右侧得到美国国立卫生研究院 (R01HL116546 和 R01 AR064241) 的支持。

本议定书中使用的动物是根据动物使用指南在俄亥俄州立大学实验室动物资源中保存的。所有动物研究都由俄亥俄州立大学的机构动物保育、使用和审查委员会授权。
1. gRNAs CRISPR 载体的设计与克隆
<ol><li>选择 2 gRNAs 靶向肌营养不良蛋白内含子20和内含子 23 (i20 和 i23) 为 SaCas9: i20: 5 '-GGGCGTTGAAATTCTCATTAC CAGAGT和 i23: 5 '-CACCGATGAGAGGGAAAGGTC CTGAAT (PAM 序列带下划线和斜体)。 注: i20 和 i23 的目标地点约为23公斤基对 (kbp)。为了扰乱编码基因, 推荐两个 gRNAs 以 NNGRRT PAM 序列为 SaCas9 (优选 NNGAAT、NNGGGT 和 NNGAGT) 和距离在 20 kbp 内的共同外显子。</li>
	<li>使用以下 PCR 参数在1x 退火缓冲器中退火 gRNA 寡核苷酸 (100 µmol/升) (5x 退火缓冲液中含有0.5 摩尔/升 K 醋酸盐, 150 毫摩尔/升 HEPES, 10 毫摩尔/升镁乙酸酯, pH7.4):95 °c 10 分钟, 90 个周期95到59°c 以0.4 °c 减少每周期为二十年代, 90 周期59到32°c 以0.3 °c 减退每周期为二十年代, 20 个周期32到26°c 以0.3 °c 减退每周期为二十年代。</li>
	<li>结扎退火的 gRNA 寡核苷酸 pX601-CMV-SaCas9-mPA-U6-gRNA 使用 BsaI 和 T4 DNA 连接酶在一个反应 (1 µL 50 ng/µL pX601-CMV-SaCas9-mPA-U6-gRNA, 1 µL 退火寡核苷酸, 1.5 µL 10x T4 dna 连接酶缓冲, 0.5 µL T4 dna 连接酶, 1 µL BsaI, 10 µL ddH2O), 以以下反应情况在 PCR 循环仪: 5 周期 [37 °c 5 分钟和16°c 10 分钟];37°c 20 分钟;80°c 5 分钟。</li>
	<li>将15µL 结扎产品转化为30µL 合格细胞, 将琼脂板中的细胞与100µg/毫升氨苄西林结合, 并以37摄氏度为 24 h。</li>
	<li>在100µg/毫升氨苄西林在37摄氏度, 250 rpm 的5-10 个殖民地和文化, 在一个振动筛孵化器为 3-4 h。</li>
	<li>通过 pcr 筛选菌落:10 µL 2x pcr 大师组合, 8 µL ddH2O, 1 µL 大肠杆菌培养, 1 µL 10 µmol/L U6-forward 底漆, 1 µL i20 或 i23 gRNA 反向底漆。PCR 循环参数:95 °c 5 分钟, 35 周期95°c 三十年代, 61 °c 三十年代, 72 °c 三十年代。</li>
	<li>通过加入4毫升新鲜的 LB 培养基 containing100 µg/毫升氨苄西林, 并在37摄氏度, 250 转每夜培养5毫升的阳性克隆培养。</li>
	<li>用合适的质粒提取试剂盒纯化质粒 DNA, 用 U6-forward 底漆测定质粒。</li>
	<li>DMEM 的培养 C2C12 细胞补充10% 只血清和1% 支笔链球菌, 直到达到70% 融合, 以检测质粒的基因编辑功效。</li>
	<li>根据制造商的协议, Electroporate 共5µg SaCas9/gRNA 质粒混合物1:1 摩尔比 1 x 106 C2C12 细胞。</li>
	<li>经过48小时后转染, 收获 C2C12 细胞和提取基因组 DNA。</li>
	<li>用 100 ng 基因组 DNA 作为模板对小鼠肌营养不良部位进行 PCR 反应: 前底漆 5 '-GGCCAAAGCAAACTCTGGTA (1 µL 10 µmol/升), 反向底漆 5 '-TTTAATCCCACGTCATGCAA (1 µL 10 µmol/升), 10 µL 2x 绿色 PCR 主混合, 7 µL ddH2O, 1 µL 基因组 DNA (100吴)。使用 PCR 循环条件作为95°c 5 分钟, 35 周期 [95 °c 三十年代, 61 °c 三十年代, 72 °c 三十年代], 72 °c 2 分钟。</li>
</ol>2. rAAV 生产
<ol><li>培养细胞, 种子 AD293 细胞到20到40的15厘米菜肴和维持在 DMEM 补充10% 血清和1% 笔链球菌, 直到达到 ~ 90% 融合转染。</li>
	<li>稀释3粒, 200 µL 降低血清培养基 (每盘): 1) 10 µg pHelper;2) 5 µg pXR (rh 74 或其他血清型) 和 3) 共5µg pX601-i20 和 pX601-i23 混合物 (1:1 比)。聚乙烯亚胺 (裴) 与80µL 的混合和在室温下孵育10分钟进行转染. 以滴状的方式将混合物添加到 AD293 细胞培养基上。</li>
	<li>对于 AAV 病毒的生产, 72 小时后转染后, 使用一个细胞刮刀收集的媒体和 AD293 细胞颗粒离心在 1100 x g 10 分钟。</li>
	<li>并用重悬15毫升 rAAV 裂解缓冲液中的细胞颗粒 (50 毫摩尔/升三基, 150 毫摩尔/升氯化钠, pH 8.5), 其次为3个冻融循环, 在液氮和42°c 水浴中交替孵化。如果不立即处理, 则将单元格裂解物存储在-80 摄氏度内以供将来处理。</li>
	<li>用0.45 µm 过滤器过滤培养基, 并在2.5 摩尔/升氯化钠中添加 40% PEG8000, 最后浓度为8%。将混合物在4摄氏度孵育1小时, 离心机在 1100 x g 处孵化15分钟。</li>
	<li>并用重悬 rAAV 裂解缓冲液中的颗粒, 并与步骤2.3 中的细胞裂解物结合, 其次是 benzonase (50 U/毫升) 治疗37°c 1 小时。</li>
	<li>离心机 benzonase 处理的 rAAV 含有混合物 1100 x g 15 分钟4摄氏度, 并保存上清 (粗 rAAV 制剂)。</li>
</ol>3. rAAV 纯化和浓缩
<ol><li>按以下顺序将粗 rAAV 制备到 ultracentrifuge 管的 iodixanol 梯度上: 粗 AAV 裂解 (~ 15 毫升), 8 毫升 15% iodixanol, 6 毫升 25% iodixanol, 5 毫升 40% iodixanol, 5 毫升 58% iodixanol。使用 1x DPBS 将管的剩余空容积填满顶部。</li>
	<li>离心样品在 23万 x g 在 ultracentrifuge 转子120分钟在10°c 和收集 3-4 毫升的40% 梯度分数包含 rAAV 微粒与 18 g 针。</li>
	<li>用离心过滤装置 (截留分子量 100 kDa) 稀释 1x DPBS 和离心机的 rAAV 颗粒。重复此步骤3-5 次, 最后将 rAAV 准备工作集中到300-500 µL。</li>
</ol>4. rAAV Titering
<ol><li>从2µL 浓缩 rAAV 中提取基因组 DNA, 在乙醇中沉淀3米 NaOAc, 并用重悬20µL ddH2O。</li>
	<li>通过 pX601 质粒的串联稀释获得 rAAV 定量的标准曲线: 1 x 109, 1 x 108,1 x 107, 1 x 106, 1 x 105拷贝在每10µL ddH2o 稀释 DNAase 处理 rAAV样本为1:10、1:50、1:100 和1:1000。</li>
	<li>用 qPCR 试剂盒定量实时 pcr (qPCR) 测定 rAAV 效价根据制造商在实时 pcr 系统中的指示: 1 µL 10 µmol/升每个底漆 (5 '-GGATTTCCAAGTCTCCACCC 和 5 '-TCCCACCGT ACACGCCTAC), 5 µL 2x PCR 大师组合, 1 µL 稀释rAAV 样品和2µL ddH2o 用以下循环参数运行反应:95 °c 3 分钟, 45 个周期 [95 °c 5 秒和61°c 20 秒]。</li>
</ol>5. 静脉和腹腔注射 rAAVrh 74-CRISPR 入新生儿mdx/utrophin+/鼠
<ol><li>将mdx/utrophin+/ 鼠标幼崽 (3 天) 通过放置在冰上1分钟, 然后通过一个复古轨道方法或腹腔注射, 在12µL 中使用 1 x 1050病毒粒子进行系统管理。</li>
	<li>让老鼠恢复并回到他们的笼子里。在 rAAV 管理十周后, 弄死对小鼠进行组织收集的联合2的接触。</li>
	<li>用于基因组 DNA 和 RNA 提取的快速冷冻组织, 利用液氮中的冷却异戊烷, 以最佳的切削温度化合物 cryosectioning 冷冻组织。</li>
</ol>6. 目标基因编辑结果分析
<ol><li>
基因组 DNA PCR 分析
	<ol>
<li>用或不 rAAV 治疗的mdx/utrophin+/- 小鼠的心脏组织中分离出总基因组 DNA。</li>
		<li>在步骤1.12 中使用相同的协议进行 PCR 分析。</li>
	</ol>
</li>
	<li>
抗肌营养不良蛋白表达的 rt-pcr 分析
	<ol>
<li>根据制造商手册, 从心脏组织提取总 rna 和 rna 提取试剂盒。</li>
		<li>要进行反向转录, 前处理总 RNA 与无 RNase DNase, 并使用5µg 处理 rna 作为模板的第一链 cDNA 合成与反向转录试剂盒。</li>
		<li>用反转录产物对肌营养不良蛋白的表达进行 rt-pcr 分析: 5 '-GGCTAGAGTATCAAACCAACATCAT 和 5 '-TGGAGGCTTACGGTTTTATCC。</li>
	</ol>
</li>
	<li>
qPCR 分析评价基因编辑效能
	<ol>
<li>应用正向底漆 (外显子 21): 5 '-TTGTCAGCTCTTCAGCCTCAAA, 反向底漆 (外显子 23), 用定量 rt-pcr 方法估计基因编辑的功效, 以检测外显子22包含转录的减少: 5 '-AAACTCTCCTTCACAGTGTCACT。使用 Gapdh 作为参考基因与向前底漆: 5 '-AGGTTGTCTCCTGCGACTTC 和反向底漆: 5 '-GGTGGTCCAGGGTTTCTTACT。</li>
		<li>根据制造商在实时 pcr 系统中的指令和2步反应条件, 使用 qPCR 套件运行定量实时 pcr (qPCR):95 °c 3 分钟, 45 循环95°c 5 秒和61°c 30 秒。</li>
	</ol>
</li>
	<li>
免疫荧光染色分析肌萎缩蛋白的表达
	<ol>
<li>用恒温器在10µm 的厚度上切开冷冻组织. 用4% 多聚甲醛在室温下固定冰冻切片15分钟。</li>
		<li>洗涤2x 与 PBS 和孵化以阻拦的解答 (10% 马血清) 为 1 h。</li>
		<li>孵化抗肌营养不良蛋白原抗体, 在4摄氏度过夜。</li>
		<li>用 PBS 3 x 5 分钟冲洗幻灯片, 室温下用荧光二次抗体 (1:500) 孵育1小时。</li>
		<li>使用安装介质与 DAPI 和成像的倒置共聚焦显微镜登上幻灯片。</li>
	</ol>
</li>
</ol>7. T7E1 试验的非靶向分析
<ol><li>使用在线 CRISPR 设计工具 (如 < http://crispr.mit.edu >) 预测潜在的非目标站点。</li>
	<li>通过 pcr 扩增覆盖前5-10 个潜在靶点的基因组区域: 基因组 dna 200, 1 µL 高保真 dna 聚合酶, 5 µL 10x PCR 缓冲混合, 1.5 µL 10 µmol/升的现场特定正向和反向引物, 调整总体积到50µL 与ddH2O。</li>
	<li>用凝胶萃取试剂盒, 通过电泳、提取和纯化 DNA 来运行 PCR 产品。使用光度计测量浓度。</li>
	<li>利用 PCR 循环仪在缓冲2.1 中缓慢地通过变性和重新退火纯化的 amplicons 来促进 heteroduplex 的形成。使用步骤1.2 中详细说明的相同循环条件。</li>
	<li>用0.5 µL T7E1 酶对30分钟的再退火产物进行消化, 并用1-2% 琼脂糖凝胶对电泳反应进行了分析。 注: amplicons 还可以通过目标深度测序进行分析。</li>
</ol>

<ol>
	<li>Makarova, K. S., Koonin, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annotation+and+Classification+of+CRISPR-Cas+Systems.">Annotation and Classification of CRISPR-Cas Systems.</a> Methods Mol Biol. 1311, 47-75 (2015).</li><li>Zetsche, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cpf1+is+a+single+RNA-guided+endonuclease+of+a+class+2+CRISPR-Cas+system.">Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.</a> Cell. 163, 759-771 (2015).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339, 819-823 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Lieber, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+double-strand+DNA+break+repair+by+the+nonhomologous+DNA+end-joining+pathway.">The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway.</a> Annual review of biochemistry. 79, 181-211 (2010).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature protocols. 8, 2281-2308 (2013).</li><li>Zhang, X., Wang, J., Cheng, Q., Zheng, X., Zhao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+gene+regulation+by+CRISPR-ddCpf1.">Multiplex gene regulation by CRISPR-ddCpf1.</a> Cell discovery. 3, 17018 (2017).</li><li>Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., Yang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-target+Effects+in+CRISPR/Cas9-mediated+Genome+Engineering.">Off-target Effects in CRISPR/Cas9-mediated Genome Engineering.</a> Molecular therapy. Nucleic acids. 4, e264 (2015).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-mediated+genome+editing+restores+dystrophin+expression+and+function+in+mdx+mice.">CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice.</a> Mol Ther. 24, 564-569 (2015).</li><li>Long, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+genome+editing+partially+restores+dystrophin+expression+in+a+mouse+model+of+muscular+dystrophy.">Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.</a> Science. 351, 400-403 (2016).</li><li>Long, C. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+muscular+dystrophy+in+mice+by+CRISPR/Cas9-mediated+editing+of+germline+DNA.">Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.</a> Science. 345, 1184-1188 (2014).</li><li>Nelson, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome+editing+improves+muscle+function+in+a+mouse+model+of+Duchenne+muscular+dystrophy.">In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.</a> Science. 351, 403-407 (2016).</li><li>Ousterout, D. G. K., Thakore, A. M., Majoros, P. I., Reddy, T. E. W. H., Gersbach, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+CRISPR/Cas9-based+genome+editing+for+correction+of+dystrophin+mutations+that+cause+Duchenne+muscular+dystrophy.">Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.</a> Nat Commun. 6, 6244 (2015).</li><li>Tabebordbar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+editing+in+dystrophic+mouse+muscle+and+muscle+stem+cells.">In vivo gene editing in dystrophic mouse muscle and muscle stem cells.</a> Science. 351, 407-411 (2016).</li><li>Bengtsson, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-specific+CRISPR/Cas9+dystrophin+gene+editing+ameliorates+pathophysiology+in+a+mouse+model+for+Duchenne+muscular+dystrophy.">Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.</a> Nat Commun. 8, 14454 (2017).</li><li>Hoffman, E. P., Brown, R. H., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin:+the+protein+product+of+the+Duchenne+muscular+dystrophy+locus.">Dystrophin: the protein product of the Duchenne muscular dystrophy locus.</a> Cell. 51, 919-928 (1987).</li><li>Nowak, K. J., Davies, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duchenne+muscular+dystrophy+and+dystrophin:+pathogenesis+and+opportunities+for+treatment.">Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment.</a> EMBO reports. 5, 872-876 (2004).</li><li>Mendell, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence-based+path+to+newborn+screening+for+Duchenne+muscular+dystrophy.">Evidence-based path to newborn screening for Duchenne muscular dystrophy.</a> Annals of neurology. 71, 304-313 (2012).</li><li>Mendell, J. R., Lloyd-Puryear, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+of+MDA+muscle+disease+symposium+on+newborn+screening+for+Duchenne+muscular+dystrophy.">Report of MDA muscle disease symposium on newborn screening for Duchenne muscular dystrophy.</a> Muscle Nerve. 48, 21-26 (2013).</li><li>Spurney, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyopathy+of+Duchenne+muscular+dystrophy:+current+understanding+and+future+directions.">Cardiomyopathy of Duchenne muscular dystrophy: current understanding and future directions.</a> Muscle Nerve. 44, 8-19 (2011).</li><li>Moriuchi, T., Kagawa, N., Mukoyama, M., Hizawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autopsy+analyses+of+the+muscular+dystrophies.">Autopsy analyses of the muscular dystrophies.</a> Tokushima J Exp Med. 40, 83-93 (1993).</li><li>McNally, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+in+the+therapy+of+cardiomyopathy+in+muscular+dystrophy.">New approaches in the therapy of cardiomyopathy in muscular dystrophy.</a> Annual review of medicine. 58, 75-88 (2007).</li><li>Baker, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approvals,+Submission,+and+Important+Labeling+Changes+for+US+Marketed+Pharmaceuticals.">Approvals, Submission, and Important Labeling Changes for US Marketed Pharmaceuticals.</a> Hosp Pharm. 52, 306-307 (2013).</li><li>Baker, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eteplirsen.">Eteplirsen.</a> Hosp Pharm. 52, 302-305 (2017).</li><li>Sicinski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+muscular+dystrophy+in+the+mdx+mouse:+a+point+mutation.">The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.</a> Science. 244, 1578-1580 (1989).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-mediated+Genome+Editing+Restores+Dystrophin+Expression+and+Function+in+mdx+Mice.">CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 24, 564-569 (2016).</li><li>El Refaey, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Genome+Editing+Restores+Dystrophin+Expression+and+Cardiac+Function+in+Dystrophic+Mice.">In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.</a> Circ Res. 121, 923-929 (2017).</li><li>Tabebordbar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+editing+in+dystrophic+mouse+muscle+and+muscle+stem+cells.">In vivo gene editing in dystrophic mouse muscle and muscle stem cells.</a> Science. 351, 407-411 (2016).</li><li>Pozsgai, E. R., Griffin, D. A., Heller, K. N., Mendell, J. R., Rodino-Klapac, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Sarcoglycan+gene+transfer+decreases+fibrosis+and+restores+force+in+LGMD2E+mice.">beta-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice.</a> Gene therapy. 23, 57-66 (2016).</li><li>Chicoine, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmapheresis+eliminates+the+negative+impact+of+AAV+antibodies+on+microdystrophin+gene+expression+following+vascular+delivery.">Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery.</a> Mol Ther. 22, 338-347 (2014).</li><li>Chicoine, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+delivery+of+rAAVrh74.MCK.GALGT2+to+the+gastrocnemius+muscle+of+the+rhesus+macaque+stimulates+the+expression+of+dystrophin+and+laminin+alpha2+surrogates.">Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin alpha2 surrogates.</a> Mol Ther. 22, 713-724 (2014).</li><li>Vouillot, L., Thelie, A., Pollet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T7E1+and+surveyor+mismatch+cleavage+assays+to+detect+mutations+triggered+by+engineered+nucleases.">Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.</a> G3. 5, 407-415 (2015).</li><li>Tsai, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUIDE-seq+enables+genome-wide+profiling+of+off-target+cleavage+by+CRISPR-Cas+nucleases.">GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.</a> Nat Biotechnol. 33, 187-197 (2015).</li></ol>

不存在利益冲突。

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?...

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。

<table><tbody><tr><td>Alexa Fluor 555 goat anti-rabbit IgG</td><td>Thermo Fisher Scientific</td><td>A-21428</td><td></td></tr><tr><td>Benzonase Nuclease</td><td>Sigma-Aldrich</td><td>E1014</td><td></td></tr><tr><td>Bio Safety Cabinets</td><td>Thermo Fisher Scientific</td><td>1347</td><td>1300 Series A2 Class II, Type A2</td></tr><tr><td>BsaI</td><td>New England BioLabs Inc</td><td>RO535S</td><td></td></tr><tr><td>Competent cells</td><td>Agilent Technologies</td><td>200314</td><td></td></tr><tr><td>Cell culture dishes, 150mm</td><td>Nest Scientific USA</td><td>715001</td><td></td></tr><tr><td>Cryostat</td><td>Leica Biosystems</td><td></td><td>Leica CM3050S</td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>MT10017CV</td><td>Corning cellgro</td></tr><tr><td>Dystrophin antibody</td><td>Spring Bioscience</td><td>E2660</td><td></td></tr><tr><td>Fast-Ion Midi Plasmdi Kits</td><td>IBI Scientific</td><td>IB47111</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher Scientific</td><td>26-140-079</td><td></td></tr><tr><td>Filter Unit, 0.45&amp;mu;m</td><td>Thermo Fisher Scientific</td><td>166-0045</td><td></td></tr><tr><td>GoTaq Master Mixes</td><td>Promega</td><td>M7122</td><td></td></tr><tr><td>High-speed plasmid mini kit</td><td>IBI Scientific</td><td>IB47102</td><td></td></tr><tr><td>Horse serum</td><td>Abcam</td><td>ab139501</td><td></td></tr><tr><td>Isotemp 2239 Water Bath</td><td>Thermo Fisher Scientific</td><td>15-460-11Q</td><td></td></tr><tr><td>Isotemp Heat Block</td><td>Thermo Fisher Scientific</td><td>11-718</td><td></td></tr><tr><td>Inverted confocal microscope</td><td>Carl Zeiss Microscopy, LLC</td><td></td><td>LSM 780</td></tr><tr><td>LightCycler 480 instrument</td><td>Roche</td><td>5015243001</td><td>LightCycler 480 Instrument II</td></tr><tr><td>Large Capacity Centrifuge</td><td>Thermo Fisher Scientific</td><td>46-910</td><td>Thermo Scientific Sorvall RC 6 Plus</td></tr><tr><td>Microcentrifuge</td><td>Thermo Fisher Scientific</td><td>75002445</td><td>Sorvall Legend Micro 21R</td></tr><tr><td>Nanodrop spectrophotometers</td><td>Thermo Scientific</td><td>ND2000CLAPTOP</td><td>NanoDrop&amp;trade; 2000/2000c</td></tr><tr><td>Oligos for i20-F</td><td>Integrated DNA Technologies</td><td></td><td>CACCgGGCGT&lt;br/&gt;TGAAATTCTCATTAC</td></tr><tr><td>Oligos for i20-R</td><td>Integrated DNA Technologies</td><td></td><td>AAAC GTAATGAGAATTTCAACGCCc;</td></tr><tr><td>Oligos for i23-F</td><td>Integrated DNA Technologies</td><td></td><td>CACCgCACCGATGAGAGGG&lt;br/&gt;AAAGGTC</td></tr><tr><td>Oligos for i23-R</td><td>Integrated DNA Technologies</td><td></td><td>GACCTTTCCCTCTCATCGGTGc</td></tr><tr><td>Oligos</td><td>Integrated DNA Technologies</td><td></td><td></td></tr><tr><td>OptiPrep Density Gradient Medium</td><td>Sigma-Aldrich</td><td>D1556-250ML</td><td></td></tr><tr><td>OptiMEM</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>PEG8000</td><td>Sigma-Aldrich</td><td>89510-1kg-F</td><td></td></tr><tr><td>Polyethylenimine</td><td>Polysciences</td><td>23966</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>P2308</td><td></td></tr><tr><td>Quick-Seal centrifuge tube</td><td>Beckman Coulter Inc</td><td>342414</td><td></td></tr><tr><td>QIAquick Gel Extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>Radiant SYBR Green Hi-ROX qPCR Kits</td><td>Alkali Scientific</td><td>QS2005</td><td></td></tr><tr><td>RevertAid RT Reverse Transcription Kit</td><td>Thermo Fisher Scientific</td><td>K1691</td><td></td></tr><tr><td>Rotor</td><td>Beckman Coulter Inc</td><td>337922</td><td>Type 70Ti</td></tr><tr><td>T4 DNA ligase</td><td>New England BioLabs Inc</td><td>M0202S</td><td></td></tr><tr><td>Thermal Cycler</td><td>Bio-rad</td><td>1861096</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific</td><td>15596026</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter Inc</td><td>8043-30-1192</td><td>Optima le-80k</td></tr><tr><td>Ultra centrifugal filter unit</td><td>MilliporeSigma</td><td>UFC510096</td><td></td></tr><tr><td>VECTASHIELD Mounting Medium with DAPI</td><td>Vector Laboratories, Inc</td><td>H-1200</td><td></td></tr></tbody></table>

adeno-associated virus-mediated delivery of crispr for cardiac gene editing in mice

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。

gRNAs 的有效性, 以诱导删除的目标基因组 DNA 区域应评估在细胞培养之前, 包装成 rAAV 的活体研究。为此, gRNAs 和 SaCas9-expressing 构造转成 C2C12 细胞, 并通过 PCR 与靶点侧面的引物对基因组 DNA 进行分析 (图 1)。500 bp 的 PCR 产物表明, 成功删除基因编辑产生的目标基因组 dna, 而控制细胞不应该产生一个带, 由于大的基因组 dna 的大小。
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Watch this Scientific Journal Video about Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice at JoVE.com

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

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。

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

adeno-associated-virus-mediated-delivery-crispr-for-cardiac-gene

Research

JoVE Journal

Medicine

8.1K 次观看.  Ohio State University Wexner Medical Center. 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。

视频: Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

在小鼠心脏rAAV9的制备过表达或敲除基因

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

科研

遗传学

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

癌症研究

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation

This protocol presents an optimized approach for producing genetically modified rat models. Adeno-associated virus (AAV) is used to deliver a DNA repair template, and electroporation is used to deliver CRISPR-Cas9 reagents to complete the genome editing process in 2-cell embryo.

使用腺相关病毒 （AAV） 和 2 细胞胚胎电穿孔对大鼠胚胎进行 CRISPR-Cas9 基因组编辑

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

发育生物学

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

神经科学

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

免疫与感染

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

生物学

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

使用CRISPR/Cas9的人类B细胞基因组工程

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9介导的斑马鱼心脏中的精确敲入编辑

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...

Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive molecules. The creation of BAT-specific knockout mice is one of the most popular approaches for understanding the contribution of a gene of interest to BAT-mediated energy regulation. The conventional gene targeting strategy utilizing the Cre-LoxP system has been the principal approach to generate tissue-specific knockout mice. However, this approach is time-consuming and tedious. Here, we describe a protocol for the rapid and efficient knockout of a gene of interest in BAT using a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The interscapular BAT is located in the deep layer between the muscles. Thus, the BAT must be exposed in order to inject the AAV precisely and directly into the BAT within the visual field. Appropriate surgical handling is crucial to prevent damage to the sympathetic nerves and vessels, such as the Sultzer's vein that connects to the BAT. To minimize tissue damage, there is a critical need to understand the three-dimensional anatomical location of the BAT and the surgical skills required in the technical steps. This protocol highlights the key technical procedures, including the design of sgRNAs targeting the gene of interest, the preparation of AAV-sgRNA particles, and the surgery for the direct microinjection of AAV into both BAT lobes for generating BAT-specific knockout mice, which can be broadly applied to study the biological functions of genes in BAT.

In this protocol, we describe the technical procedures to generate brown adipose tissue (BAT)-specific knockout mice leveraging a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The described steps include the design of the sgRNAs, the preparation of the AAV-sgRNA particles, and the microinjection of AAV into the BAT lobes.

使用组合的 Cre-LoxP、CRISPR-Cas9 和腺相关病毒单向导 RNA 系统生成棕色脂肪特异性敲除小鼠

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive ...

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

摘要

探索更多视频

此视频中的章节

在小鼠心脏rAAV9的制备过表达或敲除基因

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

使用腺相关病毒（AAV）和 2 细胞胚胎电穿孔对大鼠胚胎进行 CRISPR-Cas9 基因组编辑

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

使用CRISPR/Cas9的人类B细胞基因组工程

CRISPR-Cas9介导的斑马鱼心脏中的精确敲入编辑

使用组合的 Cre-LoxP、CRISPR-Cas9 和腺相关病毒单向导 RNA 系统生成棕色脂肪特异性敲除小鼠

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

摘要

探索更多视频

此视频中的章节

在小鼠心脏rAAV9的制备过表达或敲除基因

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

使用腺相关病毒 （AAV） 和 2 细胞胚胎电穿孔对大鼠胚胎进行 CRISPR-Cas9 基因组编辑

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

使用CRISPR/Cas9的人类B细胞基因组工程

CRISPR-Cas9介导的斑马鱼心脏中的精确敲入编辑

使用组合的 Cre-LoxP、CRISPR-Cas9 和腺相关病毒单向导 RNA 系统生成棕色脂肪特异性敲除小鼠

使用腺相关病毒（AAV）和 2 细胞胚胎电穿孔对大鼠胚胎进行 CRISPR-Cas9 基因组编辑