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Sep 2019

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PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System
CRISPR/Cas9系统的PCR介导的一日合成向导RNA   

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Abstract

Nowadays, CRISPR (clustered regularly interspaced short palindromic repeats) and the CRISPR-associated protein (Cas9) system play a major role in genome editing. To target the desired sequence of the genome successfully, guide RNA (gRNA) is indispensable for the CRISPR/Cas9 system. To express gRNA, a plasmid expressing the gRNA sequence is typically constructed; however, construction of plasmids involves much time and labor. In this study, we propose a novel procedure to express gRNA via a much simpler method that we call gRNA-TES (gRNA-transient expression system). This method employs only PCR, and all the steps including PCR and yeast transformation can be completed within 1 day. In comparison with the plasmid-based gRNA delivery system, the performance of gRNA-TES is more effective, and its total time and cost are significantly reduced.

Keywords: CRISPR/Cas9 (CRISPR/Cas9), Genome editing (基因组编辑), Guide RNA (向导RNA), PCR-based (基于PCR的 ), Yeast (酵母)

Background

Developing genome editing techniques is one of the central issues of genome science. For the past decade, the CRISPR/Cas9 system has contributed to easier and more precise genome editing as compared with previously developed techniques such as ZFN (zinc finger nuclease) and TALEN (transcription activator-like effector nuclease). For successful CRISPR/Cas9 engineering, design, expression, and delivery of the guide RNA (gRNA) components are the key factors (Stovicek et al., 2017). For prokaryotes like Escherichia coli and eukaryotes such as Saccharomyces cerevisiae, the most commonly employed method for expressing gRNA is to use a plasmid (Jiang et al., 2013; Li et al., 2015; DiCarlo et al., 2013; Bao et al., 2015; Jakočiunas et al., 2015a and 2015b); however, plasmid construction, including cloning steps for necessary components, is laborious, costly, and time-consuming. To express gRNA more simply, in this study we developed a method that we call gRNA-transient expression system (gRNA-TES), where gRNA is expressed from the PCR product. gRNA-TES is very fast and effective: it takes only 5-6 h to complete the whole process, including preparation of PCR products for expression of gRNA in yeast cells and yeast transformation. By contrast, it takes at least 3-4 days to construct a plasmid expressing gRNA including verification. As expected, when applied to replacement of desired chromosome regions in yeast, gRNA-TES effectively replaces single and multiple chromosomal regions (Easmin et al., 2019 and 2020). Therefore, we believe that gRNA-TES will be useful for other types of genome editing including segmental deletion, duplication, and splitting of chromosomes. Lastly, gRNA-TES is effective in yeast; therefore, it should be emphasized that gRNA-TES may also be efficacious in other organisms if suitable gene promoters are incorporated.


Materials and Reagents

  1. 0.1-10 μl pipette tips (BMBio, catalog number: BMT-10GXLR)

  2. 20-200 μl pipette tips (BMBio, catalog number: BMT-200R)

  3. PCR tubes (Axygen, catalog number: SKPCRF)

  4. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, catalog number: 43803)

  5. p414-TEF1p-Cas9-CYC1t (Addgene, catalog number: 43802)

  6. Escherichia coli DH5α competent cells (NIPPON GENE, catalog number: 316-06233)

  7. DNA, MB-grade from fish sperm (Roche Diagnostics, catalog number: 11467140001)

  8. KOD plus neo (TOYOBO, catalog number: KOD-401)

  9. 2 mM dNTP solution (included with KOD plus neo)

  10. 25 mM magnesium sulfate (MgSO4) (included with KOD plus neo)

  11. Oligonucleotide Primer Fw A for construction of fragment A (5’-GTTCGAAACTTCTCCGCAGT GAAAGATAAATGATCN20GTTTTAGAGCTAGAAATAGCAAG-3’) (synthesized by Hokkaido System Science, Japan) (N20 represents the 20-nt upstream sequence of the target PAM sequence)

  12. Oligonucleotide primer Rv A for construction of fragment A (5’- ACTCACAAATTAGAGCTTCA -3’) (synthesized by Hokkaido System Science, Japan)

  13. Oligonucleotide primer Fw B for construction of fragment B (5’- CGAACGACCGAGCGCAGCGA-3’) (synthesized by Hokkaido System Science, Japan)

  14. Oligonucleotide primer Rv B for construction of fragment B (5’- TTTATCTTTCACTGCGGAGAAGTTTCGAAC-3’) (synthesized by Hokkaido System Science, Japan)

  15. Ex Taq® DNA polymerase (TaKaRa, catalog number: RR001A)

  16. 10× Ex Taq buffer (Mg2+ plus) (included with Ex Taq® DNA Polymerase)

  17. 2.5 mM each dNTP mix (included with Ex Taq® DNA Polymerase)

  18. Ampicillin (Nacalai tesque, catalog number: 02739-74)

  19. Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883-250G)

  20. Polyethylene glycol 3,350 (Sigma-Aldrich, catalog number: P4338-500G)

  21. Sodium hydroxide (NaOH) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 192-15985)

  22. Control primer 1 for amplifying the CNE1 region (5’-TCACAGGGTCGATTGCAAGG-3’) (synthesized in Hokkaido System Science, Japan)

  23. Control primer 2 for amplifying the CNE1 region (5’-CTGGTGGTTCAGTGCCATCT-3’) (synthesized in Hokkaido System Science, Japan)

  24. Oligonucleotide primer 1 for checking replacement (Use the 200-176 nt upstream sequence of the target region)

  25. Oligonucleotide primer 2 for checking replacement (Use the 66-90 nt downstream reverse sequence of the target region)

  26. Prime Star Max Premix 2× (TaKaRa, catalog number: R045A)

  27. Agar (FUJIFILM Wako Pure Chemical Corporation, catalog number: 010-08725)

  28. Gene Ladder Wide 2 (Nippon Gene, catalog number: 310-06971)

  29. Glucose (FUJIFILM Wako Pure Chemical Corporation, catalog number: 043-31163)

  30. Yeast nitrogen base without amino acids (BD, Difco, catalog number: DF0919-15-3)

  31. Peptone (BD, BactoTM, catalog number: 211677)

  32. Yeast extract (BD, BactoTM, catalog number: 288620)

  33. Adenine HCL (FUJIFILM Wako Pure Chemical Corporation, product code: 016-00802)

  34. Synthetic Complete (SC) medium (see Recipes)

  35. YPDA medium (see Recipes)

Procedure

  1. Designing the 20-nt gRNA target sequence

    To design the gRNA target sequence, it is essential to have an appropriate PAM sequence. To design a target sequence with a PAM sequence for Cas9 cleavage (5’-NGG-3’ or 5’-CCN-3’ for opposite strand) close to the target region, we use the free software CRISPRdirect (https://crispr.dbcls.jp/). For S. cerevisiae, we select the S. cerevisiae representative strain S288C and paste 50-100 nt of the sequence located near the target site into the software. Then, CRISPRdirect quickly outputs the appropriate 20-nt gRNA target sequence with a PAM sequence.


  2. Preparation of PCR fragments A and B

    We use p426-SNR52p-gRNA.CAN1.Y-SUP4t as a common template plasmid to construct a PCR fragment (fragment C) expressing gRNA harboring the designed 20-nt target sequence. It contains the yeast promoter SNR52, which is responsible for expressing gRNA in yeast cells. If you want to express gRNA in another organism, you will need to use a template plasmid harboring a suitable promoter sequence obtained from that respective organism to prepare fragment C. Here, we explain the case in which gRNA is expressed from fragment C in a S. cerevisiae host. Before preparing fragment C, fragments A and B must be synthesized in two separate steps by first-round PCR.


    Fragment A: For preparation of fragment A, design the forward primer (Fw A) to include 35 nt of the sequence from part of the SNR52 promoter region (3,855-3,889 nt) of the template plasmid p426-SNR52p-gRNA.CAN1.Y-SUP4t (full sequence is available at Addgene repository, https://www.addgene.org/43803/sequences/), followed by the 20-nt gRNA target sequence, and a further 23 nt of the sequence encoding the 5’ part of the gRNA scaffold from the template plasmid (3,910-3,932 nt) (Figure 1A). Design the reverse primer sequence (Rv A) based on 5,001-5,020 nt of the reverse sequence (5’-ACTCACAAATTAGAGCTTCA-3’) of the template plasmid.


    Fragment B: Since fragment A does not contain the full sequence of the SNR52 promoter and it is essential to have this complete region for proper expression of gRNA, we need to incorporate the whole SNR52 promoter region with fragment A via fragment B. Therefore, we use a forward primer (Fw B) consisting of a 20-nt sequence (5’-CGAACGACCGAGCGCAGCGA-3’) and a reverse primer (Rv B) consisting of a 30-nt sequence (5’-TTTATCTTTCACTGCGGAGAAGTTTCGAAC-3’) of the template plasmid (3,301-3,320 nt and 3,855-3,884 nt in reverse sequence, respectively) to synthesize the whole SNR52 promoter sequence as fragment B (Figure 1A). The complementary sequence of the last 30 nt in fragment B is the same as the first 30 nt of fragment A in order to allow fragments A and B to be annealed in the second-round (overlap) PCR.



    Figure 1. Overview of gRNA-TES. A. In gRNA-TES, we prepare three fragments, namely fragments A, B, and C, by PCR. Fragments A and B are prepared by a first PCR using plasmid p426-SNR52p-gRNA.CAN1.Y-SUP4t (DiCarlo et al., 2013) as a common template. Next, fragment C, for expressing the gRNA sequence, is synthesized by a second PCR (overlap PCR) using fragments A and B as templates. In gRNA-TES, a new fragment A is always needed because it contains the 20-nt unique gRNA target sequence. The 20-nt unique gRNA target sequence is changed depending on the target region. By contrast, fragment B is solely the SNR52 promoter region; therefore, it is not necessary to synthesize a new fragment B with a different sequence every time. Using this protocol, it is not possible to design a primer sequence that can amplify the target sequence along with the whole SNR52 promoter region since it is difficult to chemically synthesize a good-quality oligonucleotide with more than 100 nucleotides. Thus, we cannot amplify fragment C by a single round of PCR. B. gRNA-TES can be used for various genome manipulations such as chromosome splitting, replacement, deletion, and duplication. Only one fragment C, which delivers one gRNA, is necessary for splitting. Double-strand break (DSB) significantly increases the frequency of homologous recombination between the target sequence (red and green box) on the chromosome and its corresponding DNA modules synthesized from plasmids p3121 (Sugiyama et al., 2005) and pSJ69 (Easmin et al., 2019). As a consequence, a high frequency of splitting is thought to occur. Two kinds of fragment C, which deliver two independent gRNAs, are necessary for replacement, deletion, and duplication. For replacement, we prepare one DNA module harboring an appropriate marker gene (here Candida glabrata LEU2, CgLEU2) flanked with the target homology sequences using plasmid pSJ69 (Easmin et al., 2019). For segmental deletion and duplication, we need two DNA modules harboring the target homology sequences: one should have a centromere, and the other should contain the marker gene. In our experiments, we use p3121 (Sugiyama et al., 2005) harboring CEN4 and pSJ69 harboring CgLEU2 as templates to synthesize the DNA modules. After successful double-strand break, a high frequency of replacement, deletion, and duplication is expected. In panel B, the black circle on the chromosome denotes the native centromere; the yellow circle on the DNA modules denotes artificially supplied CEN4. The blue arrow indicates the artificially supplied telomere; the gray box represents CgLEU2. The plasmids containing yellow- and gray-colored curved boxes are p3121 and pSJ69, respectively.


    First-round PCR reaction


  3. Preparation of fragment C by overlap PCR

    To prepare fragment C, a 100-fold dilution (×1/100) of fragment A is mixed with the same dilution of fragment B and used as a template for the second-round PCR (overlap PCR) to generate fragment C with forward primer (Fw B) 5’-CGAACGACCGAGCGCAGCGA-3’ and reverse primer (Rv A) 5’-ACTCACAAATTAGAGCTTCA-3’.


    Second-round PCR reaction


    Note: We recommend using Ex Taq® DNA polymerase for gRNA-TES, especially for the second-round PCR (overlap PCR). Although we tested various DNA polymerases, we observed that those DNA polymerases frequently produced multiple unexpected bands, especially during the overlap PCR used to prepare fragment C.


    Application of gRNA-TES to various genome manipulations

    gRNA-TES has a variety of applications from splitting to segmental replacement, deletion, and duplication of chromosomes (Figure 1B). Depending on the genome engineering technique, up to two kinds of fragment C delivering two types of gRNA are necessary. After DSB, the frequency of splitting, replacement, deletion, and duplication is expected to be increased.


    1. Preparation of DNA modules

      To split, delete, replace, or duplicate target chromosomal regions, it is necessary to incorporate DNA modules into gRNA-TES. The type of DNA module that is constructed depends upon the purpose (splitting, deletion, replacement, or duplication) of genome editing. For replacement, only one DNA module is sufficient, while for splitting, deletion, or duplication, two DNA modules are needed (Figure 1B). For replacement, design and purchase oligonucleotide primers to amplify any genetic marker flanked with the homology sequence corresponding to the first and last 30 bp of the target region. For splitting and deletion, design forward primers to amplify the centromere or marker gene flanked with 50 bp of the homology sequence corresponding to the upstream or downstream sequence of the target splitting and deletion point. For duplication, design forward primers to amplify the marker gene or centromere flanked with the homology sequence corresponding to the first or last 50 bp of the target region. To include a telomere seed sequence in all DNA modules for splitting, deletion, and duplication, we use a common reverse primer including the 5’-CCCCAACCCCAACCCCAACCCCAACCCCAACCCCAA-3’ sequence. You can use any template plasmid depending on the purpose of genome editing. We use pSJ69 (Easmin et al., 2019) and p3121 (Sugiyama et al., 2005) to synthesize DNA modules. These plasmids have the same background because they were constructed from the same plasmid pUG6 (Güldener et al., 1996) and are available upon request. For splitting, deletion, or duplication, it is necessary to select an appropriate plasmid as a template for PCR so that the newly generated chromosomes contain only one centromere.


      PCR reaction

      10× KOD plus neo buffer 5 μl
      2 mM dNTP solution 5 μl
      25 mM MgSO4 3 μl
      Template plasmid 1 μl
      Primers for Replacement

      Oligonucleotide primer 1 (15 pmol)

      5’-N30FGGCCGCCAGCTGAAGCTTCG-3’

      1.5 μl (N30F represents the first 30-bp sequence of the target region)

      Oligonucleotide primer 2 (15 pmol)

      5’-N30LAGGCCACTAGTGGATCTGAT-3’

      1.5 μl (N30L represents the last 30-bp reverse sequence of the target region)
      Primers for Splitting and Deletion

      Forward primer (15 pmol)

      5’-N50U/50DGGCCGCCAGCTGAAGCTTCG-3’

      1.5 μl (N50U/50D represents the 50-bp upstream or downstream (reverse) sequence of the target region)

      Reverse primer (15 pmol)

      5’-CCCCAACCCCAACCCCAACCCCAACCC CAACCCCAAAGGCCACTAGTGGATCTGAT-3’

      1.5 μl
      Primers for Duplication

      Forward primer (15 pmol)

      5’-N50F/50LGGCCGCCAGCTGAAGCTTCG-3’

      1.5 μl (N50F/50L represents the first (reverse) or last 50-bp sequence of the target region)

      Reverse primer (15 pmol)

      5’-CCCCAACCCCAACCCCAACCCCAACCC CAACCCCAAAGGCCACTAGTGGATCTGAT-3’

      1.5 μl
      KOD plus neo 1 μl
      Water 32 μl
      Total 50 μl
      Pre-denaturation: 94°C, 2min
      Denature: 98°C, 10 s
      Annealing: 55°C, 30 s 30 cycles
      Extension: 68°C, 2 min


    2. Yeast transformation

      1. Prepare in advance a yeast strain, for example, SJY30 (MATα ura3-52 his3-Δ200 leu2Δ1 lys2Δ202 trp1Δ63 harboring plasmid p414-TEF1p-Cas9-CYC1t [DiCarlo et al., 2013]), that expresses codon-optimized Cas9 by the introduction of p414-TEF1p-Cas9-CYC1t.

      2. Cultivate the strain overnight in YPDA liquid medium.

      3. Inoculate a fresh 5-ml aliquot of YPDA liquid medium with yeast cell pre-culture to an initial OD600 of approximately 0.2-0.3. Then incubate with a shaking speed of 140 rpm at 30°C until the OD600 reaches 0.8-1.0 (about 4-6 h).

      4. Mix the appropriate DNA modules and gRNA-expressing fragment C (e.g., we need two kinds of fragment C [Figure 1B] to target both edges of the target region and one DNA module to replace chromosomal regions) and perform transformation using the conventional LiAc/PEG method (Gietz and Schiestl, 2007).

      5. The aim of this protocol is to provide users with a procedure that is as easy as possible at every step, including colony PCR described in the next section. Therefore, the protocol was developed without measuring the DNA concentration of PCR products. For routinely performed yeast transformation, we consistently use 12 μl DNA module and 11 μl each fragment C PCR reaction mixture to replace chromosomal regions, since we have obtained sufficient transformants using such amounts (i.e., 12 μl + 11 μl + 11 μl = 34 μl) of PCR reaction mixture. Furthermore, the whole PCR reaction mixture can be directly used for yeast transformation without purification.

      6. After yeast transformation, suspend the cells in 100 μl sterilized water, spread the whole suspension onto one or two selection plates, and incubate at 30°C for 2-3 days.

    3. Confirmation of the expected chromosomal change by colony PCR and subsequent agarose gel electrophoresis

      1. To make PCR-grade genomic DNA, take a small amount of cells from each colony and suspend in 10 μl 0.02 M NaOH solution. Heat at 98°C for 10 min in a heat block and then transfer to ice.

      2. As an example, we describe how to confirm transformants obtained from a replacement experiment here. Design and purchase oligonucleotide primers for colony PCR. You can use any sequence from the upstream and downstream sequences of the target region, but we recommend designing the primer in such a way that the final PCR product will be <2 kb, since generating a larger PCR product may be problematic during colony PCR. For oligonucleotide primer 1, use a 200-176 nt upstream sequence of the target region and for oligonucleotide primer 2, use a 66-90 nt downstream reverse sequence of the target region. Since we use the CgLEU2 marker gene for replacement and the size of CgLEU2 is 1685 bp, after successful replacement of the target region, the size of the PCR product will be 200 bp + 1685 bp + 90 bp = 1975 bp (Figure 2). For example, to check the replacement of a 500-kb region in Chromosome 4 (coordinate number 494271-994270), design oligonucleotide primer 1 based on the 494071-494095 nt sequence (5’-CATATCAGTGTCTTCATCTTCATGA-3’) and oligonucleotide primer 2 based on the 994,336-994,360 nt reverse sequence (5’-TAGTGGATACGCAGGACGTGTTATC-3’) of Chromosome 4. In addition, we recommend designing control primers to check whether the PCR reaction is proceeding well. Any genomic region may be amplified as a control; we amplify the CNE1 region of Chromosome 1 as an internal control. Control primer 1 is based on the 211-230 nt sequence (5’-TCACAGGGTCGATTGCAAGG-3’) and control primer 2 on the 861-880 nt reverse sequence (5’-CTGGTGGTTCAGTGCCATCT-3’) of the CNE1 region. If the CNE1 gene is properly amplified, you will obtain a 670-bp PCR product.

      3. PCR reaction

        Prime Star Max Premix 2× 12.5 μl
        Template genomic DNA 0.5 μl

        Control primer 1 (7.5 pmol)

        5’-TCACAGGGTCGATTGCAAGG-3’

        0.75 μl

        Control primer 2 (7.5 pmol)

        5’-CTGGTGGTTCAGTGCCATCT-3’

        0.75 μl

        Oligonucleotide primer 1 (7.5 pmol)

        (Use the 200-176 nt upstream sequence of the target region)

        0.75 μl

        Oligonucleotide primer 2 (7.5 pmol)

        (Use the 66-90 nt downstream (reverse) sequence of the target region)

        0.75 μl
        Water 9 μl
        Total 25 μl

        Denature: 98°C, 10 s

        Annealing: 55°C, 5 s 30 cycles
        Extension: 72°C, 10 s


    Note: You can use any DNA polymerase for colony PCR. However, since our intention is to make this protocol as fast as possible, we use Prime Star Max Premix 2×, which requires an extension time of only 5 s/kb.



    Figure 2. Typical agarose gel electrophoresis results for chromosome replacement analysis by gRNA-TES. Lanes 1 to 9 represent independent transformants. M represents size markers (Gene Ladder Wide 2, Nippon Gene, Toyama, Japan). Lanes 1 to 3 represent transformants obtained from replacement of a 300-kb region; lanes 4 to 6 represent transformants obtained from replacement of a 400-kb region; and lanes 7 to 9 represent transformants obtained from replacement of a 500-kb region of Chromosome 4. Almost all transformants (except those in lane 3) showed the expected replacement of the respective chromosomal region and yielded the 1975-bp band. The internal control band (670 bp) was also observed in all transformants.

Data analysis

The number of transformants varies in each experiment. For example, when we targeted the replacement of smaller chromosomal regions of, for example, 150 kb and 200 kb, we obtained 263 and 287 transformants, respectively, in a single transformation (Easmin et al., 2019). When we targeted 300-kb, 400-kb, and 500-kb regions, we obtained, respectively, 51, 43, and 103 transformants in a single transformation. We tested six transformants for replacement of each of the 150-kb and 200-kb regions, and 5 transformants for each of the 300-kb, 400-kb, and 500-kb regions. The frequencies of expected replacement of the 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions were 100%, 66.6%, 80%, 100%, and 100%, respectively (Easmin et al., 2019). By contrast, a maximum of 16.6% expected frequency was observed for replacement of the 150-kb region when gRNA-TES was not applied; for replacement of the other chromosomal regions, no correct transformants were obtained when gRNA-TES was not employed.

Recipes

  1. Synthetic complete (SC) medium

    2% glucose

    0.67% yeast nitrogen base without amino acids (e.g., BD Difco)

    Note: 0.2% dropout mix containing all amino acids and nucleic acid bases lacking specific amino acids can be used for auxotrophic selection.

    For the plate assay, 2% agar is added

  2. YPDA medium

    2% glucose

    2% peptone (e.g., BD BactoTM)

    1% yeast extract (e.g., BD BactoTM)

    0.004% adenine HCl (FUJIFILM Wako Pure Chemical Corporation)

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science (JSPS)-KAKENHI, Grant-in-Aid for Scientific Research (B), Grant numbers [JP 15H04475] to S.H. This protocol was adapted from our previously published work (Easmin et al., 2019).

Competing interests

The authors declare no competing interests.

References

  1. Bao, Z., Xiao, H., Liang, J., Zhang, L., Xiong, X., Sun, N., Si, T. and Zhao, H. (2015). Homology -Integrated CRISPR–Cas (HI-CRISPR) system for one-step multi-gene disruptions in Saccharomyces cerevisiae. ACS Synth Biol 4: 585-594.
  2. DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J. and Church, G. M. (2013). Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7): 4336-4343.
  3. Easmin, F., Hassan, N., Sasano, Y., Ekino, K., Taguchi, H. and Harashima, S. (2019). gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing. J Biosci Bioeng 128(3): 373-378.
  4. Easmin, F., Sasano, Y., Kimura, S., Hassan, N., Ekino, K., Taguchi, H. and Harashima, S. (2020). CRISPR-PCD and CRISPR-PCRep: Two novel technologies for simultaneous multiple segmental chromosomal deletion/replacement in Saccharomyces cerevisiae. J Biosci Bioeng 129(2): 129-139.
  5. Gietz, R. D. and Schiestl, R. H. (2007). High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 31-34.
  6. Güldener, U., Heck, S., Fielder, T., Beinhauer, J. and Hegemann, J. (1996). A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24: 2519-2524.
  7. Jakočiunas, T., Bonde, I., Herrgård, M., Harrison, S. J., Kristensen, M., Pedersen, L. E., Jensen, M. K. and Keasling, J. D. (2015a). Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae. Metab Eng 28: 213-222.
  8. Jakočiunas, T., Rajkumar, A. S., Zhang, J., Arsovska, D., Rodriguez, A., Jendresen, C. B., Skjødt, M. L., Nielsen, A. T., Borodina, I., Jensen, M. K. and Keasling, J. D. (2015b). CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae. ACS Synth Biol 4(11): 1226-1234.
  9. Jiang, W., Bikard, D., Cox, D., Zhang, F. and Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3): 233-239.
  10. Li, Y., Lin, Z., Huang, C., Zhang, Y., Wang, Z., Tang, Y. J., Chen, T. and Zhao, X. (2015). Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab Eng 31: 13-21.
  11. Stovicek, V., Holkenbrink, C. and Borodina, I. (2017). CRISPR/Cas system for yeast genome engineering: advances and applications. FEMS Yeast Res 17(5).
  12. Sugiyama, M., Ikushima, S., Nakazawa, T., Kaneko, Y. and Harashima, S. (2005). PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae. Biotechniques 38(6): 909-914.

简介

[摘要]如今,CRISPR(clustere d定期间隔短回文重复序列)和CRISPR相关蛋白(Cas9)系统在基因组编辑中发挥着重要作用。为了成功靶向基因组的所需序列,C RISPR/Cas9 系统必不可少的就是引导核糖核酸 (gRNA) 。为了表达 gRNA,通常构建表达 gRNA 序列的质粒;^ h H但是,质粒的建设涉及很多时间和劳力。在这项研究中, 我们提出了一种通过一种更简单的方法来表达 gRNA 的新程序,我们称之为 gRNA-TES(gRNA 瞬时表达系统)。该方法仅采用PCR,PCR、酵母转化等所有步骤均可在1天内完成。在C ompar ISON与基于质粒的gRNA递送系统,gRNA-TES的性能是更有效的,并且它的总时间和成本显著降低。

[背景]开发摹Ë诺姆编辑技术是基因组学的核心问题之一。在过去的十年中,与之前开发的技术,如 ZFN(锌指核酸酶)和 TALEN(转录激活因子样效应核酸酶)相比,CRISPR/Cas9 系统有助于更容易和更精确的基因组编辑。对于成功的 CRISPR/Cas9 工程,引导 RNA (gRNA) 组件的设计、表达和传递是关键因素(Stovicek等,2017)。对于像原核生物大肠杆菌和真核生物,如酿酒酵母,表达gRNA是使用最常用的方法一质粒(姜等人,2013;李等人,2015;迪卡洛等人,2013;鲍等人. , 2015;Jako č iunas等人,2015a和2015b) ;ħ H但是,质粒构建,包括克隆步骤为必要成分,是费力的,昂贵的,并且费时。为了更简单地表达 gRNA,在本研究中,我们开发了一种称为 gRNA 瞬时表达系统 (gRNA-TES) 的方法,其中 gRNA 从 PCR 产物中表达。gRNA-TES是非常快速和有效的:只需要5 - 6小时以完成整个过程,包括制备的PCR产物的在酵母细胞和酵母转化gRNA的表达。乙ÿ相反,至少需要3 - 4天,以构建质粒表达gRNA包括核查。正如预期的那样,当应用于替换酵母中所需的染色体区域时,gRNA-TES 有效地替换了单个和多个染色体区域(Easmin等,2019和2020)。因此,我们相信 gRNA-TES 将可用于其他类型的基因组编辑,包括染色体的片段删除、复制和分裂。最后,gRNA-TES 对酵母有效;因此,应当强调的是,gRNA-TES中号AY也如果合适的基因启动子被并入在其他有机体有效。

关键字:CRISPR/Cas9, 基因组编辑, 向导RNA, 基于PCR的 , 酵母



材料和试剂


0.1 - 10 μl移液器吸头(BMBio ,目录号:BMT-10GXLR)
20 - 200 μl移液器吸头(BMBio ,目录号:BMT-200R)
PCR管小号(爱思进,目录号:SKPCRF)
p426-SNR52p-gRNA.CAN1.Y-SUP4t(Addgene ,目录号:43803)
p414-TEF1p-Cas9-CYC1t(Addgene ,目录号:43802)
大肠杆菌DH5α感受态细胞(NIPPON GENE,目录号:316-06233)
DNA,来自鱼精子的 MB 级(Roche Diagnostics,目录号:11467140001)
KOD plus neo(TOYOBO,目录号:KOD-401)
2mM的dNTP溶液(我与KOD加新ncluded)
的25mM硫酸镁(硫酸镁4 )(我ncluded与KOD加新)
用于构建片段 A 的寡核苷酸引物Fw A(5'-GTTCGAAACTTCTCCGCAGT GAAAGATAAATGATCN 20 GTTTTAGAGCTAGAAATAGCAAG-3')(由日本北海道系统科学公司合成)(N 20代表目标 PAM 序列的 20-nt 上游序列)
用于构建片段 A 的寡核苷酸引物Rv A (5'- ACTCACAAATTAGAGCTTCA -3')(由日本北海道系统科学公司合成)
用于构建片段B的寡核苷酸引物Fw B (5'-CGAACGACCGAGCGCAGCGA-3')(由日本北海道系统科学公司合成)
寡核苷酸引物器Rv乙施工的片段B(5'- TTTATCTTTCACTGCGGAGAAGTTTCGAAC-3' )(合成由北海道System Science,Japan)的
实施例的Taq ® DNA p olymerase(TaKaRa公司,目录号:RR001A)
10 ×实施例的Taq b uffer(镁2+加)(我ncluded具有Ex Taq酶® DNA聚合酶)
各2.5mM dNTP混合物(我ncluded具有Ex Taq酶® DNA聚合酶)
氨苄西林(Nacalai tesque ,目录号:02739-74)
二水醋酸锂(Sigma-Aldrich,目录号: L6883-250G)
聚乙二醇3,350(Sigma-Aldrich,目录号:P4338-500G)
氢氧化钠(NaOH)(FUJIFILM Wako Pure Chemical Corporation,目录号:192-15985)
用于扩增CNE1区域的对照引物 1 (5'-TCACAGGGTCGATTGCAAGG-3')(在日本北海道系统科学公司合成)
用于扩增CNE1区的对照引物2 (5'-CTGGTGGTTCAGTGCCATCT-3')(日本北海道系统科学合成)
用于检查替换的寡核苷酸引物 1(使用目标区域的 200 - 176 nt上游序列)
用于检查替换的寡核苷酸引物 2(使用目标区域的 66 - 90 nt下游反向序列)
Prime Star Max Premix 2×(TaKaRa ,目录号:R045A)
琼脂(FUJIFILM Wako Pure Chemical Corporation,目录号:010-08725)
Gene Ladder Wide 2(Nippon Gene,目录号:310-06971)
葡萄糖(FUJIFILM Wako Pure Chemical Corporation,目录号:043-31163)
不含氨基酸的酵母氮碱基(BD,Difco ,目录号:DF0919-15-3 )
蛋白胨(BD,Bacto TM ,目录号:211677)
酵母提取物(BD,Bacto TM ,目录号:288620)
腺嘌呤盐酸盐(FUJIFILM Wako Pure Chemical Corporation,产品代码:016-00802)
合成完全 (SC) 培养基(见配方)
YPDA培养基(见食谱)


程序


设计 20 - nt gRNA 目标序列
要设计 gRNA 目标序列,必须要有合适的 PAM 序列。为了设计一个带有 PAM 序列的目标序列,用于 Cas9 切割(5'-NGG-3' 或 5'-CCN-3' 用于对向链)靠近目标区域,我们使用免费软件CRISPRdirect (https://crispr .dbcls.jp/)。对于酿酒酵母中,我们选择了酿酒酵母代表株S288C和PASて50 - 100新台币的对位于目标位点附近序列到软件中。然后,CRISPRdirect使用 PAM 序列快速输出合适的 20-nt gRNA 目标序列。


PCR片段的制备小号甲乙
我们使用 p426-SNR52p-gRNA.CAN1.Y-SUP4t 作为通用模板质粒来构建表达含有设计的 20-nt 目标序列的 gRNA 的 PCR 片段(片段 C)。它包含的酵母启动子SNR52 ,它负责在酵母细胞中表达gRNA小号。如果您想在另一个生物体中表达 gRNA,您将需要使用包含从相应生物体获得的合适启动子序列的模板质粒来制备片段 C。这里,我们解释从S 中的片段 C 表达 gRNA 的情况. 酿酒酵母宿主。在制备片段 C 之前,片段 A 和 B 必须通过第一轮 PCR 以两个独立的步骤合成。


片段A:为了制备片段A的,设计的正向引物(FW A)为包括35个核苷酸的所述从部分序列SNR52 (3启动子区,855 - 3 ,889个核苷酸)的模板质粒P426-SNR52p的-gRNA .CAN1.Y-SUP4t(全序列可在Addgene公司库,https://www.addgene.org/43803/sequences/),其次是20-nt的gRNA靶序列和进一步的23个核苷酸的所述序列编码从gRNA支架的5'部分的模板质粒(3 ,910 - 3 ,932个核苷酸(图)URE 1A)。设计反向引物序列(器Rv根据5 A) ,001 - 5 ,020个核苷酸的反向序列(5'-ACTCACAAATTAGAGCTTCA-3' )的模板质粒。


片段 B:由于片段 A 不包含SNR52启动子的完整序列,并且必须拥有该完整区域才能正确表达 gRNA,因此我们需要通过片段 B将整个SNR52启动子区域与片段 A合并。因此,我们使用由 20-nt 序列 (5'-CGAACGACCGAGCGCAGCGA-3') 组成的正向引物 ( Fw B) 和由 30-nt 序列 (5'-TTTATCTTTCACTGCGGAGAAGTTTCGAAC-3') 组成的反向引物 ( Rv B)模板质粒(3 ,301 - 3 ,320个核苷酸和3 ,855 - 3 ,884个核苷酸中相反的顺序,分别地)来合成整个SNR52启动子序列如片段B(图1A)。片段 B 中最后 30 nt的互补序列与片段 A 的前 30 nt相同,以允许片段 A 和 B 在第二轮(重叠)PCR 中退火。






图 1. gRNA-TES 概述。A.在gRNA-TES,我们准备三个片段,即片段A,B ,和C,通过PCR。使用质粒 p426-SNR52p-gRNA.CAN1.Y-SUP4t (DiCarlo et al ., 2013) 作为通用模板,通过第一次 PCR 制备片段 A 和 B。接下来,使用片段 A 和 B 作为模板,通过第二次 PCR(重叠 PCR)合成用于表达 gRNA 序列的片段 C。在 gRNA-TES 中,总是需要一个新的片段 A,因为它包含 20-nt 独特的 gRNA 目标序列。20 - nt 独特的 gRNA 目标序列根据目标区域而变化。相比之下,片段 B 只是SNR52启动子区域;因此,没有必要以合成新的片段乙瓦特每次第i个不同的序列。使用该协议,不可能设计出可以放大目标序列以及整个SNR52启动子区域的引物序列,因为很难化学合成超过 100 个核苷酸的优质寡核苷酸。因此,我们不能通过单轮 PCR 扩增片段 C。B. gRNA-TES 可用于各种基因组操作,如染色体分裂、替换、删除和复制。分裂只需要一个片段 C,它传递一个 gRNA。双-链断裂(DSB)显著增加了从质粒合成的染色体上的靶序列(红色和绿色方框)以及其对应的DNA的模块之间的同源重组的频率小号p3121(杉山等和pSJ69(,2005)Easmin等人,2019 年)。因此,认为发生了高频率的分裂。两种片段C的,其中提供两个独立的gRNAs,是必要的替换,缺失,和重复。为了替换,我们使用质粒 pSJ69 ( Easmin et al ., 2019)制备了一个含有适当标记基因(此处为光滑念珠菌 LEU2、CgLEU2 )的DNA 模块,两侧是目标同源序列。对于片段删除和复制,我们需要两个包含目标同源序列的 DNA 模块:一个应该有一个着丝粒,另一个应该包含标记基因。在我们的实验中,我们使用p3121(杉山等人。,2005)窝藏CEN4和pSJ69窝藏CgLEU2为模板合成DNA的模块。双链断裂成功后,预计会出现高频率的替换、删除和重复。图B中,染色体上的黑色圆圈表示天然着丝粒;DNA 模块上的黄色圆圈表示人工提供的CEN4 。蓝色箭头表示人工提供的端粒;灰色框代表CgLEU2 。含有黄色质粒-和灰色-着色弯曲盒分别为p3121和pSJ69。


第一轮 PCR 反应


10 × Ex Taq 缓冲液


5微升


2.5 mM dNTP 溶液


4微升


模板质粒(约 50 ng)


1微升


寡核苷酸引物 1 (15 pmol )


1.5微升


寡核苷酸引物 2 (15 pmol )


1.5微升


Ex Taq ® DNA聚合酶


0.25微升





36.75微升


全部的


50微升


预变性:94°C,2 分钟


变性:98°C,10 秒


退火:55°C,30 秒


延伸:68°C,2 分钟




30个周期


通过重叠 PCR制备片段 C
为了制备片段 C,将片段 A 的 100 倍稀释度 ( × 1/100) 与片段 B 的相同稀释度混合,并用作第二轮 PCR(重叠 PCR)的模板,以生成带有正向引物的片段 C ( Fw B) 5'-CGAACGACCGAGCGCAGCGA-3' 和反向引物 ( Rv A) 5'-ACTCACAAATTAGAGCTTCA-3'。


第二轮 PCR 反应


10 × Ex Taq 缓冲液


5微升


2.5 mM dNTP 溶液


4微升


模板(× 1/100 片段 A)


0.5微升


模板(× 1/100 片段 B)


0.5微升


寡核苷酸引物 1 (15 pmol )


5'-CGAACGACCGAGCGCAGCGA-3'


1.5微升


寡核苷酸引物 2 (15 pmol )


5'-ACTCACAAATTTAGAGCTTCA-3'


1.5微升


Ex Taq ® DNA聚合酶


0.25微升





36.75微升


全部的


50微升


预变性:94°C,2 分钟


变性:98°C,10 秒


退火:58°C,30 秒


延伸:68°C,3 分钟




30个周期


注意:我们建议使用 Ex Taq ® DNA 聚合酶进行 gRNA-TES,尤其是第二轮 PCR(重叠 PCR)。尽管我们测试了各种 DNA 聚合酶,但我们观察到这些 DNA 聚合酶经常产生多个意想不到的条带,尤其是在用于制备片段 C 的重叠 PCR 期间。


gRNA-TES 在各种基因组操作中的应用


gRNA-TES 具有多种应用,从染色体的分裂到片段替换、删除和复制(图 1B)。根据基因组工程技术,需要提供两种类型的 gRNA 的最多两种片段 C。DSB之后,分裂、替换、删除和复制的频率有望增加。


DNA模块的制备
要拆分、删除、替换或复制目标染色体区域,必须将 DNA 模块整合到 gRNA-TES 中。构建的 DNA 模块的类型取决于基因组编辑的目的(分裂、删除、替换或复制)。对于替换,只需一个 DNA 模块就足够了,而对于分裂、删除或复制,则需要两个 DNA 模块(图 1B)。对于替换,设计和购买寡核苷酸引物以扩增侧翼与对应于目标区域的第一个和最后一个 30 bp的同源序列的任何遗传标记。对于分裂和删除,设计正向引物以扩增两侧有 50 bp同源序列的着丝粒或标记基因,该同源序列对应于目标分裂和删除点的上游或下游序列。对于复制,设计正向引物以扩增标记基因或着丝粒,两侧的同源序列对应于目标区域的第一个或最后一个 50 bp。以包括用于分离,缺失所有DNA模块端粒种子序列,和重复,我们使用了常用的反向引物包括5'-CCCCAACCCCAACCCCAACCCCAACCCCAACCCCAA-3'序列。您可以根据基因组编辑的目的使用任何模板质粒。我们使用 pSJ69(Easmin等,2019)和 p3121(Sugiyama等,2005)合成 DNA 模块。这些质粒具有相同的背景,因为它们是由相同的质粒 pUG6 构建的(Güldener等,1996),可应要求提供。用于分离,缺失或重复,有必要选择一种适当的质粒作为PCR的模板,使得新产生的染色体仅包含一个着丝粒。


PCR反应


10 × KOD 加新缓冲器


5微升


2 mM dNTP 溶液


5微升


25 mM 硫酸镁4


3微升


模板质粒


1微升


替代引物


寡核苷酸引物 1 (15 pmol )


5'-N30FGGCCGCCAGCTGAAGCTTCG-3'


1.5 μl (N30F 代表目标区域的第一个 30-bp 序列)


寡核苷酸引物 2 (15 pmol )


5'-N30LAGGCCACTAGTGGATCTGAT-3'


1.5 μl (N30L代表目标区域的最后30-bp反向序列)


分裂和删除引物


正向引物 (15 pmol )


5'-N50U/50DGGCCGCCAGCTGAAGCTTCG-3'


1.5 μl (N50U/50D代表目标区域的50-bp上游或下游(反向)序列)


反向引物 (15 pmol )


5'- CCCCAACCCCAACCCCAACCCCAACCC CAACCCCAA AGGCCACTAGTGGATCTGAT-3'


1.5微升


复制引物


正向引物 (15 pmol )


5'-N50F/50LGGCCGCCAGCTGAAGCTTCG-3'


1.5 μl (N50F/50L 代表目标区域的第一个(反向)或最后一个 50-bp 序列)


反向引物 (15 pmol )


5'- CCCCAACCCCAACCCCAACCCCAACCC CAACCCCAA AGGCCACTAGTGGATCTGAT-3'


1.5微升


KOD 加新


1微升





32微升


全部的


50微升


预变性:94°C,2分钟


变性:98°C,10 秒


退火:55°C,30 秒


30个周期


延伸:68°C,2 分钟




酵母转化
预先准备酵母菌株,例如,SJY30 ( MATα ura3-52 his3-Δ200 leu2Δ1 lys2Δ202 trp1Δ63窝藏质粒 p414-TEF1p-Cas9-CYC1t [ DiCarlo et al. , 2013] ), 优化表达 Castrodu9 by the codon p414-TEF1p-Cas9-CYC1t 的作用。
在 YPDA 液体培养基中培养过夜。
用酵母细胞预培养物接种新鲜的 5 毫升 YPDA 液体培养基,初始 OD 600约为 0.2 - 0.3。然后在 30°C 下以 140 rpm 的振荡速度孵育,直到 OD 600达到 0.8 - 1.0(约 4 - 6小时)。
混合适当的 DNA 模块和表达 gRNA 的片段 C(例如,我们需要两种片段 C [图 1B]来针对目标区域的两个边缘和一个 DNA 模块来替换染色体区域)并使用传统的LiAc / PEG 方法(Gietz和Schiestl ,2007 年)。
该协议的目的是为用户提供一个在每一步都尽可能简单的程序,包括下一节中描述的菌落 PCR。因此,该协议的制定没有测量 PCR 产物的 DNA 浓度。对于常规进行的酵母转化,我们始终使用 12 μl DNA 模块和 11 μl每个片段 C PCR 反应混合物来替换染色体区域,因为我们已经使用这样的量获得了足够的转化体(即12 μl + 11 μl + 11 μl = 34 μl ) PCR 反应混合物。此外,整个 PCR 反应混合物无需纯化即可直接用于酵母转化。
酵母转化之后,悬浮在100细胞微升灭菌水,散布在整个悬浮液到一个或两个选择板,并在30℃下孵育2 - 3天。
                                                        通过菌落 PCR 和随后的琼脂糖凝胶电泳确认预期的染色体变化
      要制备 PCR 级基因组 DNA,请从每个菌落中取出少量细胞并悬浮在 10 μl 0.02 M NaOH 溶液中。在加热块中在 98°C 下加热 10 分钟,然后转移到冰上。
      作为一个例子,我们在这里描述了如何确认从替换实验中获得的转化体。设计和购买用于菌落 PCR 的寡核苷酸引物。您可以使用目标区域上游和下游序列中的任何序列,但我们建议以最终 PCR 产物 <2 kb 的方式设计引物,因为在菌落 PCR 期间产生更大的 PCR 产物可能会出现问题。对于寡核苷酸引物1,使用200 - 176 NT目标区域的上游序列和寡核苷酸引物2,使用66 - 90 NT目标区域的下游的反向序列。由于我们使用CgLEU2标记基因进行替换,CgLEU2的大小为1685 bp,因此目标区域替换成功后,PCR产物的大小将为200 bp + 1685 bp + 90 bp = 1975 bp(图2)。例如,要检查染色体 4 中 500-kb 区域的替换(坐标编号 494271 - 994270),设计基于 494071 - 494095 nt序列(5'-CATATCAGTGTCTTCATCTTCATGA-3')和2oligonucleotide引物的寡核苷酸引物1在994 ,336 - 994 ,360个核苷酸的反向序列染色体4的(5'-TAGTGGATACGCAGGACGTGTTATC-3' )此外,我们建议设计对照引物以检查PCR反应是否顺利进行。任何基因组区域都可以作为对照进行扩增;我们放大了染色体 1的CNE1区域作为内部对照。控制引物1是基于211 - 230 NT '序列(5'-TCACAGGGTCGATTGCAAGG-3)在861和控制引物2 - 880 NT所述的(反向序列5'-CTGGTGGTTCAGTGCCATCT-3)' CNE1区域。如果CNE1基因被正确扩增,您将获得 670-bp 的 PCR 产物。
      PCR反应
Prime Star Max 预混料 2 ×


12.5微升


模板基因组DNA


0.5微升


对照底漆 1 (7.5 pmol )


5'-TCACAGGGGTCGATTGCAAGG-3'


0.75微升


对照底漆 2 (7.5 pmol )


5'- CTGGTGGTTCAGTGCCATCT -3'


0.75微升


寡核苷酸引物 1 (7.5 pmol )


(使用目标区域的 200 - 176 nt上游序列)


0.75微升


寡核苷酸引物 2 (7.5 pmol )


(使用目标区域的 66 - 90 nt下游(反向)序列)


0.75微升





9微升


全部的


25微升


变性:98°C,10 秒


退火:55°C,5 秒


30个周期


延伸:72°C,10 秒




注意:您可以使用任何 DNA 聚合酶进行菌落 PCR。然而,由于我们的目的是让这个协议尽可能快,我们使用 Prime Star Max Premix 2 × ,它只需要 5 s/kb 的扩展时间。


C:\Users\Madandan\Desktop\2003790--1803 SatoshiHarashima 952696\Figs tif\图2.tif


图2.典型的琼脂糖凝胶电泳结果小号用于通过gRNA-TES染色体置换分析。泳道1至9代表独立的转化体。M 代表尺寸标记(Gene Ladder Wide 2, Nippon Gene, Toyama, Japan)。泳道1至3代表从300-kb区域置换获得的转化体;泳道4至6代表从400-kb区域置换获得的转化体;泳道 7 至 9 代表从 4 号染色体的 500-kb 区域置换获得的转化体。几乎所有转化体(除了第 3 道中的那些)都显示出预期的相应染色体区域的置换并产生了 1975-bp 条带。在所有转化体中也观察到内部对照带(670 bp)。


数据分析


每个实验中转化体的数量不同。例如,当我们针对较小的染色体区域(例如 150 kb 和 200 kb)进行替换时,我们在一次转化中分别获得了 263 和 287 个转化体(Easmin等,2019)。当我们针对 300-kb、400-kb 和 500-kb 区域时,我们在一次转化中分别获得了 51、43 和 103 个转化体。我们测试了 6 个转化体来替换 150-kb 和 200-kb 区域中的每一个,并测试了 300-kb、400-kb 和 500-kb 区域中的每一个替换体。预期替换150-kb的,200-kb的,300-kb的,400-kb的,并且500-kb的区域的频率分别为100%,66.6%,80%,100%和100%,分别(Easmin等人., 2019)。相比之下,当不应用 gRNA-TES 时,观察到 150-kb 区域替换的最大预期频率为 16.6%;为了替换其他染色体区域,当不使用 gRNA-TES 时,没有获得正确的转化体。


食谱


合成完全 (SC) 培养基
2%葡萄糖


0.67% 不含氨基酸的酵母氮碱(例如BD Difco )


注意:含有所有氨基酸和缺乏特定氨基酸的核酸碱基的 0.2% dropout 混合物可用于营养缺陷型选择。


对于该板测定中,2%琼脂中加入


YPDA培养基
2%葡萄糖


2% 蛋白胨(例如BD Bacto TM )


1% 酵母提取物(例如BD Bacto TM )


0.004%腺嘌呤HC升(FUJIFILM和光纯药公司)


致谢


这项工作得到了日本科学促进会 (JSPS)-KAKENHI, 科学研究资助 (B) 的支持,资助号 [JP 15H04475] 到 SH该协议改编自我们以前发表的工作 ( Easmin等,2019)。


利益争夺


作者声明没有竞争利益。


参考


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引用:Hassan, N., Easmin, F., Ekino, K. and Harashima, S. (2021). PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System. Bio-protocol 11(13): e4082. DOI: 10.21769/BioProtoc.4082.
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