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Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters
利用核酶和RNA聚合酶II启动子体内及体外合成CRISPR系统的引导RNA   

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Abstract

CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct tissue/cell specific gene editing.

Keywords: Ribozyme(核酶 ), CRISPR(CRISPR), RNA polymerase II promoter(RNA聚合酶II启动子 ), Genome editing(基因组编辑), RNA transcription(RNA转录)

Background

Almost all of the reported cases of CRISPR-mediated gene editing used promoters of small nuclear RNAs such as the U6 and U3 snRNA promoters to drive the production of gRNAs in vivo (Cong et al., 2013; Mali et al., 2013). However, the U6 and U3 promoters have several major limitations: 1) They are constitutively active and not tunable; 2) They lack cell/tissue specificities; 3) They have not been well defined in many organisms; 4) U6 requires a G and U3 requires an A for transcription initiation, thus limiting target selections; 5) They are not suitable for in vitro transcriptions because of the lack of commercial RNA polymerase III. Unfortunately, RNA polymerase II promoters, which constitute the majority of the characterized promoters, cannot be directly used for gRNA production in vivo because of the following reasons: 1) The primary transcripts of RNA polymerase II promoters undergo extensive processing such as 5’-end capping, 3’-end polyadenylation, and splicing out of the introns. Some of the modifications may render the designed gRNA non-functional. 2) The mature RNA molecules are transported into cytosol; thus they are physically separated from the intended targets that are located in the nucleus. That is why production of gRNA in vivo using U6 and U3 snRNA promoters has been the dominant method (Gao and Zhao, 2014; Yoshioka et al., 2015). In this protocol, we use a ribozyme-based strategy to overcome the aforementioned limitations of RNA polymerase III promoters, enabling gRNA production from any promoters and in any organisms. We design an artificial gene named RGR (Ribozyme-gRNA-Ribozyme) that, once transcribed, generates an RNA molecule with ribozyme sequences flanking both ends of the designed gRNA (Gao and Zhao, 2014). We show that the primary transcripts of RGR undergo self-catalyzed cleavage to precisely release the desired gRNA, which can efficiently guide sequence‐specific cleavage of DNA targets in vitro and in vivo (Gao and Zhao, 2014). RGR can be transcribed from any promoters and thus allows for cell‐and tissue‐specific genome editing if appropriate promoters are chosen.

Materials and Reagents

  1. E. coli DH5a and Agrobacterium tumefaciens strain GV3101
  2. pRS316-RGR-GFP plasmid (Addgene, catalog number: plasmid 51056 )
  3. pHDE-35S-Cas9-mCherry-UBQ plasmid
  4. Primers (Table S1)
  5. Gibson assembly reagents
    You can either purchase commercial kits from (New England Biolabs, catalog number: E5510S ), or prepare your own with the following individual reagents:
    1. 5x isothermal (ISO) reaction buffer (25% PEG-8000; 500 mM Tris-HCl, pH 7.5; 50 mM MgCl2; 50 mM DTT; 1 mM each of the 4 dNTPs; and 5 mM NAD)
    2. T5 exonuclease (Epicentre, catalog number: T5E4111K )
    3. Phusion DNA polymerase (New England Biolabs, catalog number: M0530 L)
    4. Taq DNA ligase (New England Biolabs, catalog number: M0208L )
  6. Phusion High-Fidelity PCR Kit (New England Biolabs, catalog number: E0553L )
  7. LB medium
  8. Appropriate antibiotics
  9. QIAGEN Plasmid Mini Kit
  10. MfeI (New England Biolabs, catalog number: R0589S )
  11. 10x CutSmart® buffer
  12. T7, SP6 or T3 RNA polymerase with transcription buffer
    For SP6/T7 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1320 )
    For T3 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1316 )
  13. 5x transcription buffer
  14. 1 M DTT (Thermo Fisher Scientific, catalog number: P2325 )
  15. 20 U/µl RNase inhibitor (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: N8080119 )
  16. 10 mM NTP mix (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18109017 )
  17. rNTPs
  18. Inorganic pyrophosphatase
  19. EDTA
  20. 12% denaturing urea polyacrylamide gels
  21. Ethidium bromide

Equipment

  1. 37 °C water bath (Temperature-controlled water bath) (Bio-Rad Laboratories, catalog number: 1660524 )
  2. Thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: Applied Biosystems® 2720, catalog number: 4359659 )
  3. DNA electrophoresis apparatus (Bio-Rad Laboratories, model: PowerPacTM Basic Power Supply, catalog number: 1645050EDU )
  4. Microcentrifuges (Eppendorf, model: 5424 )
  5. UV transilluminator (Bio-Rad Laboratories, model: UViewTM Mini Transilluminator, catalog number: 1660531 )

Procedure

  1. Design, assemble, and clone an RGR (ribozyme-gRNA-ribozyme) unit
    We design an artificial gene named RGR, whose primary transcripts would be flanked by ribozymes at both ends (Figure 1) (Gao and Zhao, 2014).


    Figure 1. A schematic presentation of a ribozyme-flanked gRNA (RGR, ribozyme-gRNA-ribozyme) molecule. Once transcribed, the corresponding RGR DNA will produce an RNA molecule with a ribozyme at both the 5’- and 3’-end. The primary transcripts will undergo self-cleavage to release the mature gRNA. The RGR design allows the production of a functional gRNA molecule from any promoter, enabling production of gRNA molecules in vitro, in vivo, and in tissue/cell specific manner.

    1. Select a target sequence for editing the gene of interest
      The target should be 23 bp long and should contain the NGG Protospacer Adjacent Motif (PAM) site at the 3’-end. However, the NGG PAM site should not be included in the gRNA sequence itself. N refers to any nucleotide in the target N1N2N3N4N5N6N7N8N9N10N11N12N13N14N15N16N17N18N19N20NGG. Multiple web-based resources are publically available for selecting appropriate gRNA targets (http://cbi.hzau.edu.cn/crispr/; http://www.rgenome.net/cas-offinder/; http://www.genome.arizona.edu/crispr/CRISPRsearch.html). 

    2. Design an Ribozyme-gRNA-Ribozyme (RGR) unit

      1. We place a Hammerhead ribozyme at the 5’-end of the gRNA and a HDV ribozyme at the 3’-end of the gRNA (Figure 2). The complete sequences of the ribozymes were described in the plasmid pRS316-RGR-GFP (Gao and Zhao, 2014). Both the plasmid and the complete sequence of pRS316-RGR-GFP are available at Addgene.
      2. The RGR unit can then be placed under the control of a promoter for the production of the designed gRNA molecules. Any promoters including both RNA polymerase II and RNA polymerase III promoters can be used. For in vitro production of gRNAs, the RGR unit can be driven by an SP6 or T7 or T3 promoter, which can be transcribed using commercially available RNA polymerases (Gao and Zhao, 2014).
      3. Linearize the vector of your choice with a restriction enzyme and determine an 18 to 24 bp overlapping region (termed adaptor sequence) based on the Gibson assembly principle (Gibson et al., 2009). Gibson assembly method seamlessly assembles overlapping DNA molecules into one molecule by the concerted action of a 5’ exonuclease, a PHUSION DNA polymerase and a heat-stable Taq DNA ligase (Gibson et al., 2009). Additional information about Gibson assembly can be found at Addgene (http://www.addgene.org/protocols/gibson-assembly/). Assembly kits are commercially available at New England Biolabs (https://www.neb.com/products/e5510-gibson-assembly-cloning-kit).
      4. If the adaptor sequences are XXXXXXXXXXXXXXXXXXXX (upstream adaptor) and YYYYYYYYYYYYYYYYYYYY (downstream adaptor), design two forward primers and one reverse primer as shown below. Adaptor sequences including X and Y provide the necessary overlapping sequences with your linearized vector so that the RGR unit can be assembled into the vector through Gibson assembly principle. The length of adaptor sequences varies depending on the actual sequences, but usually 17 bp to 24 bp is sufficient as long as the Tm value is above 50 °C.
        1. Forward primer 1: GENE-RGR-F1
          XXXXXXXXXXXXXXXXXXXXM6M5M4M3M2M1CTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC
          The sequence X (bold) refers to the upstream adaptor sequence, which is complementary to the vector sequence. M sequence is reverse complementary to the first 6 bp of the target sequence. The italicized sequence is part of the Hammerhead ribozyme.
        2. Forward primer 2: GENE-RGR-F2
          GACGAAACGAGTAAGCTCGTCN1N2N3N4N5N6N7N8N9N10N11N12N13N14N15N16N17N18N19N20GTTTTAGAGCTAGAAATAGCAAG. N1N2N3N4N5N6 and M6M5M4M3M2M1 are reverse complementary to ensure the correct secondary structure of the Hammerhead ribozyme. We designed the two overlapping PCR primers to avoid the synthesis of expensive long primers. The target sequence is N1N2N3N4N5N6N7N8N9N10N11N12N13N14N15N16N17N18N19N20NGG.
          Note: The PAM site NGG is not included in the primer. The italicized sequence is part of the core gRNA sequence.
        3. Design a universal RGR-Reverse primer for the vector of your choice: UNIVERS-RGR-R: ZZZZZZZZZZZZZZZZZZZZGTCCCATTCGCCATGCCGAAGC. The UNIVERS-RGR-R primer should be reverse complementary to GCTTCGGCATGGCGAATGGGACYYYYYYYYYYYYYYYYYYYY, where YYYYYYYYYYYYYYYYYYYY is the downstream adaptor sequence (Y and Z are reverse complementary). The italicized sequence is complementary to the 3’ end of the HDV ribozyme.


          Figure 2. Molecular design of an RGR unit and the assembly of a complete RGR unit by PCR. The top panel shows the complete sequence of an RGR unit that produces a gRNA that targets (N)20NGG sequence. The underlined sequences are adaptor sequences from the vector and are used for cloning the RGR unit into the final CRISPR vector. Note that the adaptor sequences vary accordingly if different vectors are chosen. N1 to N20 (in red) are the target sequence prior to the NGG PAM site. It is essential that the M6M5M4M3M2M1 and N1N2N3N4N5N6 are reverse complementary so that the ribozyme can undergo self-cleavage as designed. Sequence highlighted in green is the core gRNA sequence. Sequence highlighted in yellow is the Hammerhead ribozyme. The HDV ribozyme is highlighted in purple. The primers are marked by different colors: black (Gene-RGR-F1), light blue (Gene-RGR-F2), and Red (UNIVERS-RGR-R). The Universal primer depends on the vector sequences and needs to be changed if different vectors are used. The complete RGR unit can be easily assembled by two over-lapping PCR reactions.

    3. Assemble the Ribozyme-gRNA-Ribozyme (RGR) unit
      1. An RGR unit for targeting a new gene is assembled through two rounds of overlapping PCR reactions (Figure 2). There is a 21-bp overlap between the two forward primers (Figure 2).
      2. First PCR (PCR1): Use primer pair GENE-RGR-F2 + UNIVERS-RGR-R. Template: any existing RGR construct (e.g., pRS316-RGR-GFP, available from Addgene. The expected PCR product is 233 bp).
      3. The second-round PCR (PCR2): Use primer pair GENE-RGR-F1 + UNIVERS-RGR-R. Gel-purified PCR product from PCR1 is used as the template. The expected PCR2 product is 277 bp, which is the complete RGR unit (Figure 2).
      A routine PCR reaction is set up according to the manufacturer’s recommendation (https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530). Briefly, the PCR components are mixed as described in Table 1.

      Table 1. PCR setup for amplification of the RGR unit


      Table 2. PCR conditions for amplification of the RGR unit


    4. Ligate the final RGR product into a desired vector - clone the RGR (ribozyme-gRNA-ribozyme) unit
      The complete RGR unit assembled through two rounds of PCR is cloned into the final CRISPR vector using Gibson assembly protocol (Gibson et al., 2009). Note that the PCR primers GENE-RGR-F1 and UNIVERS-RGR-R contain sequences that match part of the sequences in the vector to facilitate the in vitro assembly of the RGR unit into the vector (Figure 2).
      1. Incubate the DNA assembly mixture (Table 3) for 1 h at 50 °C. DNA assembly was conducted by following the manufacturer’s recommended protocol (Table 3). (https://www.neb.com/products/e2611-gibson-assembly-master-mix#tabselect2).

        Table 3. In vitro DNA assembly


      2. Then transform 3 µl ligated product to E. coli DH5α competent cells.
        The ligated plasmid could be transformed into E. coli competent cells following routine cloning procedure (Sambrook and Russell, 2001).
        1. Transform E. coli DH5α competent cells (homemade or commercially available products) using 3 μl of ligation product.  
        2. Inoculate 2 to 4 colonies in LB medium with appropriate antibiotics.
        3. Purify plasmids from the transformed DH5α cells using QIAGEN Plasmid Mini Kit.
      3. Verify the positive clones by Sanger sequencing before introducing the final plasmid into your cell/organism of choice (Yoshioka et al., 2015).

  2. An example of constructing an RGR unit for CRISPR-mediated gene editing
    1. Select a target gene and a vector
      We show the construction of an RGR unit to target the Arabidopsis gene Auxin Binding Protein 1 (ABP1) (Figure 3) as an example (Gao et al., 2015). We use pHDE-35S-Cas9-mCherry-UBQ plasmid as our CRISPR vector (Gao et al., 2016), which has been used for editing genes in Arabidopsis (the plasmid can be obtained through Addgene).
    2. Linearize the plasmid
      We digest pHDE-35S-Cas9-mCherry-UBQ plasmid with MfeI (Figure 3). The MfeI-linearized plasmid is used for assembling the RGR unit into the vector through Gibson assembly. The complete plasmid can express the ABP1-RGR unit from the Arabidopsis UBQ10 promoter in plants (Figure 3).

      Table 4. Linearize the vector by restriction digestion


      Mix the components shown in Table 4 and digest the vector overnight at 37 °C. Then heat inactivation the enzyme at 80 °C for 15 min. The linearized plasmid was16,084 bp.
    3. Design the PCR primers
      The upstream adaptor sequence is GTTTTTCTGATTAACAGCTCGC. The downstream adaptor we used is GACCTAACTGAGTAAGCTAGC (Figure 3).
      1. Forward primer 1: ABP1-RGR-F1:
        GTTTTTCTGATTAACAGCTCGCAGCTCCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC. The 6 bp sequence in bold is reverse complementary to the first 6 bp of the gRNA target. The underlined sequence is part of the Hammerhead ribozyme.
      2. Forward primer 2: ABP1-RGR-F2:
        GACGAAACGAGTAAGCTCGTCGGAGCTCCTTGTCCCATCAAGTTTTAGAGCTAGAAATAGCAAG. The underlined sequence is part of the Hammerhead ribozyme. Sequence in bold is the gRNA target sequence, and the italicized sequence is part of the core gRNA sequence.
      Note: The bold sequence in ABP1-RGR-F1 is reverse complementary to the first 6 bp of the bold sequence in ABP1-RGR-F2. ABP1-RGR-F1 and ABP1-RGR-F2 share the following overlapping sequences: GACGAAACGAGTAAGCTCGTC, which allows us to conduct two overlapping PCR reactions.
    4. For cloning the ABP1-RGR unit into the MfeI site in 35S-Cas9-mCherry-UBQ plasmid using Gibson assembly, the UNIVERS-RGR-R primer is:
      GCTAGCTTACTCAGTTAGGTCGTCCCATTCGCCATGCCGAAGC
      Note: The bold sequence is complementary to the downstream adaptor sequence. The italicized sequence matches the 3’ end of the HDV ribozyme.
    5. The complete RGR unit for producing a gRNA targeting the ABP1 gene is shown in Figure 3. The RGR unit has led to successful isolation of several null alleles of abp1 in Arabidopsis (Gao et al., 2015 and 2016).


      Figure 3. A Molecular design of an RGR unit for targeting the ABP1 gene in Arabidopsis. The RGR unit is cloned into the pHDE-35S-Cas9-mCherry-UBQ plasmid at the MfeI site using Gibson assembly. The adaptor sequences are underlined. Note that the downstream adaptor sequence has to be converted to reverse complementary sequence when designing the Universal primer. The target sequence is in red. The Hammerhead ribozyme and the HDV ribozyme are highlighted in yellow and purple, respectively.

  3. Guide RNA production by in vitro transcription
    Once an RGR unit is assembled and cloned into the final vector as described above, gRNAs can be easily produced by in vitro transcription using T7, SP6, or T3 RNA polymerases, which are commercially available. 
    1. Prepare a DNA template for in vitro transcription
      1. The templates for in vitro transcription are amplified by PCR from the assembled RGR constructs using two universal primers (any RGR units cloned into the same vector can be amplified using the same pair of primers). For example, RGR units cloned into the MfeI site in pHDE-35S-Cas9-mCherry-UBQ can be amplified using the following pair of primers:
        SP6-P1: GTCACTATTTAGGTGACACTATAGAAGCGGTTTTTCTGATTAACAGCTCGC and Univers-P2: GCTAGCTTACTCAGTTAGGTC.
      2. The Underlined sequence is the SP6 promoter and the bolded G in primer SP6-P1 is the transcription initiation site for SP6 RNA polymerase. If T7 or T3 RNA polymerases are used, the underlined 5’-end of the primer SP6-P1 needs to be changed to GTCACTAATACGACTCACTATAGGGAGA and GTCACAATTAACCCTCACTAAAGGGAGA respectively.
      3. PCR conditions are described in Table 1 and Table 2.
    2. In vitro transcription
      1. Add the following reagents at room temperature in the order listed in Table 5:

        Table 5. In vitro transcription of RGR to produce gRNA molecules

        Note: For higher yield, add 1 U inorganic pyrophosphatase into the reaction and incubate overnight.

      2. Incubate the mixture for 2 to 8 h at 37 °C (T7, T3) or 42 °C (SP6).
      3. Add 1 µl 0.5 M EDTA to terminate the reaction.
        Note: There is no need to further purify the RNA transcripts for in vitro CRISPR cleavage assays (for in vitro cleavage assay example, follow the link: https://www.neb.com/products/m0386-cas9-nuclease-s-pyogenes. The quality of in vitro transcription and self-processing can be analyzed by electrophoresis in 12% denaturing urea polyacrylamide gels. The RNA bands are stained with ethidium bromide and visualized using a UV transilluminator. To analyze the DNA cleavage products generated by Cas9 and the gRNA from the in vitro transcription, regular 1% agarose gel is adequate.

  4. Transformation of the RGR plasmid into Arabidopsis
    The final plasmid with the RGR construct was transformed into Arabidopsis through Agrobacterium-mediated floral dipping method (Clough and Bent, 1998). The transgenic T1 plants were identified either as hygromycin-resistant or producing mCherry fluorescence (Gao et al., 2015 and 2016). T1 plants were screened for editing events using PCR and restriction digestion as described in Gao et al., 2015 and 2016.

Data analysis

Our RGR-based production of functional gRNAs has been successful both in vitro and in vivo (Gao and Zhao, 2014; Gao et al., 2015 and 2016). We initially produced a gRNA targeting GFP using the RGR design. The gRNA was produced in vitro from the SP6 promoter using the commercially available SP6 RNA polymerase (Gao and Zhao, 2014). In vitro digestion assays indicated that the gRNA was fully functional (Gao and Zhao, 2014). We used the same RGR design to produce gRNA in yeast using the ADH promoter, an RNA polymerase II promoter (Gao and Zhao, 2014). Such a construct led to successful editing of the GFP gene in yeast. We further introduced an RGR design in Arabidopsis to produce a gRNA targeting the Arabidopsis ABP1 gene. Multiple stable heritable mutations in the ABP1 gene have been obtained (Gao et al., 2015 and 2016). For example, we made a construct that uses RGR for one gRNA production and the U6 promoter to produce another gRNA. The two gRNA molecules target two discrete sites of the ABP1 gene in Arabidopsis and were designed to generate a 771 bp deletion (Gao et al., 2016). We detected the intended deletions in 5 out of 61 T1 plants. Two of the T1 plants produced Cas9-free, stable homozygous mutants at the T2 generation (Gao et al., 2016).
The RGR design has been adapted for editing genes in other organisms (Nissim et al., 2014; Yoshioka et al., 2015).

Acknowledgments

This protocol was initially developed at University of California San Diego when YG was a graduate student there. The protocol was further improved at Huazhong Agricultural University. Authors would like to thank support from the National Gene Transformation grant #2016ZX08010002-002 to RW and NIH grant R01GM114660to YZ.

References

  1. Clough, S. and Bent, A. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6): 735–743
  2. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. and Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121): 819-823.
  3. Gao, X., Chen, J., Dai, X., Zhang, D. and Zhao, Y. (2016). An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol 171(3): 1794-1800.
  4. Gao, Y., Zhang, Y., Zhang, D., Dai, X., Estelle, M. and Zhao, Y. (2015). Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development. Proc Natl Acad Sci 112(7): 2275-2280.
  5. Gao, Y. and Zhao, Y. (2014). Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 56(4): 343-349.
  6. Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd and Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5): 343-345.
  7. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E. and Church, G. M. (2013). RNA-guided human genome engineering via Cas9. Science 339(6121): 823-826.
  8. Nissim, L., Perli, S. D., Fridkin, P., and Lu, T. K. (2014). Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell 54(4): 698-710
  9. Sambrook, J. and Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press pp: 1105-1111.
  10. Yoshioka, S., Fujii, W., Ogawa, T., Sugiura, K., and Naito, K. (2015). Development of a mono-promoter-driven CRISPR/Cas9 system in mammalian cells. Sci Rep 5:18341.

简介

CRISPR / Cas9介导的基因组编辑依赖于指导性RNA(gRNA)分子来产生序列特异性DNA切割,这是基因编辑的前提条件。在这里,我们建立了一种能够从任何启动子,任何生物体内和体外生产gRNA的方法(Gao和Zhao,2014)。该方法也可以进行组织/细胞特异性基因编辑。

背景 几乎所有报道的CRISPR介导的基因编辑病例都使用小核RNA如U6和U3 snRNA启动子的启动子来驱动体内生产gRNAs(Cong等人,2013; Mali等人,2013)。然而,U6和U3启动子有几个主要的局限性:1)它们是组成型活性的,不可调谐的; 2)它们缺乏细胞/组织特异性; 3)它们在许多生物体中没有明确定义; 4)U6需要G和U3需要A用于转录起始,从而限制目标选择; 5)由于缺乏商业RNA聚合酶III,它们不适合体外转录。不幸的是,构成大部分特征启动子的RNA聚合酶II启动子不能直接用于体内的gRNA生产,原因如下:1)RNA聚合酶II启动子的主要转录物经历广泛的加工如5'-端封端,3'末端聚腺苷酸化和拼接出内含子。一些修饰可能使设计的gRNA无功能。 2)将成熟的RNA分子转运到细胞质中;因此它们与位于细胞核中的预期目标物理分离。这就是为什么使用U6和U3 snRNA启动子在体内生产gRNA已经是主要的方法(Gao和Zhao,2014; Yoshioka等人,2015)。在本协议中,我们使用基于核酶的策略来克服RNA聚合酶III启动子的上述局限性,从任何启动子和任何生物体中产生gRNA。我们设计了一个名为“RGR”(Ribozyme-gRNA-Ribozyme)的人工基因,一旦转录,就会产生一个具有侧翼于设计的gRNA两端的核酶序列的RNA分子(Gao和Zhao,2014)。我们显示,RGR 的主要转录物经历自催化裂解以精确释放所需的gRNA,其可以有效地指导体外DNA靶标的序列特异性切割。 体内(高和赵,2014)。可以从任何启动子转录RGR,因此如果选择适当的启动子,则允许细胞和组织特异性基因组编辑。

关键字:核酶 , CRISPR, RNA聚合酶II启动子 , 基因组编辑, RNA转录

材料和试剂

  1. E。大肠杆菌DH5a和根癌土壤杆菌菌株GV3101
  2. pRS316-RGR-GFP质粒(Addgene,目录号:质粒51056)
  3. pHDE-35S-Cas9-mCherry-UBQ质粒
  4. 引录(表S1 )< br />
  5. 吉布森装配试剂
    您可以从(New England Biolabs,目录号:E5510S)购买商品包,或者使用以下各种试剂制备您自己的商品:
    1. 5x等温(ISO)反应缓冲液(25%PEG-8000; 500mM Tris-HCl,pH7.5; 50mM MgCl 2); 50mM DTT; 4mM dNTPs中的1mM; 5mM NAD)
    2. T5外切核酸酶(Epicentre,目录号:T5E4111K)
    3. Phusion DNA polymerase(New England Biolabs,目录号:M0530L)
    4. Taq DNA连接酶(New England Biolabs,目录号:M0208L)
  6. Phusion高保真PCR试剂盒(New England Biolabs,目录号:E0553L)
  7. LB培养基
  8. 合适的抗生素
  9. QIAGEN质粒迷你套件
  10. Mfe I/I(New England Biolabs,目录号:R0589S)
  11. 10x CutSmart ®缓冲区
  12. T7,SP6或T3 RNA聚合酶与转录缓冲液 对于SP6/T7(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1320)
    对于T3(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1316)
  13. 5x转录缓冲液
  14. 1 M DTT(Thermo Fisher Scientific,目录号:P2325)
  15. 20 U /μlRNA酶抑制剂(Thermo Fisher Scientific,Applied Biosystems TM,目录号:N8080119)
  16. 10mM NTP混合物(Thermo Fisher Scientific,Invitrogen TM,目录号:18109017)
  17. rNTPs
  18. 无机焦磷酸酶
  19. EDTA
  20. 12%变性尿素聚丙烯酰胺凝胶
  21. 溴化乙锭

设备

  1. 37℃水浴(温度控制水浴)(Bio-Rad Laboratories,目录号:1660524)
  2. 热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:Applied Biosystems 2720,目录号:4359659)
  3. DNA电泳装置(Bio-Rad Laboratories,型号:PowerPac TM 基本电源,目录号:1645050EDU)
  4. 微量离心机(Eppendorf,型号:5424)
  5. 紫外透射仪(Bio-Rad Laboratories,型号:UView TM 迷你透射仪,目录号:1660531)

程序

  1. 设计,组装和克隆RGR(核酶-GRNA-核酶)单位
    我们设计一个名为"RGR"的人造基因,其主要成绩单位于两端的核酶两侧(图1)(Gao和Zhao,2014)。


    图1.核酶侧翼gRNA(RGR,核酶-RNA-核酶)分子的示意图。一旦转录,相应的RGR DNA将在5'末端产生具有核酶的RNA分子, - 和3'末端。主要转录本将经历自我裂解以释放成熟的gRNA。 RGR设计允许从任何启动子生产功能性gRNA分子,使得能够在体外体内和/或组织/细胞特异性方式产生gRNA分子。 />
    1. 选择编辑感兴趣的基因的目标序列 靶标应为23 bp长,应包含3'末端的NGG Protospacer相邻基序(PAM)位点。然而,NGG PAM位点不应该被包含在gRNA序列本身中。 N指目标N 1中的任何核苷酸,N N N 3 N /sup> N 6 N N 8 N 9 N sup> 11 N N 13 N 14 N 20 N N 17 N 20 多个基于网络的资源公开可用于选择适当的gRNA目标( http: /cbi.hzau.edu.cn/crispr/; http ://www.rgenome.net/cas-offinder/; http://www.genome.arizona.edu/crispr/CRISPRsearch.html )。
    2. 设计核酶-RNA-核酶(RGR)单位
      1. 我们在gRNA的5'末端放置一个Hammerhead核酶,在gRNA的3'末端放置一个HDV核酶(图2)。在质粒pRS316-RGR-GFP中描述了核酶的完整序列(Gao和Zhao,2014)。 pRS316-RGR-GFP的质粒和完整序列均可在 Addgene
      2. 然后将RGR单元置于用于产生设计的gRNA分子的启动子的控制下。可以使用包括RNA聚合酶II和RNA聚合酶III启动子的任何启动子。对于gRNA的体外生产,RGR单位可以由SP6或T7或T3启动子驱动,其可以使用市售RNA聚合酶转录(Gao和Zhao,2014)。
      3. 使用限制酶线性化您选择的载体,并根据Gibson装配原理(Gibson等人,2009)确定18至24bp的重叠区域(称为衔接子序列)。吉布森装配方法通过5'外切核酸酶,PHUSION DNA聚合酶和热稳定的Taq DNA连接酶(Gibson等人,2009)的共同作用,将重叠的DNA分子无缝地组装成一个分子。有关Gibson装配的更多信息,请参见Addgene( http://www.addgene.org/protocols/gibson-assembly/)。装配套件可在New England Biolabs上购买( https://www.neb.com/products/e5510-gibson-assembly-cloning-kit )。
      4. 如果适配器序列为 XXXXXXXXXXXXXXXXXXXX (上游适配器)和 YYYYYYYYYYYYYYYYYYYYYY (下游适配器),请设计两个正向引物和一个反向引物,如下所示。包括X和Y的适配器序列为您的线性化矢量提供必要的重叠序列,以便RGR单元可以通过吉布森装配原理组装成向量。衔接子序列的长度根据实际序列而有所不同,但通常17 bp至24 bp的值足够大,只要T值高于50°C。
        1. 正向引物1:GENE-RGR-F1
          XXXXXXXXXXXXXXXXXXXX M 6 5 3 2 M 1 CTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC
          序列X(粗体)是指与向量序列互补的上游适配器序列。 M序列与靶序列的前6 bp反向互补。斜体序列是Hammerhead核酶的一部分。
        2. 正向引物2:GENE-RGR-F2
          GACGAAACGAGTAAGCTCGTCN 1 N N N 4 N N N 7 N 8 N 9 N 10 N N 12 N 13 N 14 N N N /sup> N 18 N 19 N 20 GTTTTAGAGCTAGAAATAGCAAG 。 N 1 N 2 N N 4 N N 和M 5 M 5 M 3 M 3 M M 1 是互补的,以确保Hammerhead核酶的正确二级结构。我们设计了两个重叠的PCR引物,以避免昂贵的长引物的合成。目标序列为N N 2 N N 4 N N 6 N 7 N N 9 N N /sup> N 12 N N 14 N 15 N sup> 17 N 18 N 19 N 20 NGG。
          注意:PAM网站NGG不包括在底漆中。斜体序列是核心gRNA序列的一部分。
        3. 为您选择的载体设计通用的RGR反向引物:UNIVERS-RGR-R: ZZZZZZZZZZZZZZZZZZZ GTCCCATTCGCCATGCCGAAGC 。 UNIVERS-RGR-R引物应与GCTTCGGCATGGCGAATGGGAC 反向互补,其中 YYYYYYYYYYYYYYYYYYYYY 是下游衔接子序列(Y和Z是反向互补的)。斜体序列与HDV核酶的3'末端互补。


          图2. RGR单元的分子设计和通过PCR组装完整的RGR单元。顶部图显示了产生靶向(N)的gRNA的RGR单元的完整序列20 序列。带下划线的序列是来自载体的衔接子序列,用于将RGR单元克隆到最终的CRISPR载体中。请注意,如果选择不同的载体,衔接子序列将相应变化。 N 1 至N 20(红色)是NGG PAM位点之前的靶序列。重要的是,M 5 M 5 M 3 M 2 M 1 和N <1> N N 3 N N 5 N 6反向互补,使得核酶可以如所设计的那样进行自我切割。以绿色突出显示的序列是核心gRNA序列。以黄色突出显示的序列是锤头核酶。 HDV核酶以紫色突出显示。引物用不同的颜色标记:黑色(Gene-RGR-F1),浅蓝色(Gene-RGR-F2)和红色(UNIVERS-RGR-R)。通用引物取决于载体序列,如果使用不同的载体,则需要改变。完整的RGR单元可以通过两次重叠PCR反应轻松组装。

    3. 组装核酶-RNA-核酶(RGR)单位
      1. 用于靶向新基因的RGR单元通过两轮重叠PCR反应进行组装(图2)。两个正向引物之间存在21-bp重叠(图2)
      2. 第一次PCR(PCR1):使用引物对GENE-RGR-F2 + UNIVERS-RGR-R。模板:任何现有的RGR构建体(例如,pRS316-RGR-GFP,可从 Addgene ,预期PCR产物为233 bp)
      3. 第二轮PCR(PCR2):使用引物对GENE-RGR-F1 + UNIVERS-RGR-R。使用PCR1凝胶纯化的PCR产物作为模板。预期的PCR2产物是277bp,这是完整的RGR单元(图2)。
      根据制造商的建议( https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530 )。简言之,如表1所述混合PCR组分。

      表1.用于扩增RGR单位的PCR设置


      表2.扩增RGR单位的PCR条件


    4. 将最终的RGR产物连接到所需的载体中 - 克隆RGR(核酶-GRNA-核酶)单位
      通过两轮PCR组装的完整的RGR单元使用 Gibson汇编协议(Gibson等人,2009)。注意,PCR引物GENE-RGR-F1和UNIVERS-RGR-R含有与载体中的序列的一部分相匹配的序列,以促进RGR单元在载体中的组装(图2 )。
      1. 在50℃孵育DNA组装混合物(表3)1小时。按照制造商推荐的方案进行DNA装配(表3)。 ( https://www.neb .com/products/e2611-gibson-assembly-master-mix#tabselect2 )。

        表3. DNA组装


      2. 然后将3μl连接的产物转化成E。大肠杆菌DH5α感受态细胞。
        连接的质粒可以转化到E中。常规克隆程序后的大肠杆菌感受态细胞(Sambrook和Russell,2001)。
        1. 变换E。大肠杆菌使用3μl连接产物的DH5α感受态细胞(自制或商业产品)。  
        2. 在含有适当抗生素的LB培养基中接种2至4个菌落。
        3. 使用QIAGEN Plasmid Mini Kit从转化的DH5α细胞中纯化质粒。
      3. 在将最终质粒引入您选择的细胞/生物体之前,通过Sanger测序验证阳性克隆(Yoshioka等人,2015)。

  2. 构建CRISPR介导的基因编辑的RGR单元的一个例子
    1. 选择一个靶基因和一个载体
      我们展示以"拟南芥"为基础的RGR单元的构建以生物素结合蛋白1(ABP1)(图3)为例(Gao等, em>,,2015)。我们使用pHDE-35S-Cas9-mCherry-UBQ质粒作为我们的CRISPR载体(Gao等人,2016),其已被用于在拟南芥中编辑基因质粒可以通过 Addgene 获得。
    2. 线性化质粒
      我们用Mfe I消化pHDE-35S-Cas9-mCherry-UBQ质粒(图3)。通过Gibson装配将MGRI-linearized质粒用于将RGR单元组装到载体中。完整的质粒可以从植物中的拟南芥UBQ10启动子表达ABP1-RGR单元(图3)。

      表4.通过限制消化
      对向量进行线性化

      混合表4所示的组分,并在37℃下消化载体过夜。然后将酶在80℃加热灭活15分钟。线性化质粒为16084 bp。
    3. 设计PCR引物
      上游转接器序列是GTTTTTCTGATTAACAGCTCGC。我们使用的下游适配器是GACCTAACTGAGTAAGCTAGC(图3)
      1. 正向引物1:ABP1-RGR-F1:
        GTTTTTCTGATTAACAGCTCGC AGCTCC CTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC 。粗体的6bp序列与gRNA靶的前6bp反向互补。下划线的序列是Hammerhead核酶的一部分。
      2. 正向引物2:ABP1-RGR-F2:
        GACGAAACGAGTAAGCTCGTC GGAGCTCCTTGTCCCATCAA GTTTAGAGCTAGAAATAGCAAG 。下划线序列是Hammerhead核酶的一部分。粗体字是gRNA靶序列,斜体序列是核心gRNA序列的一部分。
      注意:ABP1-RGR-F1中的粗体序列与ABP1-RGR-F2中的粗体序列的前6bp反向互补。 ABP1-RGR-F1和ABP1-RGR-F2共享以下重叠序列:GACGAAACGAGTAAGCTCGTC,其允许我们进行两个重叠的PCR反应。
    4. 为了使用Gibson装配将ABP1-RGR单元克隆到35S-Cas9-mCherry-UBQ质粒中的MfeI I位点,UNIVERS-RGR-R引物是:
      GCTAGCTTACTCAGTTAGGT C GTCCCATTCGCCATGCCGAAGC
      注意:粗体序列与下游适配器序列互补。斜体序列与HDV核酶的3'末端相符。
    5. 用于产生靶向ABP1基因的gRNA的完整的RGR单元显示在图3中.RGR单元导致在中成功分离了几个无效等位基因的abp1 拟南芥(高等等,2015年和2016年)。


      图3.用于靶向拟南芥中ABP1基因的RGR单元的分子设计。将RGR单位克隆到pHDE-35S-Cas9-mCherry-UBQ质粒中使用Gibson程序集的Mfe I站点。适配器序列带下划线。注意,当设计通用引物时,下游衔接子序列必须转换为反向互补序列。目标序列为红色。 Hammerhead核酶和HDV核酶分别以黄色和紫色突出显示
  3. 通过体外转录导向RNA的生成
    一旦RGR单元被组装并克隆到如上所述的最终载体中,可以使用可商购的T7,SP6或T3 RNA聚合酶通过体外转录来容易地产生gRNA。
    1. 为体外准备DNA模板转录
      1. 通过使用两个通用引物的组装的RGR构建体通过PCR扩增体外转录的模板(克隆到相同载体中的任何RGR单位可以使用相同的引物对扩增)。例如,可以使用以下一对引物扩增克隆到pHDE-35S-Cas9-mCherry-UBQ中的MfeI I位点的RGR单元:
        SP6-P1:GTCACTATTTAGGTGACACTATA G AAGCG GTTTTTCTGATTAACAGCTCGC和Univers-P2:GCTAGCTTACTCAGTTAGGTC。
      2. 下划线的序列是SP6启动子,引物SP6-P1中的粗体G是SP6 RNA聚合酶的转录起始位点。如果使用T7或T3 RNA聚合酶,引物SP6-P1的下划线的5'-末端需要改变为GTCACTAATACGACTCACTATA GGAGA和GTCACAATTAACCCTCACTAAA > G GGAGA。
      3. PCR条件描述于表1和表2中。
    2. 转录
      1. 按照表5中列出的顺序在室温下加入以下试剂:

        表5:体外转录RGR产生gRNA分子

        注意:为了获得更高的产量,在反应中加入1U无机焦磷酸酶并孵育过夜。

      2. 将混合物在37℃(T7,T3)或42℃(SP6)下孵育2至8小时。
      3. 加入1μl0.5 M EDTA终止反应 注意:不需要进一步纯化用于体外CRISPR切割测定的RNA转录物(对于体外切割测定实施例,请参见以下链接: https://www.neb.com/products/m0386-cas9-nuclease-s-pyogenes 。可以通过在12%变性尿素聚丙烯酰胺凝胶中电泳分析体外转录和自身处理的质量,用溴化乙锭染色RNA条带,并使用UV透射仪进行显色,分析Cas9产生的DNA裂解产物和来自体外转录,常规的1%琼脂糖凝胶是足够的。

  4. 将RGR质粒转化成拟南芥
    通过土壤杆菌介导的花浸法(Clough and Bent,1998)将具有RGR构建体的最终质粒转化到拟南芥中。将转基因T1植物鉴定为抗潮霉素或产生mCherry荧光(Gao等人,2015和2016)。使用PCR和限制性消化筛选T1植物进行编辑,如Gao等人,2015和2016所述。

数据分析

我们基于RGR的功能性gRNA的生产在体外和体内成功(Gao和Zhao,2014; Gao等人, 2015年和2016年)。我们最初使用RGR设计产生了靶向GFP的gRNA。使用市售的SP6 RNA聚合酶从SP6启动子体外产生gRNA(Gao和Zhao,2014)。消化测定表明gRNA是完全功能的(Gao和Zhao,2014)。我们使用相同的RGR设计,使用ADH启动子RNA聚合酶II启动子(Gao和Zhao,2014)在酵母中产生gRNA。这样的结构导致在酵母中成功地编辑了GFP 基因。我们进一步在拟南芥中引入了RGR设计以产生靶向拟南芥ABP1基因的gRNA。已经获得了ABP1基因中的多个稳定的可遗传突变(Gao等人,2015和2016)。例如,我们制作了一种使用RGR进行一个gRNA生产的构建体,并且U6启动子产生另一个gRNA。两个gRNA分子靶向拟南芥中ABP1基因的两个离散位点,并设计为产生771bp的缺失(Gao等人, 2016)。我们检测到61个T1植物中有5个的预期缺失。两个T1植物在T2代产生了无Cas9的稳定的纯合突变体(Gao等人,2016)。
RGR设计已经适应于编辑其他生物体中的基因(Nissim等人,2014; Yoshioka等人,2015)。

致谢

该协议最初在加州大学圣地亚哥分校开发,当时YG是研究生。华中农业大学的议案进一步完善。作者要感谢国家基因转化授权#2016ZX08010002-002至RW和NIH授权R01GM114660至YZ的支持。

参考文献

  1. Clough,S.and Bent,A.(1998)。  花卉浸泡:拟南芥的农杆菌介导转化的简化方法植物J > 16(6):735-743
  2. Cong,L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA and Zhang,F 。(2013)。使用CRISPR/Cas的多重基因组工程系统。 科学 339(6121):819-823。
  3. Gao,X.,Chen,J.,Dai,X.,Zhang,D. and Zhao,Y。(2016)。  可靠地分离由CRISPR/Cas9介导产生的遗传和Cas9 -free 拟南芥突变体的有效策略基因组编辑。植物生理学 171(3):1794-1800。
  4. Gao,Y.,Zhang,Y.,Zhang,D.,Dai,X.,Estelle,M. and Zhao,Y。(2015)。  体内和导向RNA。/em> 56(4):343-349。
  5. Gibson,DG,Young,L.,Chuang,RY,Venter,JC,Hutchison,CA,3rd and Smith,HO(2009)。  DNA分子的酶装配达几百千碱基。方法6(5):343-345 。
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  8. Sambrook,J.and Russell,DW(2001)。< a class ="ke-insertfile"href ="https://www.mendeley.com/research/molecular-cloning-laboratory-mannual-3rd-ed/"target ="_ blank"> Molecular Cloning:A Laboratory Manual,3rd ed。 Cold Spring Harbor Laboratory Press pp:1105-1111。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, T., Gao, Y., Wang, R. and Zhao, Y. (2017). Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters. Bio-protocol 7(4): e2148. DOI: 10.21769/BioProtoc.2148.
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