选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除
Summary
遗传操纵体细胞系的能力的最新进展对于基础和应用研究具有巨大的潜力。在这里，我们提出了CRISPR / Cas9在哺乳动物细胞系中产生敲除生产和筛选的两种方法，使用和不使用选择标记。
Abstract
CRISPR / Cas9基因组工程系统通过允许精确的基因组编辑，很少的努力，彻底改变了生物学。在引导特异性的单指导RNA（sgRNA）的指导下，Cas9蛋白在靶位点切割两条DNA链。 DNA断裂可以触发非同源末端连接（NHEJ）或同源性定向修复（HDR）。 NHEJ可以引入导致帧转移突变的小缺失或插入，而HDR允许更大和更精确的扰动。在这里，我们提出了通过将建立的CRISPR / Cas9方法与下游选择/筛选的两个选项相结合来产生敲除细胞系的方案。 NHEJ方法使用单个sgRNA切割位点和选择无关筛选，其中以高通量方式通过点免疫印迹评价蛋白质产生。 HDR方法使用跨越感兴趣基因的两个sgRNA切割位点。与提供的HDR模板一起，该方法可以实现删除几十kb，由插入的可选电阻标记辅助。讨论每种方法的适用应用和优点。
Introduction
稳定的遗传改变比细胞扰动的瞬时方法提供了一个优点，这可以在其效率和持续时间上变化。由于目标特异性核酸酶的发展，如锌指核酸酶1,2,3,4,5，转录激活物样效应核酸酶（TALEN） 6,7,8,9 ，近年来，基因组编辑越来越普及和衍生自聚集的，定期交织的短回文重复（CRISPR）系统的RNA引导的核酸酶10 。
CRISPR / Cas9编辑机器改编自细菌和古菌用于防御病毒感染的免疫系统ass =“xref”> 11，12，13。在这个过程中，入侵病毒序列的短的20-30nt片段被引入到基因组基因座中，作为侧重于重复单元14,15的 “间隔物”。随后的转录和RNA加工产生小的CRISPR相关RNA 16 （crRNA），其与反式激活的crRNA17（tracrRNA）一起与效应物Cas9核酸内切酶组装。因此，crRNA提供对Cas9靶向的特异性，指导复合物切割互补的病毒DNA序列并防止进一步的感染18,19 。目标DNA中的任何“原始孢子”序列都可以作为crRNA的来源，只要它直接与短原型相邻基序（PAM）5'相连，在化脓性链球菌 Cas9 <sup class =“xref”> 20。宿主CRISPR基因座中间隔物附近的PAM序列的缺失区分自身和非自身，防止宿主的靶向。由于其普遍性和灵活性，该生物系统已经被强力地适应于基因组编辑，使得几乎任何PAM相邻的DNA位点都可以被靶向。在该版本中，进一步的修改将crRNA和tracrRNA融合到装载到Cas9蛋白21中的单个引导RNA（sgRNA）组分中。
在真核细胞中Cas9和sgRNA表达后，Cas9蛋白在目标基因座处切割两条DNA链。在没有合适的同源序列区域的情况下，细胞通过非同源末端连接（NHEJ） 22,23,24修复了该断裂，其通常引入小的缺失或很少插入。定位开放时阅读框，修复可能导致产生非功能蛋白质产物的翻译移码。相比之下，当提供具有大的同源区域的外源模板时，细胞可以通过同源性定向修复25,26固定双链断裂。该途径允许在基因组中进行更大的精确的缺失，置换或插入，以及引入可切除的选择标记27 。
在这里，我们提出了通过这两种CRISPR / Cas9方法产生敲除细胞系的方案（ 图1A ）。 NHEJ方法使用单个sgRNA切割位点和选择无关筛选，因此需要很少的前期准备。当使用这种方法时，必须设计指导与最有可能产生敲除的转录物5'末端附近的外显子互补的RNA。自从修改在这种情况下，到基因组的离子量很小，筛选克隆克隆基于斑点印迹，其中以高通量方式评估蛋白质产物。我们使用ELAV样1蛋白（ELAVL1）敲除生产线的生成作为例子。第二种方法依赖于同源性定向修复（HDR），并使用跨越感兴趣的基因或区域的两个sgRNA切割位点，允许几十kb的缺失。在切割位点侧面具有两个具有同源性区域的质粒提供替代模板（ 图1B ），引入可选择的抗性标记，其提高敲除产生效率。该方法也可适用于引入具有适当设计的同源臂的基因修饰。在这种情况下，新的DNA片段的整合允许基于PCR的筛选（ 图1C ）。在这里，我们使用Pumilio RNA结合家族成员2（PUM2）敲除生产线的生成作为例子。
Protocol
Representative Results
Discussion
CRISPR / Cas9系统允许有效地产生稳定的基因组修饰，这提供了与其他瞬时操作方法更一致的替代方案。在这里，我们提出了两种在哺乳动物细胞系中快速鉴定CRISPR / Cas9基因敲除的方法。这两种方法都需要很少的细胞材料，因此可以在克隆培养的早期阶段进行测试，节省时间和试剂。为了提高两种方法的效率，我们建议测试多种sgRNA，因为效率因序列和基因组位置而异。此外，使用多个sgRNA可用于通…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢Gissell Sanchez，Megan Lee和Jason Estep的实验帮助，Weifeng Gu和Xuemei Chen分享试剂。
Materials
| Competent E. coli cells | |||
| plasmid prep kit | |||
| pSpCas9(BB) plasmid | Addgene | 42230 | Ran et al 2013, cloning sgRNAs |
| BbsI enzyme | ThermoFisher | FD1014 | Ran et al 2013, cloning sgRNAs |
| T7 DNA ligase | NEB | M0318L | Ran et al 2013, cloning sgRNAs |
| Tango Buffer | ThermoFisher | BY5 | Ran et al 2013, cloning sgRNAs |
| PlasmidSafe exonuclease | Epicentre | E3105K | Ran et al 2013, cloning sgRNAs |
| Q5 hot start high fidelity polymerase | NEB | M0494A | HA amplification |
| pUC19-BsaI | Modified pUC19 plasmid, mutated existing BsaI site and inserted two outward facing BsaI sites after BamHI/EcoRI digestion | ||
| pGolden-Neo | Addgene | 51422 | Resistance cassette |
| pGolden-Hygro | Addgene | 51423 | Resistance cassette |
| BsaI enzyme | NEB | R3535S | Homology arm contruction |
| T7 DNA ligase | NEB | M0318L | |
| PlasmidSafe exonuclease | Epicentre | E3105K | |
| Toothpicks | bacterial PCR | ||
| Taq polymerase | bacterial colony PCR | ||
| HEK293 human cells | |||
| DMEM | Corning | 10-013-CV | |
| FBS | Corning | MT35010CV | |
| Penicillin-Strep (opt.) | Gibco | 15140-122 | |
| 6 well plates | BioLite | 12556004 | |
| TransIT-LTI Transfection Reagent | Mirus | MIR2300 | for lipofection only |
| Opti-MEM | ThermoFisher | 31985062 | for lipofection only |
| G418 (Neomycin) | Sigma Aldrich | A1720-5G | |
| Hygromycin | Sigma Aldrich | H3274-250MG | |
| 96 well plates | ThermoFisher | 12556008 | |
| Passive Lysis Buffer, 5x | Promega | ||
| 1xSDS loading buffer | recipe decribed in protocol | ||
| Nitrocellulose Membrane | Bio-Rad | 162-0115 | |
| TBST | recipe decribed in protocol | ||
| Dehydrated milk | |||
| SuperSignal West Dura Extended Duration Substrate | ThermoFisher | 34075 | for HRP-conjugated secondary antibodies |
| Extracta DNA prep for PCR | Quantabio | 95091-025 | |
| KOD polymerase | Novagen | 71316 |
References
- Carroll, D. Genome engineering with zinc-finger nucleases. 유전학. 188 (4), 773-782 (2011).
- Kim, Y. G., Cha, J., Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A. 93 (3), 1156-1160 (1996).
- Bibikova, M., et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol. 21 (1), 289-297 (2001).
- Bibikova, M., Golic, M., Golic, K. G., Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. 유전학. 161 (3), 1169-1175 (2002).
- Porteus, M. H., Cathomen, T., Weitzman, M. D., Baltimore, D. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol Cell Biol. 23 (10), 3558-3565 (2003).
- Bogdanove, A. J., Voytas, D. F. TAL effectors: customizable proteins for DNA targeting. Science. 333 (6051), 1843-1846 (2011).
- Miller, J. C., et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 29 (2), 143-148 (2011).
- Moscou, M. J., Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science. 326 (5959), 1501 (2009).
- Christian, M., et al. Targeting DNA double-strand breaks with TAL effector nucleases. 유전학. 186 (2), 757-761 (2010).
- Jiang, W., Marraffini, L. A. CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems. Annu Rev Microbiol. 69, 209-228 (2015).
- Barrangou, R., et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 315 (5819), 1709-1712 (2007).
- Karginov, F. V., Hannon, G. J. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol Cell. 37 (1), 7-19 (2010).
- Wiedenheft, B., Sternberg, S. H., Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 482 (7385), 331-338 (2012).
- Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J., Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 60 (2), 174-182 (2005).
- Pourcel, C., Salvignol, G., Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 151 (Pt 3), 653-663 (2005).
- Brouns, S. J., et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 321 (5891), 960-964 (2008).
- Deltcheva, E., et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 471 (7340), 602-607 (2011).
- Garneau, J. E., et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 468 (7320), 67-71 (2010).
- Marraffini, L. A., Sontheimer, E. J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 322 (5909), 1843-1845 (2008).
- Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 109 (39), E2579-E2586 (2012).
- Jinek, M., et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337 (6096), 816-821 (2012).
- Davis, A. J., Chen, D. J. DNA double strand break repair via non-homologous end-joining. Transl Cancer Res. 2 (3), 130-143 (2013).
- Guirouilh-Barbat, J., et al. Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells. Mol Cell. 14 (5), 611-623 (2004).
- Moore, J. K., Haber, J. E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol. 16 (5), 2164-2173 (1996).
- Liang, F., Han, M., Romanienko, P. J., Jasin, M. Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc Natl Acad Sci U S A. 95 (9), 5172-5177 (1998).
- Gratz, S. J., et al. Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. 유전학. 196 (4), 961-971 (2014).
- Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339 (6121), 819-823 (2013).
- Engler, C., Kandzia, R., Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. 3 (11), e3647 (2008).
- Ran, F. A., et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 8 (11), 2281-2308 (2013).
- Luo, Y., Lin, L., Bolund, L., Sorensen, C. B. Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning. Biotechniques. 56 (5), 263-268 (2014).
- Green, M. R., Sambrook, J., Sambrook, J. . Molecular cloning: a laboratory manual. , (2012).
- Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife. 3, e04766 (2014).
- Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24 (6), 1012-1019 (2014).
