Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
通过近端dCas9靶向提高CRISPR基因编辑效率   

评审
匿名评审
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Nature Communications
3-Apr 2017

 

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of the chromatin, were also found to impede target DNA access and cleavage by SpCas9 in vitro (Knight et al., 2015; Hinz et al., 2015; Horlbeck et al., 2016; Isaac et al., 2016). Moreover, many CRISPR-Cas systems characterized to date often exhibit inactivity in mammalian cells and are thus precluded from gene editing applications even though they are active in bacteria or on purified DNA substrates. Thus, there is a need to devise a means to alleviate chromatin inhibition to increase gene editing efficiency, especially on difficult-to-access genomic sites, and to enable use of otherwise inactive CRISPR-Cas nucleases for gene editing need. Here we describe a proxy-CRISPR protocol for restoring nuclease activity of various class 2 CRISPR-Cas nucleases on otherwise inaccessible genomic sites in human cells via proximal targeting of a catalytically dead Cas9 (Chen et al., 2017). This protocol is exemplified here by using Campylobacter jejuni Cas9 (CjCas9) as nuclease and catalytically dead SpCas9 (SpdCas9) as proximal DNA binding protein to enable CjCas9 to cleave the target for gene editing using single stranded DNA oligo templates.

Keywords: CRISPR-Cas nuclease (CRISPR-Cas核酸酶), Cas9 (Cas9), dCas9 (dCas9), Cell culture (细胞培养), Transfection (转染), Double strand breaks (双链断裂), Gene editing (基因编辑)

Background

By creating targeted chromosomal DNA double strand breaks (DSBs) or single strand breaks (nicks) or serving as a DNA binding module for other DNA modification effectors, programmable endonucleases have become an important tool for genome modification in eukaryotic cells (Gaj et al., 2013). In response to targeted DNA breaks, host cells can invoke various repair pathways to mend the damages to maintain the genome integrity. Insertions and/or deletions derived from NHEJ repair errors can be capitalized for gene knockout and homologous recombination can be exploited for introducing pre-determined changes on gene of interest by providing a DNA donor. In addition to these more traditional gene editing applications, catalytically inactive forms of programmable endonucleases are increasingly used as DNA binding modules for other DNA modification effectors, such as cytidine deamination enzymes (Komor et al., 2016). However, no matter which forms of programmable nucleases are utilized, target site binding is the prerequisite step and local chromatin structure can determine whether or not or how efficiently a programmable nuclease can bind the target site (Knight et al., 2015; Hinz et al., 2015; Horlbeck et al., 2016; Isaac et al., 2016, Chen et al., 2017). We hypothesize that binding at proximal locations by a programmable DNA binding protein could change the local chromatin structure and render an otherwise inaccessible target site accessible for binding.

Previous generations of programmable nucleases, such as meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), solely rely on protein structure to recognize target sites, and thus re-targeting of these nucleases requires rather laborious protein structural change. In contrast, class 2 CRISPR-Cas effector nucleases use protein structure to recognize a protospacer adjacent motif (PAM) and employ CRISPR RNA (crRNA) to bind the target site adjacent to the PAM. Because PAM is typically a short DNA sequence, such as 5’-NGG-3’ for SpCas9, and thus occurs frequently in a genome, re-targeting of CRISPR-Cas nucleases is a simple process of changing the crRNA sequence by molecular cloning or chemical synthesis. This targeting modality makes CRISPR-Cas systems very suitable for use as nucleases or as DNA binding proteins. This protocol combines these two utilities together to expand CRISPR gene editing capability. The CRISPR-Cas system used as nuclease must be orthogonal to the CRISPR-Cas system used as DNA binding protein to avoid binding site sharing. In general, different subtypes of class 2 CRISPR-Cas systems (e.g., type II-A, type II-B, type II-C, and type V) are orthogonal to one another. Within each subtype, some systems are highly divergent and could be also orthogonal to one another, but they need to be experimentally verified. Currently, it is highly recommended to use SpdCas9 as proximal DNA binding protein, for SpCas9 is the most robust system in mammalian cells to date, although it can also be inactive at certain genomic sites. However, it is anticipated that more robust Cas9 systems will be developed for use as DNA binding proteins.

Materials and Reagents

  1. Pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 2140 , 2149 , 2065E , 2069 , and 2079E )
  2. 75 cm2 U-shaped canted neck cell culture flask with vent cap (Corning, catalog number: 430641U )
  3. Microcentrifuge tubes, 1.5 ml (Sigma-Aldrich, catalog number: T6649 )
  4. Tissue culture plate, 6-well (Corning, Costar®, catalog number: 3516 )
  5. Serological pipettes
    5 ml (Corning, Costar®, catalog number: 4051 )
    10 ml (Corning, Costar®, catalog number: 4488 )
    25 ml (Corning, Costar®, catalog number: 4489 )
  6. Cuvettes
  7. Coverslip
  8. 50-ml conical centrifuge tube (Corning, catalog number: 430828 )
  9. Human K562 cells (ATCC, catalog number: CCL-243 )
  10. Catalytically inactive Streptococcus pyogenes Cas9 (SpdCas9) plasmid DNA (from MilliporeSigma; see Figure 1A)
  11. SpCas9 sgRNA plasmid constructs with the guide sequences 5’-CCAAGGGTGAGGCCGGGAAG-3’ and 5’-CATCTCCCCCATGTACACCT-3’ for binding two human POR target sites (from MilliporeSigma; see Figure 1B)
  12. Campylobacter jejuni Cas9 (CjCas9) plasmid DNA (from MilliporeSigma; see Figure 1C)
  13. CjCas9 sgRNA plasmid construct with the guide sequence 5’-TTCGCCAGTACGAGCTTGTG-3’ for binding a human POR target site (from MilliporeSigma; see Figure 1D)


    Figure 1. Vector maps

  14. Single stranded DNA oligo donor for introducing a diagnostic EcoRI site at the CjCas9 cleavage site in POR. The oligo sequence is: 5’-CACCCTTGGTCTCCCCTTTCCAGCATTCGCCAGTACGAGCGAATTCTTGTGGTCCACACCGACATAGATGCGGCCAAGGTGTACATGG-3’ (Underlined: EcoRI site)
    Note: Synthesize the oligo at 0.2 µmol scale and purify by PAGE. Re-suspend the oligo in 10 mM Tris buffer (pH 7.6) at 200 µM.
  15. PCR primers for amplification of the targeted POR genomic region: forward 5’-CTCCCCTGCTTCTTGTCGTAT-3’, reverse 5’-ACAGGTCGTGGACACTCACA-3’
  16. Alcohol
  17. Hank’s balanced salt solution (Sigma-Aldrich, catalog number: H6648 )
  18. Nucleofector instrument (Lonza, catalog number: AAB-1001 )
  19. Amaxa Cell Line Nucleofector Kit V (Lonza, catalog number: VCA-1003 )
  20. GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, catalog number: NA2010 )
  21. JumpStart Taq ReadyMix (Sigma-Aldrich, catalog number: P2893 )
  22. GenElute PCR Clean-Up Kit (Sigma-Aldrich, catalog number: NA1020 )
  23. EcoRI restriction enzyme (New England Biolabs, catalog number: R0101S )
  24. 10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl (Bio-Rad Laboratories, catalog number: 4561036 )
  25. Tris buffered saline (Sigma-Aldrich, catalog number: T5912 )
  26. Gel loading buffer (Sigma-Aldrich, catalog number: G2526 )
  27. DirectLoad Wide Range DNA Marker (Sigma-Aldrich, catalog number: D7058 )
  28. Iscove’s modified Dulbecco’s medium (Sigma-Aldrich, catalog number: I3390 )
  29. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
  30. L-glutamine, 200 mM (Sigma-Aldrich, catalog number: G7513 )
  31. K562 culture medium (see Recipes)

Equipment

  1. Water bath (Polyscience)
  2. Laminar flow hood for sterile tissue culture, biosafety level 2 approved (Thermo Fisher Scientific)
  3. Microbiological incubator with 37 °C, atmospheric CO2 (Thermo Fisher Scientific)
  4. Pipettes (Gilson)
  5. Bench-top centrifuge (Eppendorf, model: 5417 C )
  6. Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories, catalog number: 1658004 )
  7. PCR thermocycler (Bio-Rad Laboratories, catalog number: 1861096 )

Software

  1. ImageJ (https://imagej.nih.gov/ij/)

Procedure

  1. Target site selection and oligo design for sgRNA expression
    1. Nuclease cleavage site selection will be dependent on gene editing objective. For gene knockout, it is recommended to select cleavage sites in the first two coding exons or in protein functional domains. For gene correction or homologous recombination (HR), the cleavage site should be as close as possible to the nucleotide to be changed or to the target integration site to maximize the efficiency. For cytidine deamination-based editing, the nicking site will be dependent on the cytidine deamination window.
    2. Select a proximal dCas9 binding site upstream and downstream of the cleavage site. The dCas9 binding sites each must be separated from the nuclease binding site by about 10 bp to avoid potential steric hindrance, but beyond the 10 bp separation the dCas9 binding sites also should be kept as close as possible to the nuclease binding site to maximize the effects. The dCas9 binding sites can be on either strand of the DNA.
    3. One of the advantages of CRISPR-Cas effector nucleases over previous generations of programmable nucleases as genome editing tools is the simplicity of nuclease retargeting via straightforward oligo design and sgRNA expression plasmid cloning once target sites are selected. In this illustrative experiment, the two SpdCas9 proximal binding sites are: 5’-CCAAGGGTGAGGCCGGGAAGCGG-3’, and 5’-CAAGGTGTACATGGGGGAGATGG-3’; and the CjCas9 binding site is: 5’-TTCGCCAGTACGAGCTTGTGGTCCACAC-3’; where the underlined nucleotides are protospacer adjacent motifs (PAMs). Therefore, the DNA oligo sequences for sgRNA expression plasmid cloning are: forward 5’-ACCGCCAAGGGTGAGGCCGGGAAG-3’ and reverse 5’-AAACCTTCCCGGCCTCACCCTTGG-3’ for SpdCas9 binding site 1; forward 5’-ACCGCAAGGTGTACATGGGGGAGA-3’ and reverse 5’-AAACTCTCCCCCATGTACACCTTG-3’ for SpdCas9 binding site 2; and forward 5’-ACCGTTCGCCAGTACGAGCTTGTG-3’ and reverse 5’-AAACCACAAGCTCGTACTGGCGAA-3’ for the CjCas9 binding site; where the underlined nucleotides in the forward sequences are a constant overhang for ligation to a human U6 promoter and the underlined nucleotides in the reverse sequences are a constant overhang for ligation to a SpdCas9 sgRNA scaffold or a CjCas9 sgRNA scaffold in cloning vectors.

  2. Transfection and gene editing analysis
    1. Culture K562 cells in Iscove’s modified Dulbecco’s medium, supplemented with 10% FBS and 2 mM L-glutamine. Maintain the culture at 37 °C and 5% CO2. Split the culture every 2-3 days or when the culture reaches 1-2 million cells per ml by transferring 1/10 or 1/5 of the culture to a new culture flask containing fresh medium pre-warmed to 37 °C to make a 1:10 or a 1:5 split (see Note 1).
    2. Split the culture to 0.25 million cells per ml one day before transfection. The culture will reach about 0.5 million cells per ml for transfection the next day (see Note 1).
    3. Prepare three plasmid DNA solutions for transfection in 1.5-ml microcentrifuge tubes (see Note 2):
      1. 5 µg of SpdCas9 plasmid DNA, 4.2 µg of CjCas9 plasmid DNA, 3 µg each of the two SpdCas9 sgRNA plasmid constructs and the CjCas9 sgRNA plasmid construct, and 1.5 µl of 200 µM of single stranded DNA oligo donor.
      2. 4.2 µg of CjCas9 plasmid DNA, 3 µg of the CjCas9 sgRNA plasmid construct, and 1.5 µl of 200 µM of single stranded DNA oligo donor as no proximal dCas9 targeting control.
      3. 1.5 µl of 200 µM of single stranded DNA oligo donor as oligo-only control. Keep the tubes on ice before transfection.
    4. Add 2 ml of fresh culture medium to each well of a 6-well plate and equilibrate the medium at 37 °C and 5% CO2 for at least 20 min before transfection.
    5. Set the Nucleofector program to T-016 (see Note 3). Unseal the cuvettes and disposable transfer pipettes and place them inside a cell culture hood.
    6. Count the cells with a glass hemocytometer:
      1. Clean the hemocytometer and coverslip with alcohol before use.
      2. Gently swirl the flask to make sure the cells are evenly distributed. Take 10 ul of cells and add to the chamber under the coverslip. Cells will be drawn out by capillary action and spread evenly on the hemocytometer.
      3. Count the cells with 10x objective under a microscope. Using a hand tally counter, count the cells in all four corner squares with 16 smaller squares. Only count the cells within a square or on the right-hand or bottom boundary line.
      4. Average the total number by four and then multiply by 10,000 (104). This will be the number of cells per ml in the culture.
    7. Transfer the required amount of cells to a sterile 50-ml conical centrifuge tube. Each transfection reaction will require 1.0 million cells (see Note 4). Centrifuge the cells at 200 x g at room temperature for 5 min. Carefully remove the culture medium by aspiration.
    8. Resuspend the cell pellet in 20 ml of Hank’s balanced salt solution. Centrifuge at 200 x g at room temperature for 5 min and carefully remove the wash fluid by aspiration. Gently re-suspend the cell pellet in nucleofection solution V to 1.0 million cells per 100 µl (see Note 5).
    9. Pipette 100 µl of the cells into a 1.5-ml tube containing a plasmid DNA solution and mix thoroughly but gently by pipetting up and down 3-5 times. Transfer the whole content to a nucleofection cuvette and perform the transfection immediately using the T-016 program.
    10. Add a pipette full of pre-equilibrated culture medium to the transfected cells inside the cuvette with a disposable transfer pipette and immediately transfer the whole content into a well containing pre-equilibrated culture medium. Culture the cells at 37 °C and 5% CO2 immediately after nucleofection.
    11. Extract genomic DNA 3 days after transfection using GenElute Mammalian Genomic DNA Miniprep Kit. PCR amplify the POR genomic region using JumpStart Taq ReadyMix with the forward primer 5’-CTCCCCTGCTTCTTGTCGTAT-3’ and the reverse primer 5’-ACAGGTCGTGGACACTCACA-3’ and the following cycling condition: 98 °C for 2 min for initial denaturation; 34 cycles of 98 °C for 15 sec, 62 °C for 30 sec, and 72 °C for 45 sec; and a final extension at 72 °C for 5 min.
    12. Purify PCR products with GenElute PCR Clean-Up Kit. Digest purified PCR products (about 500 ng per digestion) with EcoRI (20 U) at 37 °C for 2 h. Resolve digestion products on a pre-cast 10% acrylamide gel from Bio-Rad Laboratories using DirectLoad Wide Range DNA Marker as markers. Figure 2 illustrates an example of expected results.


      Figure 2. ssDNA oligo templated gene editing in K562 cells by CjCas9 using proximal dCas9 targeting. A. Campylobacter jejuni Cas9 (CjCas9) and catalytically inactive Streptococcus pyogenes Cas9 (SpdCas9) target sites in the human POR locus and an ssDNA oligo donor carrying a diagnostic EcoRI site. Targets are indicated by bars and PAMs are highlighted in dark blue (CjCas9) and purple (SpdCas9). B. EcoRI digestion analysis of ssDNA oligo templated gene editing. EcoRI site integration efficiency (%) was determined by ImageJ. The two EcoRI restriction fragments are indicated by yellow arrows. The sgRNA numbers correspond to the target numbers in (A). ND, not determined. M, wide-range DNA markers.

Data analysis

Use ImageJ to measure the band intensity for the uncut band and EcoRI digested bands. Sequence integration efficiency (%) is determined by the formula: 100 x (b + c)/(a + b + c), where a is the integrated intensity of the undigested PCR product, and b and c are the integrated intensities of each restriction digestion product.

Notes

  1. When working with adherent cells, split the culture every 2-3 days or when the culture reaches approximately 80% confluency (approximately 80% of the surface is occupied by cells). Split the culture two days before transfection. Seed the culture at about 20% confluency so that the culture will be at about 60-80% confluency at the time of cell preparation for transfection.
  2. Prepare plasmid DNA at a concentration ≥ 2 µg/µl to limit the total volume to ≤ 10 µl for each transfection.
  3. Different cell lines require different nucleofection programs. Follow the instructions from the manufacturer when working with other cell lines.
  4. Up to 2 million K562 cells can be used per nucleofection, but it may reduce the transfection efficiency slightly.
  5. Do not leave cells in the nucleofection solution for a prolonged time (30 min).

Recipes

  1. K562 culture medium
    500 ml Iscove’s modified Dulbecco’s medium
    50 ml fetal bovine serum
    5 ml 200 mM L-glutamine

Acknowledgments

The authors wish to thank Patrick Sullivan for R&D administrative support and the financial support from MilliporeSigma, a business of Merck KGaA, Darmstadt, Germany. This protocol was originally published as part of Chen et al. (2017)

References

  1. Barrangou, R. and Marraffini, L. A. (2014). CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell 54(2): 234-244.
  2. Chen, F., Ding, X., Mei, Y., Seebeck, T., Jiang, Y. and Davis, G. (2017). Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting. Nat Commun 8(14958).
  3. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. and Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121): 819-823.
  4. Gaj, T., Gersbach, C. A. and Barbas, C. F., 3rd (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7): 397-405.
  5. Hinz, J. M., Laughery, M. F. and Wyrick, J. J. (2015). Nucleosomes inhibit Cas9 endonuclease activity in vitro. Biochemistry 54(48): 7063-7066.
  6. Horlbeck, M. A., Witkowsky, L. B., Guglielmi, B., Replogle, J. M., Gilbert, L. A., Villalta, J. E., Torigoe, S. E., Tjian, R. and Weissman, J. S. (2016). Nucleosomes impede Cas9 access to DNA in vivo and in vitro. Elife 5.
  7. Isaac, R. S. Jiang, F., Doudna, J. A., Lim, W. A., Narlikar, G. J. and Almeida, R. (2016). Nulceosome breathing and remodeling constrain CRISPR-Cas9 function. eLife 5.
  8. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816-821.
  9. Knight, S. C., Xie, L., Deng, W., Guglielmi, B., Witkowsky, L. B., Bosanac, L., Zhang, E. T., El Beheiry, M., Masson, J. B., Dahan, M., Liu, Z., Doudna, J. A. and Tjian, R. (2015). Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science 350(6262): 823-826.
  10. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. and Liu, D. R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533(7603): 420-424.
  11. 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.
  12. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V. and Zhang, F. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163(3): 759-771.

简介

集群定期间隔短回归重复(CRISPR)和CRISPR相关(Cas)系统作为细菌和古菌中的适应性免疫系统,用于防御入侵病毒和质粒(Barrangou和Marraffini,2014)。来自某些2类CRISPR-Cas系统的效应核酸酶已被重新用于真核细胞中的异源靶向(Jinek et al。,2012; Cong等人,2013; Mali ,2013; Zetsche等人,2015)。然而,真核生物的基因组环境与CRISPR-Cas系统发展的原核生物的基因组环境有明显的不同。发现哺乳动物异染色质是通过化脓性链球菌Cas9(SpCas9)靶向DNA接近的障碍,并且还发现染色质的基本单位的核小体阻碍了通过SpCas9的靶DNA进入和切割[ (Knight等人,,2015; Hinz等人,2015; Horlbeck等人,2016年) ; Isaac等,,2016)。此外,许多CRISPR-Cas系统的特征是在哺乳动物细胞中经常表现出不活动,因此即使它们在细菌或纯化的DNA底物中是活性的,因此也被排除在基因编辑应用之外。因此,需要设计一种减轻染色质抑制以提高基因编辑效率的方法,特别是在难以接近的基因组位点上,并且能够使用否则无活性的CRISPR-Cas核酸酶进行基因编辑的需要。在这里,我们描述了一种代理CRISPR方案,用于通过催化死亡的Cas9的近靶定位恢复人类细胞中其他不可接近的基因组位点的各种2型CRISPR-Cas核酸酶的核酸酶活性(Chen等人,2017 )。该方案在此通过使用空肠弯曲杆菌Cas9(CjCas9)作为核酸酶和催化死亡的SpCas9(SpdCas9)作为近端DNA结合蛋白,以使得CjCas9能够使用单链DNA寡核苷酸模板切割用于基因编辑的靶标。
【背景】通过产生靶向的染色体DNA双链断裂(DSB)或单链断裂(nicks)或用作其他DNA修饰效应子的DNA结合模块,可编程内切核酸酶已成为真核细胞中基因组修饰的重要工具(Gaj et al。等等,2013)。响应靶向的DNA断裂,宿主细胞可以调用各种修复途径来修复损伤,以维持基因组的完整性。从NHEJ修复错误导出的插入和/或缺失可以用于基因敲除,并且可以利用同源重组来通过提供DNA供体来引入目标基因的预​​先确定的变化。除了这些更传统的基因编辑应用之外,越来越多地使用催化无活性形式的可编程内切核酸酶作为其它DNA修饰效应子的DNA结合模块,例如胞苷脱氨酶(Komor et al。,2016)。然而,无论使用哪种形式的可编程核酸酶,靶位点结合是前提步骤,并且局部染色质结构可以确定可编程核酸酶是否能够有效地结合靶位点(Knight等人 2015年; Hinz等人,2015年; Horlbeck等人,2016; Isaac等人,2016,Chen et al。,2017)。我们假设通过可编程DNA结合蛋白在近端位置的结合可以改变局部染色质结构,并使另一种不可接近的靶位点可用于结合。
 前代的可编程核酸酶,如大范围核酸酶,锌指核酸酶(ZFN)和转录激活物样效应核酸酶(TALENs),仅依靠蛋白质结构来识别靶位点,因此这些核酸酶的重新靶向需要费力的蛋白质结构变化。相比之下,2级CRISPR-Cas效应核酸酶使用蛋白质结构来识别原始相邻基序(PAM),并使用CRISPR RNA(crRNA)结合与PAM相邻的靶位点。因为PAM通常是短的DNA序列,例如用于SpCas9的5'-NGG-3',因此经常在基因组中发生,CRISPR-Cas核酸酶的重新靶向是通过分子克隆来改变crRNA序列的简单过程化学合成。这种靶向模式使CRISPR-Cas系统非常适合用作核酸酶或DNA结合蛋白。该协议将这两个实用程序组合在一起,以扩展CRISPR基因编辑功能。用作核酸酶的CRISPR-Cas系统必须与用作DNA结合蛋白的CRISPR-Cas系统正交,以避免结合位点共享。一般来说,2类CRISPR-Cas系统(例如,II-A型,II-B型,II-C型和V型)的不同亚型彼此正交。在每个子类型中,一些系统是高度发散的,并且也可以彼此正交,但是它们需要经过实验验证。目前,强烈建议使用SpdCas9作为近端DNA结合蛋白,SpCas9是目前哺乳动物细胞中最强大的系统,尽管它也可能在某些基因组位点处无活性。然而,预计将开发更强大的Cas9系统用作DNA结合蛋白。

关键字:CRISPR-Cas核酸酶, Cas9, dCas9, 细胞培养, 转染, 双链断裂, 基因编辑

材料和试剂

  1. 移液器吸头(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2140,2149,2065E,2069和2079E)
  2. 75厘米 2具有通气帽的U形斜颈颈细胞培养瓶(Corning,目录号:430641U)
  3. 微量离心管,1.5 ml(Sigma-Aldrich,目录号:T6649)
  4. 组织培养板,6孔(Corning,Costar ®,目录号:3516)
  5. 血清移液管
    5 ml(Corning,Costar ®,目录号:4051)
    10 ml(Corning,Costar ®,目录号:4488)
    25 ml(Corning,Costar ®,目录号:4489)
  6. 比武杯
  7. 盖子
  8. 50ml锥形离心管(Corning,目录号:430828)
  9. 人类K562细胞(ATCC,目录号:CCL-243)
  10. 催化无活性化脓性链球菌Cas9(SpdCas9)质粒DNA(来自MilliporeSigma;参见图1A)
  11. 具有引导序列5'-CCAAGGGTGAGGCCGGGAAG-3'和5'-CATCTCCCCCATGTACACCT-3'的SpCas9 sgRNA质粒构建体用于结合两个人POR 靶位点(来自MilliporeSigma;参见图1B)
  12. 空肠弯曲杆菌Cas9(CjCas9)质粒DNA(来自MilliporeSigma;参见图1C)
  13. 具有引导序列5'-TTCGCCAGTACGAGCTTGTG-3'的CjCas9 sgRNA质粒构建体用于结合人POR 靶位点(来自MilliporeSigma;参见图1D)


    图1.矢量地图

  14. 用于在POR 中的CjCas9切割位点处引入诊断性EcoRI位点的单链DNA寡核苷酸供体。寡核苷酸序列是:5'-CACCCTTGGTCTCCCCTTTCCAGCATTCGCCAGTACGAGCGAATTC TTGTGGTCCACACCGACATAGATGCGGCCAAGGTGTACATGG-3'(下划线:生态RI位点)
    注意:合成寡核苷酸0.2μmol,通过PAGE纯化。在200μM下将该寡核苷酸重新悬浮于10mM Tris缓冲液(pH 7.6)中
  15. 用于扩增靶基因组区域的PCR引物:正向5'-CTCCCCTGCTTCTTGTCGTAT-3',反向5'-ACAGGTCGTGGACACTCACA-3'
  16. 酒精
  17. 汉克平衡盐溶液(Sigma-Aldrich,目录号:H6648)
  18. Nucleofector仪器(Lonza,目录号:AAB-1001)
  19. Amaxa细胞系Nucleofector Kit V(Lonza,目录号:VCA-1003)
  20. GenElute Mammalian Genomic DNA Miniprep Kit(Sigma-Aldrich,目录号:NA2010)
  21. JumpStart Taq ReadyMix(Sigma-Aldrich,目录号:P2893)
  22. GenElute PCR清洁试剂盒(Sigma-Aldrich,目录号:NA1020)
  23. Eco限制酶(New England Biolabs,目录号:R0101S)
  24. 10%Mini-PROTEAN TGX Precast Protein Gels,15孔,15μl(Bio-Rad Laboratories,目录号:4561036)
  25. Tris缓冲盐水(Sigma-Aldrich,目录号:T5912)
  26. 凝胶加载缓冲液(Sigma-Aldrich,目录号:G2526)
  27. DirectLoad宽范围DNA标记(Sigma-Aldrich,目录号:D7058)
  28. Iscove改良的Dulbecco的培养基(Sigma-Aldrich,目录号:I3390)
  29. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442)
  30. L-谷氨酰胺,200mM(Sigma-Aldrich,目录号:G7513)
  31. K562培养基(参见食谱)

设备

  1. 水浴(Polyscience)
  2. 用于无菌组织培养的层流罩,生物安全2级认证(Thermo Fisher Scientific)
  3. 微生物培养箱,37℃,大气CO 2(Thermo Fisher Scientific)
  4. 移液器(Gilson)
  5. 台式离心机(Eppendorf,型号:5417 C)
  6. Mini-PROTEAN四立式电泳池(Bio-Rad Laboratories,目录号:1658004)
  7. PCR热循环仪(Bio-Rad Laboratories,目录号:1861096)

软件

  1. ImageJ( https://imagej.nih.gov/ij/

程序

  1. sgRNA表达的靶位点选择和寡核苷酸设计
    1. 核酸酶切割位点选择将依赖于基因编辑目标。对于基因敲除,推荐在前两个编码外显子或蛋白质功能结构域中选择切割位点。对于基因校正或同源重组(HR),切割位点应尽可能接近待改变的核苷酸或靶向整合位点以最大化效率。对于基于胞苷脱氨的编辑,切口位置将依赖于胞苷脱氨窗口。
    2. 选择切割位点上游和下游的近端dCas9结合位点。必须将dCas9结合位点与核酸酶结合位点分离约10bp,以避免潜在的位阻,但是超过10bp的分离时,dCas9结合位点也应尽可能保持与核酸酶结合位点尽可能的最大化。 dCas9结合位点可以在DNA的任一条上。
    3. 作为基因组编辑工具,CRISPR-Cas效应物核酸酶的优点是可以通过简单的寡核苷酸设计和sgRNA表达质粒克隆进行核酸酶重新靶向,简化了目标位点。在该说明性实验中,两个SpdCas9近端结合位点是:5'-CCAAGGGTGAGGCCGGGAAGGGG-3'和5'-CAAGGTGTACATGGGGGAGA TGG-3'; CjCas9结合位点为:5'-TTCGCCAGTACGAGCTTGTGGGCCACAC-3';其中下划线的核苷酸是原始相邻基序(PAM)。因此,用于sgRNA表达质粒克隆的DNA寡核苷酸序列是:对于SpdCas9结合位点的正向5'ACCG CCAAGGGTGAGGCCGGGAAG-3'和反向5'-AAACCTTCCCGGCCTCACCCTTGG-3' 1;前向5' - ACCG CAAGGTGTACATGGGGGAA-3'和反向5'-AAAC TCTCCCCCATGTACACCTTG-3'用于SpdCas9结合位点2;并向CjCas9结合位点前向5'-ACCG TTCGCCAGTACGAGCTTGTG-3'和反向5'-AAACCACAAGCTCGTACTGGCGAA-3'其中正向序列中下划线的核苷酸是连接到人U6启动子的恒定突出端,并且逆序列中的下划线的核苷酸是用于连接到克隆载体中的SpdCas9 sgRNA支架或CjCas9 sgRNA支架的恒定突出端。 >
  2. 转染和基因编辑分析
    1. 在Iscove修饰的Dulbecco培养基中培养K562细胞,补充有10%FBS和2mM L-谷氨酰胺。保持37℃和5%CO 2的培养物。每2-3天分开培养一次,或者当培养物将1/10或1/5的培养物转移到含有预热到37℃的新鲜培养基的新培养瓶中时,培养物达到1-2百万个细胞/ ml 1:10或1:5拆分(见注1)。
    2. 在转染前一天将培养物分成25万个细胞/ ml。第二天,文化将达到约50万个细胞/ ml转染(见注1)
    3. 准备三个质粒DNA溶液用于在1.5 ml微量离心管中转染(见注2):
      1. 5μgSpdCas9质粒DNA,4.2μgCjCas9质粒DNA,3μgSpdCas9sgRNA质粒构建体和CjCas9sgRNA质粒构建体,以及1.5μl200μM单链DNA寡核苷酸供体。
      2. 4.2μgCjCas9质粒DNA,3μgCjCas9 sgRNA质粒构建体和1.5μl200μM单链DNA寡核苷酸供体,作为无近端dCas9靶向对照。
      3. 将1.5μl200μM单链DNA寡核苷酸供体作为寡核苷酸对照。转染前将管保持在冰上。
    4. 在6孔板的每个孔中加入2ml新鲜培养基,并在37℃和5%CO 2下平衡培养基至少20分钟。转染前。
    5. 将Nucleofector程序设置为T-016(见注3)。开启比色皿和一次性移液器,将其放入细胞培养罩内。
    6. 用玻璃血细胞计数器计数细胞:
      1. 在使用前用酒精清洁血细胞计数器和盖玻片。
      2. 轻轻旋转烧瓶,确保细胞均匀分布。取10ul细胞,加入盖玻片下的腔室。细胞将通过毛细血管作用引出,并均匀分布在血细胞计数器上
      3. 在显微镜下用10倍物镜计数细胞。使用手提计数器,计数所有四个方格中的单元格,其中16个较小的正方形。只计算一个正方形或右边或底部边界线上的单元格。
      4. 将总数平均为4,然后乘以10,000(10 4 )。这将是培养物中每ml细胞数。
    7. 将所需量的细胞转移到无菌的50ml锥形离心管中。每个转染反应将需要100万个细胞(见注4)。将细胞在室温下以200×g离心5分钟。小心地通过抽吸去除培养基。
    8. 将细胞沉淀重悬于20ml Hank's平衡盐溶液中。在室温下以200g离心5分钟,并通过抽吸小心地除去洗涤液。将核转染溶液V中的细胞沉淀轻轻重新悬浮至每100μl100万个细胞(见注5)。
    9. 将100μl细胞移入含有质粒DNA溶液的1.5 ml管中,充分混匀,轻轻上下移动3-5次。将全部内容转移到核转染细胞比色杯中,并使用T-016程序立即进行转染
    10. 使用一次性转移移液管将装有预平衡培养基的移液管添加到比色杯内的转染细胞上,并立即将全部内容物转移到含有预平衡培养基的孔中。核转染后立即在37℃和5%CO 2培养细胞
    11. 使用GenElute Mammalian Genomic DNA Miniprep Kit转染后3天提取基因组DNA。使用具有正向引物5'-CTCCCCTGCTTCTTGTCGTAT-3'和反向引物5'-ACAGGTCGTGGACACTCACA-3'的JumpStart Taq ReadyMix扩增POR基因组区域,并且以下循环条件:98℃2 min进行初始变性; 98℃15秒,62℃30秒,72℃45秒的34个循环;最后在72℃延伸5分钟。
    12. 用GenElute PCR Clean-Up Kit纯化PCR产物。将消化纯化的PCR产物(每个消化约500ng)与EcoRI(20U)在37℃下反应2小时。使用DirectLoad Wide Range DNA Marker作为标记,从Bio-Rad Laboratories在预制的10%丙烯酰胺凝胶上解决消化产物。图2显示了预期结果的一个例子

      图2.使用近端dCas9靶向的CjCas9在K562细胞中的ssDNA寡聚模板化基因编辑。 :一种。空肠弯曲杆菌Cas9(CjCas9)和催化无活性的化脓性链球菌Cas9(SpdCas9)人POR基因座中的靶位点和携带诊断生态 RI站点。目标由酒吧指示,PAM以深蓝色(CjCas9)和紫色(SpdCas9)突出显示。 B.DNA寡核苷酸模板化基因编辑的RI消化分析。生态环保RI站点集成效率(%)由ImageJ确定。两个生态 RI限制性片段用黄色箭头表示。 sgRNA编号对应于(A)中的目标数。 ND,未确定。 M,广泛的DNA标记。<​​br />

数据分析

使用ImageJ测量未剪切带和Eco RI消化条带的带强度。序列整合效率(%)由以下公式确定:100×(b + c)/(a + b + c),其中a是未消化PCR产物的积分强度,b和c分别为限制性消化产物。

笔记

  1. 当使用贴壁细胞时,每隔2-3天或当培养物达到约80%汇合时(约80%的表面被细胞占据)分解培养物。在转染前两天分裂培养物。以约20%汇合的方式种植培养物,使得在转染细胞准备时培养物将达到约60-80%的汇合度。
  2. 制备浓度≥2μg/μl的质粒DNA,以将每个转染的总体积限制为≤10μl。
  3. 不同的细胞系需要不同的核转染程序。使用其他细胞系时,请遵循制造商的说明。
  4. 每个核转染可以使用多达200万个K562细胞,但可能会稍微降低转染效率。
  5. 不要将细胞留在核转染溶液中长时间(30分钟)。

食谱

  1. K562培养基
    500毫升Iscove修改后的Dulbecco的培养基 50ml胎牛血清
    5ml 200mM L-谷氨酰胺

致谢

作者希望感谢Patrick Sullivan的R&amp; D行政支持以及MilliporeSigma的财务支持,MilliporeSigma是德国达姆施塔特的Merck KGaA的业务。该协议最初是作为Chen等人的一部分发表的。(2017)

参考

  1. Barrangou,R.和Marraffini,LA(2014年)。 CRISPR-Cas系统:原核生物升级至适应性免疫。分子细胞 54(2):234-244。
  2. Chen,F.,Ding,X.,Mei,Y.,Seebeck,T.,Jiang,Y.and Davis,G.(2017)。&nbsp; 针对哺乳动物基因组编辑的不同CRISPR-Cas系统的靶向激活,通过近端CRISPR目标进行。 Nat Commun 8(14958 )。
  3. Cong,L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA and Zhang,F 。(2013)。使用CRISPR / Cas的多重基因组工程系统。 科学 339(6121):819-823。
  4. Gaj,T.,Gersbach,CA和Barbas,CF,3rd(2013)。&nbsp; ZFN,TALEN和基于CRISPR / Cas的基因组工程方法。 Trends Biotechnol 31(7):397-405。
  5. Hinz,JM,Laughery,MF and Wyrick,JJ(2015)。&nbsp;
  6. Horlbeck,MA,Witkowsky,LB,Guglielmi,B.,Replogle,JM,Gilbert,LA,Villalta,JE,Torigoe,SE,Tjian,R。和Weissman,JS(2016)。&lt; a class =插入文件“href =”http://www.ncbi.nlm.nih.gov/pubmed/26987018“target =”_ blank“>核小体阻碍Cas9体内获取DNA和/或体外。 Elife 5.
  7. Isaac,RS Jiang,F.,Doudna,JA,Lim,WA,Narlikar,GJ和Almeida,R。(2016)。&nbsp; Nulceosome呼吸和重塑约束CRISPR-Cas9功能。 eLife 5.
  8. Jinek,M.,Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,JA和Charpentier,E.(2012)。&nbsp; 可编程双RNA引导的DNA内切核酸酶在适应性细菌免疫中的应用。 科学 337(6096) :816-821。
  9. Knight,SC,Xie,L.,Deng,W.,Guglielmi,B.,Witkowsky,LB,Bosanac,L.,Zhang,ET,El Beheiry,M.,Masson,JB,Dahan,M.,Liu,Z 。,Doudna,JA和Tjian,R。(2015)。&nbsp; 活细胞中CRISPR-Cas9基因组询问的动力学。 350(6262):823-826。
  10. Komor,AC,Kim,YB,Packer,MS,Zuris,JA和Liu,DR(2016)。&nbsp; 可编辑基因组DNA中没有双链DNA切割的靶基因。 533(7603):420-424。 />
  11. Mali,P.,Yang,L.,Esvelt,KM,Aach,J.,Guell,M.,DiCarlo,JE,Norville,JE and Church,GM(2013)。&nbsp; 通过Cas9进行RNA指导的人类基因组工程 科学 339( 6121):823-826。
  12. Zetsche,B.,Gootenberg,JS,Abudayyeh,OO,Slaymaker,IM,Makarova,KS,Essletzbichler,P.,Volz,SE,Joung,J.,van der Oost,J.,Regev,A.,Koonin,EV和Zhang,F。(2015)。 Cpf1是一个单一RNA引导的2类CRISPR-Cas系统的内切核酸酶。细胞 163(3):759-771。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, F., Ding, X., Feng, Y., Seebeck, T., Jiang, Y. and Davis, G. D. (2017). Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting. Bio-protocol 7(15): e2432. DOI: 10.21769/BioProtoc.2432.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。