然[u1]可以在技术上提供23(或24)bp的重叠同源性,但是全基因的整体提供> 1,200 bp的同源性,极大地偏倚了该序列内发生的交叉,并使组装倾向偏移,包括[u1]位点以及中心基因。使用侧翼[u1]位点构建CDC11 (WT基因)和SHS1(删除盒)两者。
      注意:该过程也可以使用缺失盒而不是基因的WT编码序列来实现。如果标记的盒子包括可选标记,则可以在单个步骤中进行构建,因为主动选择将确保[u1]位点以及删除标记物的存在。
    4. 对于Cas9表达盒的构建(图3),我们证明了使用由于最终目的产物的复杂性(九个单独的DNA序列组装)构建的另外的中间质粒。在单个质粒组合物插入[u1](显示)或[u2]靶位点之后,第一中间体在诱导型启动子和通用终止子之间产生具有唯一限制性位点( I)的载体序列。另外一轮的体内组装允许包含Cas9基因(两个扩增片段,从Addgene质粒#43804扩增)和C末端NLS序列。
      注意:该中间整合载体(图3,#3)将允许将任何Cas9直向同源物快速包含到酵母中的[u1]或[u2]含HIS3基因座中。

      图3. Cas9表达盒的质粒组装1.与样品基因周围的侧翼[u1]位点的组装相似(图2),构建[u1]或[u2]侧翼表达盒以逐步方式(红色星号)完成。 S上。化脓性成虫Cas9受GAL1 / 10启动子的控制,并位于酵母HIS3基因的独特位点和5'UTR的下游。独特的限制性位点(RS,不是 I)被包含在起始密码子之后和在ADH1终止符序列之前。 2.将[u1]或[u2]位点置于整个MX终止子序列之后,但位于酵母HIS3 3'UTR的上游。 3.在构建每个单独的载体后,将两个片段进行PCR扩增,并使用体内结合将其合并成较大的载体以产生中间表达载体。 4.独特的不限制性酶切位点的消化可以进行质粒装配的最后一步,以插入Cas9和NLS(核定位信号),两者都以两个重叠的PCR片段在帧内与起始点扩增密码子。 5.在验证整个Cas9表达盒后,PCR扩增可以在HIS3基因座上融合到酵母基因组中(见图4)。


      图4.将[u1]侧翼基因和Cas9表达盒整合到酵母基因组中。 作为一个概念证明,我们选择使用两种天然酵母基因(和SHS1 )在酵母中试验这种方法,并将Cas9集成到一个安全的-harbor(HIS3 )位点。 CDC11 是必不可少的基因,我们的策略说明了为进行基本和非必需基因的修饰和Cas9编辑而采取的其他步骤。使用表达缺少3'UTR序列的GeneX(TM119)的WT拷贝的基于URA3的覆盖载体转化WT BY4741酵母。 2.使用标准的基于MX的缺失盒删除内源GeneX拷贝,并在含有G418的培养基上进行选择。 3.将整个[u1]侧翼的GeneX构建体(图2)扩增并转化到GeneXΔ菌株中,并在含有5-FOA的培养基上选择,以反向选择基于URA3的覆盖载体。剩下的唯一可行的酵母将整合[u1]侧翼的GeneX(WT)拷贝代替Kan R 盒,导致无标记的修饰。酵母整合事件都经过每轮连续修改后的诊断性PCR和DNA测序证实。 4.在单独的GeneY基因座( 1 ),构建了一个缺失盒,并侧接了[u1] Cas9位点(与图2中的GeneX相似的策略)并转化进入酵母以删除内源GeneY拷贝。最后,扩增整个Cas9表达盒(图3),转化成酵母,并在HIS3基因座(he3Δ1)上整合,提供菌株G418电阻。如图所示,最终菌株含有六个相同的[u1]靶位点。然而,还使用与位于Cas9盒侧面的[u1]位点不同的[u2]序列测试替代构建体,允许在HIS3 基因座处进行单独和顺序编辑。

    5. 在构建和验证所需的质粒(图2,3底)后,最终的PCR扩增允许将整个设计的盒整合到酵母基因组中(参见方法C)。
    6. 可以从质粒或染色体制剂扩增供体PCR,在DSB断裂的任一侧具有至少30bp的侧翼同源性。用作我们的概念验证的供体DNA的PCR片段(,CDC11,SHS1和HIS3 )从分离的染色体DNA准备的WT酵母与几百个碱基的侧翼UTR序列。为了扩增WT HIS3,使用SF838-1Dα基因组DNA。
    7. 可以使用多种方案构建sgRNA表达质粒。我们的sgRNA的构建包括:
      1. 在具有SNR52启动子,SUP4终止子的(DiCarlo等人,2013)之后,对sgRNA盒序列进行建模,并且包括[ u1] 20 bp靶序列和所需的结构RNA序列(79 bp)。化脓性成虫 Cas9。从Genscript(Piscataway,NJ)合成了整个盒子。
        注意:已经开发了许多方法,其允许构建包括限制性消化和连接,体内组装或吉布森克隆的sgRNA质粒(Laughery等人,2015; Ryan等人,2016)。 >
      2. 侧翼限制位点应包含在合成的sgRNA盒(Finnigan和Thorner,2016)的图1C中。将我们的合成基因(Genscript)克隆到pUC57中,并含有侧翼的BamHI和XhoI位点。高拷贝的pRS425酵母质粒和[u1] sgRNA质粒应该在37℃和孵育器两端用BamHⅠ和XhoⅠ消化过夜,分离出线性化的片段在1%琼脂糖凝胶上,并使用Qiaquick凝胶提取试剂盒(QIAGEN)萃取。
      3. 使用T4 DNA连接酶(NEB),纯化的DNA应在16℃连接16小时。通过在65℃下孵育10分钟(在PCR块中进行)停止反应。
      4. 应用10μl连接反应转化100μlTOP10感受态大肠杆菌,在冰上孵育15分钟,在42℃热冲击45秒(水浴),回收到冰2分钟,轻轻混合500μlSOC培养基(参见食谱),37℃孵育1小时(慢转培养箱)。
      5. 应将细胞接种在LB + AMP平板上(参见食谱),并在37℃孵育过夜。将克隆分离物在液体LB + AMP培养基(37℃水浴中过夜14-16小时)培养,并用细菌质粒提取试剂盒(GeneJet)提取质粒。 sgRNA质粒的DNA测序后的诊断性消化可用于确认从pUC57到pRS425的正确亚克隆。
      6. 为了产生靶向sgRNA的质粒(除了20bp的靶序列以外,其相同),连续的修改的快速PCR(Zheng等,2004)可以是用于将[u1]序列变为[u2]。 (这可以在较小的pUC57载体,然后是上述亚克隆步骤或最终的pRS425载体上实现)。简言之,应设计60 bp的寡核苷酸(正向和反向引物)互补并含有中心1-4碱基取代(变化不必是连续的,而应在相对较近的区域内)。
      7. 应使用高保真KOD热启动聚合酶(EMD Millipore),推荐条件,退火温度为50°C,延长时间为40 sec / Kb,将整个载体进行PCR扩增。简单地说,将含有KOD组分(Mg 2+,1×缓冲液和1×dNTP)的50μl反应(总体积)全部提供),模板DNA(100-500ng),寡核苷酸反向,最终浓度为1μM每种引物),并且应制备1μlKOD酶。初始热启动循环(95℃,2分钟),然后循环由(i)95℃,15秒,(ii)50℃,30秒和(iii)72℃,X秒(其中X是模板质粒的总大小除以40秒/ Kb)。 16个总循环后,应包括在72°C的最后10分钟延伸时间。
      8. 在DNA琼脂糖凝胶上确认后,应用37℃(培养箱),用1μl的DpnⅠ消化7.5μlPCR反应物,最终反应体积为50μl。消化与dpn I用于破坏PCR反应中的环状(甲基化)模板酵母质粒DNA,以便除去由于模板DNA而不是预期的质粒组件可能在酵母中繁殖的假阳性克隆。
      9. 将2μl的经处理的消化反应的Dpn转化为E。大肠杆菌如前所述
      10. 克隆分离物应通过DNA测序确认
  3. 将构建体整合到酵母基因组中
    1. 我们将[u1] / [u2]侧翼DNA序列置于酵母基因组中的策略说明了必需(CDC11)和非必需(SHS1)之间的能力,用这种方法操纵的基因(图4)。我们的逐步整合方案的变化可以使用不同的基因缺失或其他方法来实现,包括(i)将并行和随后的交配,二倍体形成和孢子形成中的分离的标记基因整合到相反的交配类型中(给定的操纵基因包括可选择的某些标记); (ii)整合载体如pRS300系列(Sikorski和Hieter,1989)和/或(iii)CRISPR / Cas9本身以产生双链断裂,并允许以[u1] - 片段作为供体DNA插入。我们的程序说明了构建预期酵母菌株的一种可能的策略
    2. 应使用改良的乙酸锂方案(Finnigan和Thorner,2015),用保护性“覆盖载体”(URA3标记)转化WT BY4741酵母,并在SD-URA平板上选择。
    3. 应该将PCR基因(例如,,CDC11 )的基因敲除盒(Finnigan等,2015)用PCR扩增足够的5'和3'UTR侧翼序列 - 数百个bps),在37℃下用DpnI消化过夜,转化到酵母中,并在含有G418的培养基上选择。另外,由于缺少必需基因(例如标记的覆盖质粒),对含有5-FOA的平板(对于URA3标记的覆盖质粒进行反选),菌落也应敏感, em>cdc11Δ)渲染酵母应允。
      注意:URA3标记的覆盖载体不能包括任何天然的3'UTR,否则删除盒可以删除质粒载体而不是内源基因。
    4. 为了清楚/简单,我们将参考我们关于 CDC11 必需基因,SHS1 非必需基因和Cas9本身的概念证明。具有侧翼UTR(> 300bp)(图2)的[u1] - CDC11 - [u1](必需基因)PCR片段应转化到cdc11Δ酵母接下来用“Dpn”进行治疗。由于在整合片段上不存在可选择的标记,应将酵母铺在含有5-FOA的培养基上(选择用于恢复CDC11基因座和细胞活力)。所有产生的酵母菌株均应通过诊断PCR和DNA测序进行验证
    5. 其次,应使用[u1] - shs1Δ:: Hyg R - [ u1]以标准方式通过用扩增的PCR产物转化酵母(来自步骤C4),并在含有潮霉素的培养基上进行选择(图2)。 >
    6. 第三,应该通过添加一个(或两个)版本的Cas9-表达盒 - 侧翼为[u1]或[u2]位点来修改HIS3 基因座。由于包含“HIS3”和“3'UTR”序列(500bp)的整个Cas9盒(图3)的大尺寸,可以使用Cas9基因内部的引物产生两个扩增的PCR产物(产生用于重组的至少100bp的内部重叠),用DpnI处理,共转化到酵母中(来自步骤C5),并在含有G418的培养基上选择。殖民地也应该测试潮霉素抗性;由于所有基于MX的药物盒之间的共同启动子和终止子序列(Goldstein和McCusker,1999),建议每轮选择后确认所有标记的基因座(通过生长表型并可能通过诊断性PCR),因为存在标记“交换”的倾向,可能与所需站点的集成竞争。 Cas9表达盒的整合载体可以靶向任何一种。具有大约5'和3'UTR序列(在HIS3基因的任一侧的1,000bp内)的酿酒酵母菌株。这种整合在含有完整的HIS3 基因his3Δ1或his3Δ0酵母的菌株中也是有效的。
      注意:确认最终的[u1]和[u2]侧翼菌株应包括使用高保真聚合酶,PCR纯化和DNA测序的三个操纵基因座(CDC11,SHS1和HIS3)的PCR扩增

  4. CRISPR / Cas9复合编辑在芽殖酵母中使用人造目标位点
    1. 修改的酵母转化方案用于Cas9编辑。首先,为含有所有六个[u1]位点的酵母菌株以及URA3标记的酵母菌株制备过夜培养物(合成辍学培养基减去尿嘧啶,2%棉子糖,0.2%蔗糖,参见食谱)覆盖表达WT CDC11的载体,并在30℃温育
    2. 酵母菌株应该被稀释到大约0.30的OD 600中,进入YPGal(2%半乳糖)中,并在30℃下再培养4.5小时以诱导Cas9表达。
    3. 应以7,300 xgg收获大约10 OD 600细胞,用0.5ml水洗涤,以相同的速度再次离心一次,并用无菌的方法除去水移液器。
    4. 应将酵母重悬于0.5ml 100mM无菌乙酸锂中,以7300×g离心,然后除去上清液(将细胞留在管中)。
    5. 应创建一个'PEG主混合物'(新鲜),包含以下物质:240μl50%聚乙二醇(PEG),36μl1M乙酸锂,50μlSS DNA(煮沸10分钟并在冰上冷却)和可变量的无菌水(供体DNA,质粒DNA和水应该总共34μl)。混合物(包括水)应分开涡旋,并加入酵母颗粒中
    6. Cas9编辑的DNA应包括以下内容:
      1. sgRNA质粒(总数在1000-1,500ng之间)
      2. 供体DNA(每个编辑的基因座)作为扩增的PCR片段(1,000-1,500ng总DNA,基于PCR产物的长度而变化)。
    7. Cas9编辑的对照反应应包括(i)空载不含sgRNA的pRS425载体,即使在不存在任何供体DNA的情况下也可用作转化体总数的上限; (ii)在YPDex(2%葡萄糖)(见食谱)中培养的酵母(参见食谱)而不是半乳糖 - 然而,鉴于代谢活性和总细胞数的差异,这可能产生显着不同数量的转化菌落,和/或(iii)包含的sgRNA质粒,并且没有任何种类的供体DNA(无菌水) - 这导致Cas9诱导的双链断裂,但是需要任何存活的酵母在每个DSB位置进行NHEJ(两个对照在图5中示出)。


      图5.来自基于Cas9的编辑体内实例结果。 A.生成的酵母菌株与三个位点侧翼的六个相同的[u1]人造遗址示意图(GeneX,GeneY ,和Cas9位于HIS3基因座),并且在为每个基因座加入[u1]和供体DNA的sgRNA之后,为所有编辑的基因组位置预期的替换等位基因(WT)。 B.作为概念证明,我们提供了Cas9 mCAL编辑体内的示例数据。板A包括用sgRNA转化的酵母和所有三个供体PCR片段。板B包括sgRNA但没有供体DNA。板C包括空质粒(与引导RNA质粒骨架相同)且不含供体DNA。因为DSB在酵母中是致命的,所以板B导致没有可行的菌落,而供体DNA的包含允许修复每个操纵位点和大量的菌落。所提供的实施例不使用用于整合的供体DNA的任何选择,并且仅选择覆盖载体(URA3)和单个sgRNA质粒(LEU2)的存在。 />
    8. 在将PEG母液混合物加入酵母后,应加入适量的DNA(质粒和PCR),并将管子最大旋转10秒以达到完全再悬浮。
    9. 应在42℃热休克45分钟,以7300×g离心1分钟,用移液管除去PEG混合物。
    10. 应将酵母重新悬浮于新鲜的YPGal液体中,并置于30℃过夜(14-16小时)以回收。
    11. 恢复后,细胞(和YPGal液体)应直接镀在SD-URA-LEU板上,并在30℃下孵育3天,然后成像。我们的概念证明的三个示例条件如图5所示。板A.酵母用sgRNA [u1]质粒和供体DNA转化所有三个基因座。酵母B.用sgRNA [u1]质粒转化酵母,无供体DNA。用空的pRS425载体转化酵母酵母并没有供体DNA。每个板上记录菌落总数(图5底部)。 Cas9诱导的DSB使细胞不可行(图5B),但是对于所有三个基因座包含供体DNA允许通过同源重组进行后续修复,并拯救细胞活力(图5A)。
      注意:概念证明说明了Cas9编辑与人工位点的使用,没有标记选择(缺乏选择覆盖载体的尿嘧啶,而缺乏亮氨酸选择[u1] sgRNA的存在)。但是,如果需要,可以使用选择/标记。

  5. 编辑基因组序列的验证
    1. 酵母菌Cas9编码的确认应首先从克隆分离株中制备基因组DNA(Amberg等人,2006)。
    2. 应使用基因组DNA作为模板进行诊断性PCR,以测试被操纵基因座的身份和大小(在我们的例子中,CDC11,SHS1和/ HIS3 )来评估供体DNA是否适当地整合到基因组中。作为实例,当在2%琼脂糖DNA凝胶上分离时,除去CDC11的侧翼[u1]位点(和用天然基因替换)在人造部位损失时显示出小的变化。类似地,WT SHM1基因的整合(代替缺失)允许PCR组合提供正确整合的证据。
    3. 最后,操纵基因座应该再次扩增,纯化,并进行DNA测序。
      注意:如图所示,示例策略说明了侧翼为[u1]位点的Cas9表达盒。我们还通过构建[u2]侧翼Cas9盒带以及适当的引导RNA和供体DNA(Finnigan和Thorner,2016),编辑其他位点后,对Cas9进行了顺序的消除。在这种情况下,连续的转化将允许以后的时间去除Cas9基因。

数据分析

对于质粒和酵母菌株构建的所有阶段(程序A-E),进行诊断PCR和通过DNA测序的最终确认,以评估所有组装的DNA片段(质粒或基因组)是否按预期生成。使用在线DNA校准程序(生物工作台,圣地亚哥超级计算机中心)分析桑格序列。对于图5,使用扇区法计数菌落;独立实验一式三份进行,仅提供每个试验的代表性平板( http://www.g3journal.org/content/6/7/2147.long )(Finnigan and Thorner,2016)。

笔记

我们的系统包含了一些重要的改进和新颖的用途,用于在芽殖酵母中进行基于CRISPR / Cas9的基因编辑。

  1. 使用单个sgRNA靶向多个基因座位点(减少对多个sgRNA构建体的需求)
  2. 构建复合体或途径的所有基因侧翼的所有独特位点预编程的初始酵母菌株将允许通过Cas9进行大规模并行的未来复制和菌株生成;所有的组合都是可能的,因为每个未修饰基因的WT拷贝可以很容易地被修复(如我们已经显示的)
  3. 允许无缝切除Cas9基因本身与修改其他基因座或在未来时间顺序。
  4. 任何一侧(侧翼)人造遗址编程的目的地确保整个基因/标记/盒的切除,并有助于防止混杂交叉,如果单个等位基因或类似突变体需要整合到WT拷贝的位置。
  5. 这个过程可以用来编辑基因的基因
  6. 该方法可用于全部染色体片段,基因组或多个基因的缺失或修饰
  7. 基于Cas9的编辑可以实现无标记整合(携带引导RNA的质粒在无选择压力的情况下容易丢失)。
  8. 减少/消除离靶效应 - 由于绝对控制将在基因组内靶向的序列,因此应有助于优化编辑,同时将Cas9的募集减少到相似或其他基因组位点。
  9. 由于在基因组内多次具有完全相同的独特人造部位,因此可以全面筛选/搜索可能改变Cas9编辑的局部表观遗传或位置效应,因为sgRNA和靶序列在基因组上是相同的。
  10. 合成基因组工程中的援助 - 包含Cas9靶位点将允许整个基因组的未来编辑,染色体分裂,重排,等等。

食谱

  1. YPD液体介质
    1%酵母提取物
    2%蛋白胨
    2%葡萄糖
    高压灭菌
  2. YPGal
    1%酵母提取物
    2%蛋白胨
    2%半乳糖
    YP高压灭菌,半乳糖过滤器灭菌,混合
  3. SOC媒体
    2%胰蛋白胨
    0.5%酵母提取物
    10 mM NaCl
    2.5 mM KCl
    1mM MgCl 2
    1mM MgSO 4
    20 mM葡萄糖终浓度
  4. YPD板(如果需要,包括适当的药物)
    1%酵母提取物
    2%蛋白胨
    2%葡萄糖
    20克/升Bacto琼脂
    高压灭菌
    媒体冷却后, 55°C
  5. 合成辍学媒体(例如,,-URA-LEU)
    1.7克酵母氮碱减去硫酸铵和负氨基酸
    5.0g硫酸铵
    1.1g“几乎完全”的氨基酸混合物
    剩余氨基酸混合物(全部来自Sigma-Aldrich,甲硫氨酸150mg / L;赖氨酸180mg / L;组氨酸60mg / L
    注意:如果需要,亮氨酸260 mg / L和尿嘧啶20 mg / L。
    10毫克/升色氨酸(过滤灭菌,不经高压灭菌)
    2%葡萄糖
    20克/升Bacto琼脂
    将无菌水和琼脂与剩余组分分开混合并高压灭菌
  6. 5-FOA板
    合成培养基成分(适当的氨基酸辍学,如有)
    0.5 g / L 5-FOA
    0.5 g / L尿嘧啶
    将所有组分(与水/琼脂混合物分开)加热至75℃30分钟,过滤灭菌并与高压消毒的水/琼脂混合物结合使用
  7. LB板(含适当药物)
    1%胰蛋白胨
    0.5%酵母提取物
    15 g / L琼脂
    10g / L NaCl
    4 mM NaOH
  8. TAE缓冲液(1x终浓度),pH8.4。
    40mM Tris(1M储备溶液)
    20mM冰醋酸
    1mM EDTA(0.5M储备溶液,pH 8.0)

致谢

该项目由国家卫生研究院国家综合医学研究所的机构发展奖(IDeA)得到支持,授权号为P20 GM103418,提交给G.C.F.内容完全是作者的责任,并不一定代表国家综合医学科学研究所或国家卫生研究院的官方观点。这项工作得到堪萨斯州立大学约翰逊癌症研究中心创新研究奖(G.C.F.)的支持。这项工作得到堪萨斯州立大学艺术和科学学院的本科研究奖(R.M.G.)的支持。该协议由(Finnigan和Thorner,2016)修改和修改。我们要感谢Jeremy Thorner(加利福尼亚大学伯克利分校)提供有用的建议和意见。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Giersch, R. M. and Finnigan, G. C. (2017). Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences. Bio-protocol 7(18): e2557. DOI: 10.21769/BioProtoc.2557.
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