腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递
Summary
在这里, 我们提供了一个详细的协议, 以进行体内心脏基因编辑的小鼠使用重组腺相关病毒 (rAAV) 介导的 CRISPR 交付。本协议为治疗杜氏肌营养不良症营养不良性型心肌病提供了一种有前景的治疗策略, 可用于产后小鼠心脏特异性击倒。
Abstract
群集, 定期 interspaced, 短, 回文重复 (CRISPR) 系统大大促进了基因组工程的培养细胞和生物体的各种物种。CRISPR 技术也被探索为一些人类疾病的新疗法。概念证明数据是非常鼓舞人心的例子, 最近的研究表明, 基因编辑的治疗方法的可行性和有效性的杜氏肌营养不良症 (DMD) 使用小鼠模型。特别是, 静脉注射和腹腔注射重组腺相关病毒 (rAAV) 血清 rh 74 (rAAVrh 74) 已使有效的心脏交付金黄色葡萄球菌CRISPR 相关蛋白 9 (SaCas9) 和两个引导 rna (gRNA) 删除基因组区域与突变密码子的小白鼠Dmd基因23。这同样的方法也可以用来敲出的兴趣基因, 并研究他们的心脏功能的产后小鼠时, 当 gRNA 是针对编码区域的基因。在本协议中, 我们详细介绍了如何 rAAVrh 74-CRISPR 载体的设计, 以及如何在新生小鼠中实现高效的心脏分娩。
Introduction
集群, 定期 interspaced, 短回文重复 (CRISPR) 技术代表了最新的基因组工程学的进步, 并改变了目前的基因在细胞和有机体的做法。基于 CRISPR 的基因组编辑利用一个单一的指南 RNA (gRNA) 来指导 CRISPR 相关的限制性, 如 CRISPR 相关蛋白 9 (Cas9) 和 Cpf1 的基因组 DNA 目标, 这是互补序列的 protospacer 编码由 gRNA 与具体 protospacer 相邻母题 (PAM)1,2。PAM 的变化取决于 Cas9 或 Cpf1 基因的细菌种类。从化脓链球菌(SpCas9) 中提取的最常用的 Cas9 核酸酶识别出一个 PAM 序列, 它直接位于基因组 DNA 中的目标序列下游, 非靶链上。在目标地点, Cas9 和 Cpf1 蛋白产生双链断裂 (争端), 然后通过内部细胞 DNA 修复机制 (包括非同源端连接 (NHEJ) 和同源定向修复 (HDR) 路径3) 进行修复, 4。在缺乏同源重组的 DNA 模板的情况下, 争端发生者主要是由容易出错的 NHEJ 途径修复的, 这可能导致在编码中创建了 frameshift 突变的核苷酸的插入或删除 (indels)。基因的区域5,6。因此, CRISPR 系统已被广泛用于设计各种细胞模型和整个生物体的功能损失突变, 以研究基因的功能。此外, 催化不活跃的 Cas9 和 Cpf1 已经发展到转录和 epigenetically 调节目标基因的表达7,8。
最近, SpCas9 和 SaCas9 (来源于金黄色葡萄球菌) 已被包装成重组腺相关病毒 (rAAV) 载体的产后基因矫正的动物模型的人类疾病, 特别是杜氏肌肉萎缩症 (DMD)9,10,11,12,13,14,15。dmd 是一种致命的 X 连锁隐性疾病, 由dmd基因突变引起, 它编码肌营养不良蛋白16,17, 影响约1/3500 男性出生根据新生儿筛查18,19. 它的特点是渐进性肌肉虚弱和浪费。DMD 患者通常失去在10岁到12岁之间行走的能力, 在20-30 岁20岁时死于呼吸和/或心力衰竭。值得注意的是, 心肌病在 > 90% 的 dmd 患者中发展, 代表了 dmd 患者21,22的主要死因。虽然 Deflazacort (一种抗炎药) 和 Eteplirsen (一个外显子跳过医学为跳过外显子 51) 最近已批准的 DMD 由食品和药物管理局 (FDA)23,24, 这些治疗没有纠正基因组 DNA 水平的遗传缺陷。在肌萎缩蛋白基因的外显子23上进行点突变的 mdx 鼠标被广泛用作 DMD 模型25。此外, 我们以前证明, 功能性肌萎缩蛋白表达恢复的框架内删除的基因组 DNA 覆盖外显子 21, 22 和23在骨骼肌的mdx小鼠使用肌内 Ad-SpCas9/gRNA 分娩9, 在mdx/utrophin+/- 小鼠的心脏肌肉中使用静脉注射和腹腔 rAAVrh. 74-SaCas9/gRNA 交付26。
在本议定书中, 我们详细描述了产后心脏基因编辑的每一个步骤, 以恢复mdx/utrophin+/- 小鼠的肌萎缩蛋白, 使用 rAAVrh. 74-SaCas9/gRNA 载体, 从 gRNA 设计到肌萎缩蛋白分析在心肌切片。
Protocol
Representative Results
Discussion
在本议定书中, 我们详细介绍了通过 rAAVrh 在产后小鼠体内实现心脏基因编辑的所有必要步骤. 74-介导的 SaCas9 和两 gRNAs 的交付。如前所述, 这种方法可用于恢复营养不良性小鼠肌萎缩蛋白的表达, 如我们的工作27中所描述的, 但它也可以使小鼠心脏相关基因的快速反向遗传学研究, 而不需要冗长的过程传统的挖空方法来生成和繁殖挖空鼠标模型。
为了在?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
右侧得到美国国立卫生研究院 (R01HL116546 和 R01 AR064241) 的支持。
Materials
| Alexa Fluor 555 goat anti-rabbit IgG | Thermo Fisher Scientific | A-21428 | |
| Benzonase Nuclease | Sigma-Aldrich | E1014 | |
| Bio Safety Cabinets | Thermo Fisher Scientific | 1347 | 1300 Series A2 Class II, Type A2 |
| BsaI | New England BioLabs Inc | RO535S | |
| Competent cells | Agilent Technologies | 200314 | |
| Cell culture dishes, 150mm | Nest Scientific USA | 715001 | |
| Cryostat | Leica Biosystems | Leica CM3050S | |
| DMEM | Thermo Fisher Scientific | MT10017CV | Corning cellgro |
| Dystrophin antibody | Spring Bioscience | E2660 | |
| Fast-Ion Midi Plasmdi Kits | IBI Scientific | IB47111 | |
| FBS | Thermo Fisher Scientific | 26-140-079 | |
| Filter Unit, 0.45μm | Thermo Fisher Scientific | 166-0045 | |
| GoTaq Master Mixes | Promega | M7122 | |
| High-speed plasmid mini kit | IBI Scientific | IB47102 | |
| Horse serum | Abcam | ab139501 | |
| Isotemp 2239 Water Bath | Thermo Fisher Scientific | 15-460-11Q | |
| Isotemp Heat Block | Thermo Fisher Scientific | 11-718 | |
| Inverted confocal microscope | Carl Zeiss Microscopy, LLC | LSM 780 | |
| LightCycler 480 instrument | Roche | 5015243001 | LightCycler 480 Instrument II |
| Large Capacity Centrifuge | Thermo Fisher Scientific | 46-910 | Thermo Scientific Sorvall RC 6 Plus |
| Microcentrifuge | Thermo Fisher Scientific | 75002445 | Sorvall Legend Micro 21R |
| Nanodrop spectrophotometers | Thermo Scientific | ND2000CLAPTOP | NanoDrop™ 2000/2000c |
| Oligos for i20-F | Integrated DNA Technologies | CACCgGGCGTTGAAATTCTCATTAC | |
| Oligos for i20-R | Integrated DNA Technologies | AAAC GTAATGAGAATTTCAACGCCc; | |
| Oligos for i23-F | Integrated DNA Technologies | CACCgCACCGATGAGAGGGAAAGGTC | |
| Oligos for i23-R | Integrated DNA Technologies | GACCTTTCCCTCTCATCGGTGc | |
| Oligos | Integrated DNA Technologies | ||
| OptiPrep Density Gradient Medium | Sigma-Aldrich | D1556-250ML | |
| OptiMEM | Thermo Fisher Scientific | 31985070 | |
| PEG8000 | Sigma-Aldrich | 89510-1kg-F | |
| Polyethylenimine | Polysciences | 23966 | |
| Proteinase K | Sigma-Aldrich | P2308 | |
| Quick-Seal centrifuge tube | Beckman Coulter Inc | 342414 | |
| QIAquick Gel Extraction kit | Qiagen | 28704 | |
| Radiant SYBR Green Hi-ROX qPCR Kits | Alkali Scientific | QS2005 | |
| RevertAid RT Reverse Transcription Kit | Thermo Fisher Scientific | K1691 | |
| Rotor | Beckman Coulter Inc | 337922 | Type 70Ti |
| T4 DNA ligase | New England BioLabs Inc | M0202S | |
| Thermal Cycler | Bio-rad | 1861096 | |
| TRIzol Reagent | Thermo Fisher Scientific | 15596026 | |
| Ultracentrifuge | Beckman Coulter Inc | 8043-30-1192 | Optima le-80k |
| Ultra centrifugal filter unit | MilliporeSigma | UFC510096 | |
| VECTASHIELD Mounting Medium with DAPI | Vector Laboratories, Inc | H-1200 |
References
- Makarova, K. S., Koonin, E. V. Annotation and Classification of CRISPR-Cas Systems. Methods Mol Biol. 1311, 47-75 (2015).
- Zetsche, B., et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 163, 759-771 (2015).
- Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339, 819-823 (2013).
- Mali, P., et al. RNA-guided human genome engineering via Cas9. Science. 339, 823-826 (2013).
- Lieber, M. R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annual review of biochemistry. 79, 181-211 (2010).
- Ran, F. A., et al. Genome engineering using the CRISPR-Cas9 system. Nature protocols. 8, 2281-2308 (2013).
- Zhang, X., Wang, J., Cheng, Q., Zheng, X., Zhao, G. Multiplex gene regulation by CRISPR-ddCpf1. Cell discovery. 3, 17018 (2017).
- Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., Yang, S. H. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Molecular therapy. Nucleic acids. 4, e264 (2015).
- Xu, L., et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol Ther. 24, 564-569 (2015).
- Long, C., et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 351, 400-403 (2016).
- Long, C. Z., et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 345, 1184-1188 (2014).
- Nelson, C. E., et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 351, 403-407 (2016).
- Ousterout, D. G. K., Thakore, A. M., Majoros, P. I., Reddy, T. E. W. H., Gersbach, C. A. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun. 6, 6244 (2015).
- Tabebordbar, M., et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 351, 407-411 (2016).
- Bengtsson, N. E., et al. Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nat Commun. 8, 14454 (2017).
- Hoffman, E. P., Brown, R. H., Kunkel, L. M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 51, 919-928 (1987).
- Nowak, K. J., Davies, K. E. Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment. EMBO reports. 5, 872-876 (2004).
- Mendell, J. R., et al. Evidence-based path to newborn screening for Duchenne muscular dystrophy. Annals of neurology. 71, 304-313 (2012).
- Mendell, J. R., Lloyd-Puryear, M. Report of MDA muscle disease symposium on newborn screening for Duchenne muscular dystrophy. Muscle Nerve. 48, 21-26 (2013).
- Spurney, C. F. Cardiomyopathy of Duchenne muscular dystrophy: current understanding and future directions. Muscle Nerve. 44, 8-19 (2011).
- Moriuchi, T., Kagawa, N., Mukoyama, M., Hizawa, K. Autopsy analyses of the muscular dystrophies. Tokushima J Exp Med. 40, 83-93 (1993).
- McNally, E. M. New approaches in the therapy of cardiomyopathy in muscular dystrophy. Annual review of medicine. 58, 75-88 (2007).
- Baker, D. E. Approvals, Submission, and Important Labeling Changes for US Marketed Pharmaceuticals. Hosp Pharm. 52, 306-307 (2013).
- Baker, D. E. Eteplirsen. Hosp Pharm. 52, 302-305 (2017).
- Sicinski, P., et al. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 244, 1578-1580 (1989).
- Xu, L., et al. CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice. Molecular therapy : the journal of the American Society of Gene Therapy. 24, 564-569 (2016).
- El Refaey, M., et al. In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice. Circ Res. 121, 923-929 (2017).
- Tabebordbar, M., et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 351, 407-411 (2016).
- Pozsgai, E. R., Griffin, D. A., Heller, K. N., Mendell, J. R., Rodino-Klapac, L. R. beta-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice. Gene therapy. 23, 57-66 (2016).
- Chicoine, L. G., et al. Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery. Mol Ther. 22, 338-347 (2014).
- Chicoine, L. G., et al. Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin alpha2 surrogates. Mol Ther. 22, 713-724 (2014).
- Vouillot, L., Thelie, A., Pollet, N. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3. 5, 407-415 (2015).
- Tsai, S. Q., et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 33, 187-197 (2015).
