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May 2018

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flySAM Transgenic CRISPRa System Manual
基于flySAM的CRISPRa转基因系统操作手册   

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Abstract

Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed over two decades ago. It is laborious and expensive to clone the coding DNA sequence (CDS) of a gene, especially those of large size. In addition, side effects of this method are often observed because of the ectopic expression. Also, simultaneous activation of two genes with the traditional method is often time-consuming, and few are achievable for three or more genes. In this protocol, we describe how to build an effective and convenient targeting activator system, flySAM, to activate endogenous genes in Drosophila melanogaster based on the structure-guided engineering of CRISPR-Cas9 complex.

Keywords: CRISPR-Cas nuclease (CRISPR-Cas核酸酶), dCas9 (dCas9), Drosophila (果蝇), CRISPRa (CRISPRa), flySAM (flySAM)

Background

Currently, most of the genes in Drosophila can be knocked down or knocked out through powerful genetic tools and several loss-of-function (LOF) resources have been established based on these tools (Ren et al., 2013; Qiao et al., 2018). A similar gain-of-function (GOF) resource is valuable to study gene function as it can be a complement to LOF. However, we do not have a genome-wide GOF resource until now mainly because of technical difficulties. The traditional gene overexpression system in Drosophila is the Gal4/UAS:cDNA system. It needs to clone the coding DNA sequence (CDS) of a gene downstream of the upstream activation sequence (UAS) which can be controlled by Gal4 transcription factor. It is laborious, expensive and sometimes unachievable. Therefore, an easier way to realize GOF is urgently needed. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an effective genome-editing tool. The transcriptional activation system in Drosophila based on the CRISPR/Cas9 technique, termed “CRISPR transcriptional activation (CRISPRa) system”, has been developed in recent years (Konermann et al., 2015; Lin et al., 2015; Ewen-Campen et al., 2017). Cas9 has HNH and RuvC nuclease domains which are responsible for the cutting of complementary and non-complementary DNA strand, respectively. Through introducing single amino acid mutation within HNH and RuvC domains of Cas9 (dCas9), nuclease activity of Cas9 is abolished without affecting its DNA binding ability (Dominguez et al., 2016). The CRISPR transcriptional activation CRISPRa system is generated by fusing several activation domains to dCas9 and can be recruited to the upstream of the Transcription Start Site (TSS) through single-guide RNAs (sgRNAs). The specificity of CRISPRa system is controlled by 20 bp guide sequence in sgRNA, thus one can simply use the 20 bp guide sequence against different targets. Previously, VPR-based CRISPRa system has been built in Drosophila in vivo and in vitro (Lin et al., 2015; Ewen-Campen et al., 2017). In this system, three activation domains, VP64, P65, Rta (VPR), tandemly following the dCas9. However, due to low efficiency, this system relies on two sgRNAs to boost gene transcription, which not only increased the complicacy and cost of the cloning procedure, but also increased toxicity and the chances of off-target effects. In addition, although it works both in vitro and in vivo, it often produces weaker phenotypes than the traditional Gal4/UAS:cDNA system.

To improve the efficiency, reduce the toxicity, and streamline the cloning procedure, this protocol introduces how to construct transcriptional activation lines based on flySAM system. In this system, the dCas9-VP64 and MCP-p65-HSF1 is controlled by 10x UAS and sgRNA2.0 which is driven by U6:2 promoter (Jia et al., 2018). Unlike the previous methods, single sgRNA can successfully activate gene transcription. In addition, to study the intrinsic and extrinsic roles of genes with redundant functions or involved in protein complex, we can easily clone multiple sgRNAs to target different genes.

Materials and Reagents

  1. 1.5 ml MaxyClear Microtubes (Axygen, catalog number: MCT-150-C)
  2. 0.2 ml Polypropylene PCR Tube (Axygen, catalog number: PCR-0208-C)
  3. Pipette tips (Corning, Axygen®)
  4. 0.22 μm Millipore filters (Millipore, catalog number: R7MA69670)
  5. Fly stocks (All Drosophila strains are stored in Tsinghua Fly Center)
    1. y sc v nanos-integrase; attP40 (TB00016)
    2. y sc v nanos-integrase;; attP2 (TB00018)
    3. y sc v (TB00077)
    4. y sc v; Gla Bc/CyO (TB00023)
    5. y sc v;; Dr,e/TM3,Sb (TB00139)
  6. Trans5α Chemically Competent Cell (Transgen Biotech, catalog number: CD201-01)
  7. flySAM (Ampicillin resistant, plasmid map is shown in Figure 1)
  8. BbsI (New England Biolabs, catalog number: R0539L)
  9. NheI (New England Biolabs, catalog number: R3131L)
  10. SpeI (New England Biolabs, catalog number: R3133L)
  11. T4 DNA ligase (New England Biolabs, catalog number: M0202L)
  12. Alkaline phosphatase, calf intestinal (CIP) (New England Biolabs, catalog number: M0290S)
  13. AxyPrep Plasmid Miniprep Kit (AXYGEN, catalog number: AP-MN-P-250)
  14. AxyPrep DNA Gel Extraction Kit (AXYGEN, catalog number: AP-GX-250)
  15. PurePlasmid Mini Kit (CWBiotech, catalog number: CW0500M)
  16. Forward Primer 1: 5’-TCAACAAACGaacaataggacac-3’
  17. Reverse Primer 1: 5’-aAAAAAGCACCGACTCGGTG-3’
  18. Forward Primer 2: 5’-ACATCAGGAAAGAGCAGTTGAG-3’
  19. Reverse Primer 2: 5’-TTGCTCACCTGTGATTGCTCC-3’
  20. 50x TAE (Double Helix, catalog number: P0309A)
  21. Agarose (Invitrogen, catalog number: 0000602315)
  22. LB powder (BD, catalog number: 2171199)
  23. KCl (AMRESCO, catalog number: 7447-40-7)
  24. Tris-HCl (AMRESCO, catalog number: 1185-53-1)
  25. EDTA (AMRESCO, catalog number: 60-00-4)
  26. NaCl (AMRESCO, catalog number: 7647-14-5)
  27. Ampicillin (Solarbio, catalog number: A8180)
  28. Ethidium bromide (Sigma, catalog number: E8751)
  29. GoTag Green Master Mix, 2x (Promega, catalog number: 000179370) 
  30. Sodium phosphate buffer (see Recipes)
  31. 10x Injection buffer (see Recipes)
  32. 10x Annealing buffer (see Recipes)
  33. LB (Luria Bertani) medium (see Recipes)
  34. 1x TAE (see Recipes)
  35. 1.5% agarose gel (see Recipes)


    Figure 1. The map of flySAM construct. The dCas9 is under the control of 10 x UAS and Drosophila synthetic core promoter (DSCP) and followed by VP64. dCas9-VP64 and MCP-P65-HSF1 are mediated and separated by T2A self-cleaving peptide. ftz intron is located between CRISPRa components and SV40 polyA tail. The grey block between two BbsI sites is a marker to detect whether the sgRNA is under the control of U6:2 regulatory sequence. Two gypsy elements which can boost gene expression are used to flank the CRISPRa components. The attB sequence and a vermillion+ marker are also included in this plasmid.

Equipment

  1. Pipette (Eppendorf)
  2. Microwave (SANYO, model: EM-2509EB1)
  3. Autoclave (SANYO, model: MLS-3780)
  4. PCR Thermal Cycler (Eppendorf, Mastercycler nexus GSX1)
  5. Microcentrifuge (Eppendorf, model: 5417R)
  6. DNA electrophoresis apparatus (Bio-Rad)
  7. NanoDrop 2000 Spectrophotometer (Thermo Scientific)

Procedure

  1. Oligo designation 
    1. Select 20 nt length sgRNA upstream of NGG within -100 to -400 of the target gene’s TSS
      The following parameters should be considered to ensure the quality of sgRNAs:
      1. TTTT sequence should not be in 20 nt sgRNA sequence because it is the transcriptional termination signal.
      2. To avoid off-target, ≥ 18 nt of the targeting sequence of sgRNA should not match to other genomic loci.
      3. The optimal sgRNA normally carries high GC content, usually over 50%.
    2. To synthesize the 20 nt DNA sequence, the overhang sequence 5’-TTCG-3’ from BbsI should be added into forward primer and 5’-aaac-3’ to the reverse complement primer.
      e.g., 5’-NNNAGCAATCGACATGCAAGCGGCTCGGAGCCAGGGCACCTGCNNN-3’
      (Underlined AGG is the PAM sequence)
      Forward: 5’-ttcgCATGCAAGCGGCTCGGAGCC-3’
      Reverse: 5’-aaacGGCTCCGAGCCGCTTGCATG-3’
    3. Resuspend oligos with ddH2O to final 20 µM and set up the annealing reaction:


  2. Backbone preparation
    1. BbsI is used to linearize flySAM vector.


    2. Run 1.5% agarose gel. Two bands should be visible (Figure 2), one is about 700 bp (this spacer can help to evaluate the cutting efficiency and later on to select correct clones by PCR, as the size of PCR product from correct clone is different from incorrect clone), another is about 14 kb. Cut and collect the 14 kb band and extract the backbone by AxyPrep DNA Gel Extraction kit following the standard protocol. Elute DNA with 50 µl ddH2O, measure the DNA concentration with NanoDrop 2000, should be around 50 ng/µl.


      Figure 2. An example gel of BbsI-digested flySAM2.0. M is DNA marker. flySAM2.0 is the BbsI-digested product.

  3. Ligation
    1. Ligate the annealed sgRNA product with linearized flySAM vector


      Transform 2-5 µl of the ligation product into 10 µl Trans5α Chemically Competent Cells following the standard protocol, and spread on an LB-agar plate containing 100 µg/ml ampicillin. Incubate these plates at 37 °C overnight in an inverted state.
    2. Use the following primers to perform colony PCR:
      Forward Primer 1: 5’-TCAACAAACGaacaataggacac-3’
      Reverse Primer 1: 5’-aAAAAAGCACCGACTCGGTG-3’
      Thermal cycling condition is:

      The size of PCR product from correct clone is about 300 bp, whereas either no band or 1,000 bp band is incorrect.
    3. Inoculate correct clone in LB medium with 100 µg/ml ampicillin.
    4. Culture bacteria and miniprep plasmid by using AxyPrep Plasmid Miniprep Kit.

  4. Sanger sequencing to further confirm the insertion
    Confirm flySAM-sgRNA plasmid by Sanger sequencing using below primer:
    Forward Primer 1: 5’-TCAACAAACGaacaataggacac-3’
    Notes:
    1. If only one sgRNA needs to be cloned, the next step is to go directly to Procedure J. 
    2. (Optional) If two or more genes need to be activated, in other words, two or more sgRNAs need to be cloned, the next procedures can be performed after Procedure D: 

  5. Fragment preparation
    1. Double digest flySAM (one sgRNA has been cloned into it) by NheI and SpeI.


    2. Run agarose gel. Two bands should be visible, one is about 1 kb, another is about 14 kb. Cut and collect the 1 kb band and extract the fragment by AxyPrep DNA Gel Extraction kit following the standard protocol. Elute DNA with 50 µl ddH2O, measure the DNA concentration by NanoDrop 2000, should be around 40 ng/µl.

  6. Backbone preparation
    1. Digest flySAM (another sgRNA which is different with the one in Procedure E has been cloned into it) by NheI or SpeI.


    2. Add 0.2 µl CIP and incubate at 37 °C for 8 min.
    3. Purify the digested product by AxyPrep DNA Gel Extraction kit.

  7. Ligation and transformation
    Ligate the fragment from Procedure E to backbone from Procedure F. The procedure is similar to Procedure C.

  8. Colony identification
    1. The following primers are used for colony PCR:
      Forward Primer 2: 5’-ACATCAGGAAAGAGCAGTTGAG-3’
      Reverse Primer 2: 5’-TTGCTCACCTGTGATTGCTCC-3’
      Thermal cycling condition is:

      The correct clone is about 800 bp.
    2. Inoculate the correct clone in LB medium with 100 µg/ml ampicillin.
    3. Culture bacteria and miniprep plasmid by using AxyPrep Plasmid Miniprep Kit.

  9. Further confirm the correct clone by restriction enzymes digestion
    1. Double digest the plasmid by NheI and SpeI.


    2. Run agarose gel, one band should be about 2 kb (if two sgRNAs together), another is about 13 kb if the plasmid is right.

  10. Purification and microinjection
    1. For microinjection, the plasmid (~10 mg) should be purified. PurePlasmid Mini Kit is used according to the producer’s specifications. The plasmid DNA is finally eluted in 70 µl of 1x Injection Buffer. The appropriate concentration of each sample is 100 ng/µl-200 ng/µl, which can be determined by NanoDrop 2000.
    2. Prepare the injected embryos and inject the transgene vector into the embryos of tool flies as standard procedure (Chromosome II y sc v nanos-integrase; attP40. Chromosome III y sc v nanos-integrase;; attP2) (Ni et al., 2011).
    3. The injected embryos should be kept at 25 °C and 60% humidity to adulthood (G0).

  11. Selection for transgenic fly
    Both ♂ G0 and ♀ G0 should be crossed to opposite sex of y sc v;;. Since the flySAM transgenes carry a copy of vermillion (v+), which rescues the mutant vermillion (v) allele inherited from the injection stock. Thus transformants will have a dark red wild type (v+) eye color and the v+ G1 should be picked (Figure 3).


    Figure 3. Eye color screening marker. The transformants have a dark red wild type (v+) eye color (left) while flies that haven’t been genetically modified have a light red (v-) eye color (right).

    1. For G1♂ transformants from ♂ G0 follow these sequential steps
      1. Cross the siblings to the virgin ♀ of y sc v;; Dr, e/TM3, sb (for 3rd chromosome insertions) or y sc v; Gla Bc/CyO (for 2nd chromosome insertions).
      2. Collect ♂ and virgin ♀ siblings that have v+ (wild type) eye color and CyO or TM3 and cross them to each other.
      3. Keep the homozygous flies (keep the stock as balanced heterozygotes in case the homozygous flies are unhealthy).
    2. For G1♂ transformants from ♀ G0 follow these sequential steps
      1. Cross to virgin ♀ of y sc v;;
      2. Cross the v+ ♂ to the virgin ♀ of y sc v;; Dr, e/TM3, sb (for 3rd chromosome insertions) or y sc v; Gla Bc/CyO (for 2nd chromosome insertions).
      3. Collect ♂ and virgin ♀ siblings that have v+ (wild type) eye color and CyO or TM3 and cross them to each other.
      4. Keep the homozygous flies (keep the stock as balanced heterozygotes in case the homozygous flies are unhealthy).
    3. For ♀ transformants from ♂ or ♀ G0 follow these sequential steps
      1. Cross to ♂ of y sc v;;
      2. Cross the v+ ♂ to the virgin ♀ of y sc v;;
      3. Cross the v+ ♂ to the virgin ♀ of y sc v;; Dr, e/TM3, sb (for 3rd chromosome insertions) or y sc v; Gla Bc/CyO (for 2nd chromosome insertions).
      4. Collect ♂ and virgin ♀ siblings that have v+ (wild type) eye color and CyO or TM3 and cross them to each other.
      5. Keep the homozygous flies (keep the stock as balanced heterozygotes in case the homozygous flies are unhealthy).

Data analysis

If we want to activate two or more genes, the primer used for DNA sequencing in Procedure D cannot be used to sequence because two U6:2 promoters are in one vector. Colony identification and enzymes digestion are more efficient to identify the right clone.

Recipes

  1. 0.2 M Sodium phosphate buffer (pH 6.8) (stock solution)
    51 ml 0.2 M NaH2PO4
    49 ml 0.2 M Na2HPO4
    Store at room temperature
    1. 0.2 M Na2HPO4
      Dissolve 71.6 g Na2HPO4•12H2O in 1 L of ddH2O, mix thoroughly
      Store at room temperature
    2. 0.2 M NaH2PO4
      Dissolve 31.2 g Na2HPO2•2H2O in 1 L of ddH2O, mix thoroughly
      Store at room temperature
  2. 10x Injection buffer
    1 mM sodium phosphate buffer (pH 6.8)
    50 mM KCl
    Prepare in sterile distilled water and filter through 0.22 μm Millipore filters
    It can be stored at -20 °C for one year
  3. 10x Annealing buffer
    100 mM Tris-HCl (pH 7.5)
    10 mM EDTA
    1 M NaCl
    It can be stored at room temperature for one year
  4. LB (Luria Bertani) medium
    Dissolve 25 g of the powder in 1 L of ddH2O, mix thoroughly
    Autoclave at 121 °C for 15 min
  5. 1x TAE
    20 ml 50x TAE
    980 ml ddH2O
    Store at room temperature
  6. 1.5% agarose gel
    1.5 g agarose
    100 ml 1x TAE
    Heat solution to dissolve agarose in microwave
    Add ethidium bromide to a final concentration of 0.2 μg/ml

Acknowledgments

This work was supported by NIH Grants R01GM084947 and R24OD021997 and the NIH’s Ruth L. Kirschstein National Research Service Award F32GM113395 from the NIH General Medical Sciences Division. This work was supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of the People's Republic of China (2015BAI09B03, 2016YFE0113700), and the National Natural Science Foundation of China (31571320).

Competing interests

The authors do not have any possible conflicts of interest.

References

  1. Qiao, H. H., Wang, F., Xu, R. G., Sun, J., Zhu, R., Mao, D., Ren, X., Wang, X., Jia, Y., Peng, P., Shen, D., Liu, L. P., Chang, Z., Wang, G., Li, S., Ji, J. Y., Liu, Q. and Ni, J. Q. (2018). An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila. Nat Commun 9(1): 4160.
  2. Ren, X., Sun, J., Housden, B. E., Hu, Y., Roesel, C., Lin, S., Liu, L. P., Yang, Z., Mao, D., Sun, L., Wu, Q., Ji, J. Y., Xi, J., Mohr, S. E., Xu, J., Perrimon, N. and Ni, J. Q. (2013). Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci U S A 110(47): 19012-19017.
  3. Dominguez, A. A., Lim, W. A. and Qi, L. S. (2016). Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol 17(1): 5-15.
  4. Ewen-Campen, B., Yang-Zhou, D., Fernandes, V. R., Gonzalez, D. P., Liu, L. P., Tao, R., Ren, X., Sun, J., Hu, Y., Zirin, J., Mohr, S. E., Ni, J. Q. and Perrimon, N. (2017). Optimized strategy for in vivo Cas9-activation in Drosophila. Proc Natl Acad Sci U S A 114(35): 9409-9414.
  5. Jia, Y., Xu, R. G., Ren, X., Ewen-Campen, B., Rajakumar, R., Zirin, J., Yang-Zhou, D., Zhu, R., Wang, F., Mao, D., Peng, P., Qiao, H. H., Wang, X., Liu, L. P., Xu, B., Ji, J. Y., Liu, Q., Sun, J., Perrimon, N. and Ni, J. Q. (2018). Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM. Proc Natl Acad Sci U S A 115(18): 4719-4724. 
  6. Konermann, S., Brigham, M. D., Trevino, A. E., Joung, J., Abudayyeh, O. O., Barcena, C., Hsu, P. D., Habib, N., Gootenberg, J. S., Nishimasu, H., Nureki, O. and Zhang, F. (2015). Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517(7536): 583-588.
  7. Lin, S., Ewen-Campen, B., Ni, X., Housden, B. E. and Perrimon, N. (2015). In vivo transcriptional activation using CRISPR/Cas9 in Drosophila. Genetics 201(2): 433-442.
  8. Ni, J. Q., Zhou, R., Czech, B., Liu, L. P., Holderbaum, L., Yang-Zhou, D., Shim, H. S., Tao, R., Handler, D., Karpowicz, P., Binari, R., Booker, M., Brennecke, J., Perkins, L. A., Hannon, G. J. and Perrimon, N. (2011). A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods 8(5): 405-407.

简介

可以增强基因表达的强大而通用的方法对于系统地研究基因功能是有用的。 迄今为止,与产生功能丧失突变体的方法相比,实现功能获得的方法是有限的。 Drosophila 中的整个区域严重依赖于Gal4 / UAS:二十多年前开发的cDNA过表达系统。 克隆基因的编码DNA序列(CDS)是困难且昂贵的,特别是那些大尺寸的基因。 此外,由于异位表达,经常观察到该方法的副作用。 此外,用传统方法同时激活两个基因通常是耗时的,并且很少有三个或更多基因可以实现。 在该协议中,我们描述了如何基于CRISPR-Cas9复合物的结构指导工程,构建有效且方便的靶向激活剂系统flySAM,以激活黑腹果蝇中的内源基因。

【背景】 目前, Drosophila 中的大多数基因可以通过强大的遗传工具被敲除或敲除,并且已经基于这些工具建立了几种功能丧失(LOF)资源(Ren et al。,2013; Qiao et al。,2018)。类似的功能获得(GOF)资源对于研究基因功能是有价值的,因为它可以是LOF的补充。然而,到目前为止,我们还没有基因组范围的GOF资源,主要是因为技术上的困难。 果蝇中的传统基因过表达系统是Gal4 / UAS:cDNA系统。它需要克隆上游激活序列(UAS)下游的基因的编码DNA序列(CDS),其可以由Gal4转录因子控制。这是费力的,昂贵的,有时无法实现。因此,迫切需要一种更容易实现GOF的方法。聚集的规则间隔短回文重复序列(CRISPR)/ CRISPR相关蛋白9(Cas9)系统是一种有效的基因组编辑工具。基于CRISPR / Cas9技术的 Drosophila 中的转录激活系统,被称为“CRISPR转录激活(CRISPRa)系统”,近年来已经开发出来(Konermann et al。 ,2015; Lin et al。,2015; Ewen-Campen et al。,2017)。 Cas9具有HNH和RuvC核酸酶结构域,其分别负责切割互补和非互补DNA链。通过在Cas9的HNH和RuvC结构域中引入单个氨基酸突变(dCas9),Cas9的核酸酶活性被消除而不影响其DNA结合能力(Dominguez 等,,2016)。 CRISPR转录激活CRISPRa系统通过将几个激活结构域与dCas9融合而产生,并且可以通过单指导RNA(sgRNA)募集到转录起始位点(TSS)的上游。 CRISPRa系统的特异性受sgRNA中20bp引导序列的控制,因此可以简单地使用针对不同靶标的20bp引导序列。以前,基于VPR的CRISPRa系统已经在 Drosophila 体内和体外中建立(Lin et al。 ,2015; Ewen-Campen et al。,2017)。在这个系统中,三个激活域,VP64,P65,Rta(VPR),串联在dCas9之后。然而,由于效率低,该系统依赖于两个sgRNA来促进基因转录,这不仅增加了克隆过程的复杂性和成本,而且增加了毒性和脱靶效应的机会。此外,虽然它在体外和体内都有效,但它常常比传统的Gal4 / UAS:cDNA系统产生更弱的表型。

为了提高效率,降低毒性,简化克隆程序,该协议介绍了如何构建基于flySAM系统的转录激活系。在该系统中,dCas9-VP64和MCP-p65-HSF1由10x UAS和sgRNA2.0控制,其由U6:2启动子驱动(Jia et al。,2018)。与以前的方法不同,单个sgRNA可以成功激活基因转录。此外,为了研究具有多余功能或参与蛋白质复合物的基因的内在和外在作用,我们可以容易地克隆多个sgRNA以靶向不同的基因。

关键字:CRISPR-Cas核酸酶, dCas9, 果蝇, CRISPRa, flySAM

材料和试剂

  1. 1.5 ml MaxyClear Microtubes(Axygen,目录号:MCT-150-C)
  2. 0.2 ml聚丙烯PCR管(Axygen,目录号:PCR-0208-C)
  3. 移液器吸头(Corning,Axygen ®)
  4. 0.22μmMillipore过滤器(Millipore,目录号:R7MA69670)
  5. 飞行种群(所有 Drosophila 菌株存放在清华飞行中心)
    1. y sc v nanos-integgrase; attP40 (TB00016)
    2. y sc v nanos-integrase ;; attP2 (TB00018)
    3. y sc v (TB00077)
    4. y sc v; Gla Bc / CyO (TB00023)
    5. y sc v ;;博士,e / TM3,Sb (TB00139)
  6. Trans5α化学感受态细胞(Transgen Biotech,目录号:CD201-01)
  7. flySAM(氨苄青霉素抗性,质粒图谱如图1所示)
  8. BbsI(New England Biolabs,目录号:R0539L)
  9. NheI(New England Biolabs,目录号:R3131L)
  10. SpeI(New England Biolabs,目录号:R3133L)
  11. T4 DNA连接酶(New England Biolabs,目录号:M0202L)
  12. 碱性磷酸酶,小牛肠道(CIP)(New England Biolabs,目录号:M0290S)
  13. AxyPrep Plasmid Miniprep Kit(AXYGEN,目录号:AP-MN-P-250)
  14. AxyPrep DNA凝胶提取试剂盒(AXYGEN,目录号:AP-GX-250)
  15. PurePlasmid Mini Kit(CWBiotech,目录号:CW0500M)
  16. 正向引物1:5'-TCAACAAACGaacaataggacac-3'
  17. 反向引物1:5'-aAAAAAGCACCGACTCGGTG-3'
  18. 正向引物2:5'-ACATCAGGAAAGAGCAGTTGAG-3'
  19. 反向引物2:5'-TTGCTCACCTGTGATTGCTCC-3'
  20. 50x TAE(双螺旋,目录号:P0309A)
  21. 琼脂糖(Invitrogen,目录号:0000602315)
  22. LB粉末(BD,目录号:2171199)
  23. KCl(AMRESCO,目录号:7447-40-7)
  24. Tris-HCl(AMRESCO,目录号:1185-53-1)
  25. EDTA(AMRESCO,目录号:60-00-4)
  26. NaCl(AMRESCO,目录号:7647-14-5)
  27. 氨苄青霉素(Solarbio,目录号:A8180)
  28. 溴化乙锭(Sigma,目录号:E8751)
  29. GoTag Green Master Mix,2x(Promega,目录号:000179370) 
  30. 磷酸钠缓冲液(见食谱)
  31. 10x注射缓冲液(见食谱)
  32. 10x退火缓冲液(见食谱)
  33. LB(Luria Bertani)中等(见食谱)
  34. 1x TAE(见食谱)
  35. 1.5%琼脂糖凝胶(见食谱)


    图1. flySAM构建体的图谱。 dCas9受10×UAS和 Drosophila 合成核心启动子(DSCP)的控制,其次是VP64。 dCas9-VP64和MCP-P65-HSF1由T2A自切割肽介导和分离。 ftz内含子位于CRISPRa组分和SV40 polyA尾之间。两个BbsI位点之间的灰色区块是检测sgRNA是否在U6:2调节序列控制下的标记。两种可以促进基因表达的吉普赛元素被用于侧翼CRISPRa组分。 attB序列和朱红色 + 标记也包括在该质粒中。

设备

  1. 移液器(Eppendorf)
  2. 微波炉(三洋,型号:EM-2509EB1)
  3. 高压灭菌器(三洋,型号:MLS-3780)
  4. PCR热循环仪(Eppendorf,Mastercycler nexus GSX1)
  5. 微量离心机(Eppendorf,型号:5417R)
  6. DNA电泳仪(Bio-Rad)
  7. NanoDrop 2000分光光度计(Thermo Scientific)

程序

  1. Oligo指定 
    1. 在靶基因TSS的-100至-400范围内选择NGG上游的20nt长sgRNA
      应考虑以下参数以确保sgRNA的质量:
      1. TTTT序列不应该在20nt sgRNA序列中,因为它是转录终止信号。
      2. 为避免脱靶,sgRNA的≥18nt的靶向序列不应与其他基因组位点匹配。
      3. 最佳sgRNA通常携带高GC含量,通常超过50%。
    2. 为了合成20nt DNA序列,应将来自BbsI的突出序列5'-TTCG-3'加入到正向引物中,并将5'-aaac-3'加入到反向互补引物中。
      例如,5'-NNNAGCAATCGA CATGCAAGCGGCTCGGAGCC GCACCTGCNNN-3'
      (带下划线的 AGG 是PAM序列)
      转发: 5' - ttcg CATGCAAGCGGCTCGGAGCC-3'
      反向: 5' - aaac GGCTCCGAGCCGCTTGCATG-3'
    3. 用ddH 2 O重悬寡核苷酸至最终20μM并建立退火反应:


  2. 骨干准备
    1. BbsI用于线性化flySAM载体。


    2. 运行1.5%琼脂糖凝胶。应该可以看到两个条带(图2),一个是大约700bp(这个间隔区可以帮助评估切割效率,然后通过PCR选择正确的克隆,因为正确克隆的PCR产物的大小不同于不正确的克隆) ,另一个是大约14 kb。切割并收集14kb条带,并按照标准方案通过AxyPrep DNA凝胶提取试剂盒提取骨架。用50μlddH 2 O洗脱DNA,用NanoDrop 2000测量DNA浓度,应该在50 ng /μl左右。


      图2.BbsI消化的flySAM2.0的示例凝胶。 M是DNA标记。 flySAM2.0是BbsI消化的产物。

  3. 结扎
    1. 用线性化的flySAM载体连接退火的sgRNA产物


      按照标准方案将2-5μl连接产物转化到10μlTrans5α化学感受态细胞中,并涂布在含有100μg/ ml氨苄青霉素的LB琼脂平板上。将这些板在37℃下以倒置状态孵育过夜。
    2. 使用以下引物进行菌落PCR:
      正向引物1:5'-TCAACAAACGaacaataggacac-3'
      反向引物1:5'-aAAAAAGCACCGACTCGGTG-3'
      热循环条件是:

      来自正确克隆的PCR产物的大小约为300bp,而没有条带或1,000bp条带是不正确的。
    3. 在含有100μg/ ml氨苄青霉素的LB培养基中接种正确的克隆。
    4. 使用AxyPrep Plasmid Miniprep Kit培养细菌和miniprep质粒。

  4. Sanger测序进一步确认插入
    使用以下引物通过Sanger测序确认flySAM-sgRNA质粒:
    正向引物1:5'-TCAACAAACGaacaataggacac-3'
    注意:
    1. 如果只需要克隆一个sgRNA,下一步就是直接进入程序J. 
    2. (可选)如果需要激活两个或更多基因,换句话说,需要克隆两个或更多个sgRNA,可以在程序D之后执行下一个程序: 

  5. 片段准备
    1. 由NheI和SpeI双重消化flySAM(一个sgRNA被克隆到其中)


    2. 运行琼脂糖凝胶。应该可以看到两个波段,一个约为1 kb,另一个约为14 kb。切割并收集1kb条带,并按照标准方案通过AxyPrep DNA凝胶提取试剂盒提取片段。用50μlddH 2 O洗脱DNA,用NanoDrop 2000测量DNA浓度,应该在40 ng /μl左右。

  6. 骨干准备
    1. 通过NheI或SpeI消化flySAM(另一种与程序E中不同的sgRNA已被克隆到其中)。


    2. 加入0.2μlCIP并在37°C下孵育8分钟。
    3. 通过AxyPrep DNA凝胶提取试剂盒纯化消化的产物。

  7. 结扎和转型
    将程序E中的片段连接到程序F的骨干。程序类似于程序C

  8. 殖民地鉴定
    1. 以下引物用于菌落PCR:
      正向引物2:5'-ACATCAGGAAAGAGCAGTTGAG-3'
      反向引物2:5'-TTGCTCACCTGTGATTGCTCC-3'
      热循环条件是:

      正确的克隆大约是800bp。
    2. 在含有100μg/ ml氨苄青霉素的LB培养基中接种正确的克隆。
    3. 使用AxyPrep Plasmid Miniprep Kit培养细菌和miniprep质粒。

  9. 通过限制酶消化进一步确认正确克隆
    1. 通过NheI和SpeI双重消化质粒


    2. 运行琼脂糖凝胶,一个条带应该是大约2kb(如果两个sgRNA一起),如果质粒是正确的,另一条带大约是13kb。

  10. 纯化和显微注射
    1. 对于显微注射,应纯化质粒(~10mg)。 PurePlasmid Mini Kit根据生产商的规格使用。最后将质粒DNA在70μl1x注射缓冲液中洗脱。每种样品的适当浓度为100 ng /μl-200 ng /μl,可通过NanoDrop 2000测定。
    2. 准备注射的胚胎并将转基因载体注射到工具果蝇的胚胎中作为标准程序(染色体II y sc v nanos-integrase; attP40 。染色体III y sc v nanos-integrase ;; attP2 )(Ni et al。,2011)。
    3. 注射的胚胎应保持在25°C和60%湿度至成年期(G0)。

  11. 选择转基因苍蝇
    ♂G0和♀G0都应该与 y sc v; 的异性交叉。由于flySAM转基因带有 vermillion (v + )的拷贝,其拯救了从注射液中遗传的突变朱红色(v)等位基因。因此,转化体将具有暗红色野生型(v + )眼睛颜色,并且应选择v + G1(图3)。


    图3.眼睛颜色筛查标记。 转化体有一种深红色野生型(v + )眼睛颜色(左),而未经过基因改造的果蝇有浅红色(v - )眼睛颜色(右)。

    1. 对于来自♂G0的G1♂转化体,遵循以下顺序步骤
      1. 将兄弟姐妹交给 y sc v的处女; ;; Dr,e / TM3,sb (对于3 rd 染色体插入)或 y sc v; Gla Bc / CyO (对于2 nd 染色体插入)。
      2. 收集具有v + (野生型)眼睛颜色和 CyO 或 TM3 的♂和处女♀兄弟姐妹,并将它们相互交叉。
      3. 保持纯合的苍蝇(保持股票作为平衡的杂合子,以防纯合蝇不健康)。
    2. 对于来自♀G0的G1♂转化体,遵循以下顺序步骤
      1. 越过 y sc v ;; 的处女♀
      2. 越过v + ♂到 y sc v的原始; ;; Dr,e / TM3 ,sb(对于3 rd 染色体插入)或 y sc v; Gla Bc / CyO (对于2 nd 染色体插入)。
      3. 收集具有v + (野生型)眼睛颜色和 CyO 或 TM3 的♂和处女♀兄弟姐妹,并将它们相互交叉。
      4. 保持纯合的苍蝇(保持股票作为平衡的杂合子,以防纯合蝇不健康)。
    3. 对于来自♂或♀G0的♀转化体,请遵循以下顺序步骤
      1. 越过 y sc v ;; 的♂
      2. 越过v + ♂到 y sc v的处女;;
      3. 越过v + ♂到 y sc v的原始; ;; Dr,e / TM3,sb (对于3 rd 染色体插入)或 y sc v; Gla Bc / CyO (对于2 nd 染色体插入)。
      4. 收集具有v + (野生型)眼睛颜色和 CyO 或 TM3 的♂和处女♀兄弟姐妹,并将它们相互交叉。
      5. 保持纯合的苍蝇(保持股票作为平衡的杂合子,以防纯合蝇不健康)。

数据分析

如果我们想激活两个或更多个基因,程序D中用于DNA测序的引物不能用于测序,因为两个U6:2启动子在一个载体中。菌落鉴定和酶消化更有效地鉴定正确的克隆。

食谱

  1. 0.2 M磷酸钠缓冲液(pH 6.8)(原液)
    51毫升0.2M NaH 2 PO 4
    49ml 0.2M Na 2 HPO 4
    在室温下储存
    1. 0.2 M Na 2 HPO 4
      在1L ddH 2 O中溶解71.6g Na 2 HPO 4 •12H 2 O,充分混合< br /> 在室温下储存
    2. 0.2M NaH 2 PO 4
      在1L ddH 2 O中溶解31.2g Na 2 HPO 2 •2H 2 O,充分混合< br /> 在室温下储存
  2. 10x注射缓冲液
    1 mM磷酸钠缓冲液(pH 6.8)
    50 mM KCl
    准备无菌蒸馏水,并通过0.22μmMillipore过滤器过滤
    它可以在-20°C下储存一年
  3. 10x退火缓冲区
    100mM Tris-HCl(pH 7.5)
    10 mM EDTA
    1 M NaCl
    它可以在室温下储存一年
  4. LB(Luria Bertani)中等
    将25g粉末溶于1L ddH 2 O中,充分混合
    在121℃下高压灭菌15分钟
  5. 1x TAE
    20毫升50倍TAE
    980 ml ddH 2 O
    在室温下储存
  6. 1.5%琼脂糖凝胶
    1.5克琼脂糖
    100毫升1x TAE
    加热溶液以溶解微波炉中的琼脂糖
    加入溴化乙锭至终浓度为0.2μg/ ml

致谢

这项工作得到NIH Grants R01GM084947和R24OD021997以及美国国立卫生研究院通用医学科学部NIH的Ruth L. Kirschstein国家研究服务奖F32GM113395的支持。这项工作得到了中华人民共和国科学技术部国家重点技术研究发展计划(2015BAI09B03,2016YFE0113700)和国家自然科学基金(31571320)的支持。

利益争夺

作者没有任何可能的利益冲突。

参考

  1. Qiao,HH,Wang,F.,Xu,RG,Sun,J.,Zhu,R.,Mao,D.,Ren,X.,Wang,X.,Jia,Y.,Peng,P.,Shen, D.,Liu,LP,Chang,Z.,Wang,G.,Li,S.,Ji,JY,Liu,Q。和Ni,JQ(2018)。 在果蝇中具有低毒性的高效多目标转基因RNAi技术。 Nat Commun 9(1):4160。
  2. Ren,X.,Sun,J.,Housden,BE,Hu,Y.,Roesel,C.,Lin,S.,Liu,LP,Yang,Z.,Mao,D.,Sun,L.,Wu, Q.,Ji,JY,Xi,J.,Mohr,SE,Xu,J.,Perrimon,N。和Ni,JQ(2013)。 使用种系特异性Cas9优化基因编辑技术 Drosophila melanogaster。 Proc Natl Acad Sci USA 110(47):19012-19017。
  3. Dominguez,A.A.,Lim,W。A.和Qi,L。S.(2016)。 超越编辑:重新利用CRISPR-Cas9进行精确的基因组调控和审讯。 Nat Rev Mol Cell Biol 17(1):5-15。
  4. Ewen-Campen,B.,Yang-Zhou,D.,Fernandes,VR,Gonzalez,DP,Liu,LP,Tao,R.,Ren,X.,Sun,J.,Hu,Y.,Zirin,J。 ,Mohr,SE,Ni,JQ和Perrimon,N。(2017)。 在果蝇中体内 Cas9激活的优化策略。 Proc Natl Acad Sci USA 114(35):9409-9414。
  5. Jia,Y.,Xu,RG,Ren,X.,Ewen-Campen,B.,Rajakumar,R.,Zirin,J.,Yang-Zhou,D.,Zhu,R.,Wang,F.,Mao, D.,Peng,P.,Qiao,HH,Wang,X.,Liu,LP,Xu,B.,Ji,JY,Liu,Q.,Sun,J.,Perrimon,N。和Ni,JQ(2018) )。 使用flySAM在 Drosophila 中进行下一代CRISPR / Cas9转录激活。< / a> Proc Natl Acad Sci USA 115(18):4719-4724。&nbsp;
  6. Konermann,S.,Brigham,MD,Trevino,AE,Joung,J.,Abudayyeh,OO,Barcena,C.,Hsu,PD,Habib,N.,Gootenberg,JS,Nishimasu,H.,Nureki,O。和张,F。(2015)。 通过工程CRISPR-Cas9复合物进行基因组规模的转录激活。 Nature 517(7536):583-588。
  7. Lin,S.,Ewen-Campen,B.,Ni,X.,Housden,B.E。和Perrimon,N。(2015)。使用果蝇中的CRISPR / Cas9 体内转录激活。 Genetics 201(2):433-442。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jia, Y., Shen, D., Wang, X., Sun, J., Peng, P., Xu, R., Xu, B. and Ni, J. (2019). flySAM Transgenic CRISPRa System Manual. Bio-protocol 9(2): e3147. DOI: 10.21769/BioProtoc.3147.
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