Name: using crispr/cas9 gene editing to investigate the oncogenic activity of mutant calreticulin in cytokine dependent hematopoietic cells
Uploaded: 2018-01-05T15:00:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells at JoVE.com

这项工作得到了 NIH (R01HL131835), 一个达蒙罗扬临床研究员奖和斯塔尔癌症财团的支持。

1. sgRNA 设计使用在线工具13
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设计 sgRNAs 的目标基因的兴趣使用免费的在线工具。
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<li>复制并粘贴感兴趣基因的 NCBI 参考序列到宽广的学院 sgRNA 设计师网页工具: (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design)。 注意: 此工具可识别显内的 sgRNA 序列和跨越内含子/外显子边界但仍在外显子内进行劈裂的那些。</li>
		<li>下载并打开 excel 中的文本输出文件。</li>
		<li>关注于用 sgRNA 序列和 sgRNA 上下文序列填充的列。请注意, sgRNA 序列不包含 protospacer 相邻的主题 (PAM), 但上下文序列是这样的。</li>
		<li>请注意, 在目标的功效评分列出了预测的劈裂效率评分的0至 1, 其中评分1表示更高的劈裂效率。</li>
		<li>使用 excel 的 "排序" 功能, 可以通过 "目标切口%" 列的效果评分或目标基因内的位置来排列目标。 注意: 根据基因中的位置进行分类对识别特定域或感兴趣的外显子的 sgRNAs 是有用的。</li>
		<li>选择 3-6 sgRNAs, 目标的领域与高 (> 0.6) 的目标效果评分。它可能是有用的, 知道什么样的突变可能负责的表型, 是需要的 (请参见图 1解释的目标策略用于钙)。 注: sgRNAs 低于建议的0.6 效果评分阈值时, 应考虑到缺乏其他良好的候选人。</li>
		<li>使用 MIT gRNA 分析 web 工具来筛选潜在的不相干效果 (http://crispr.mit.edu/)。对于选定的每个 sgRNA, 运行目标序列 (包括 PAM)。 注: 广泛的研究所 sgRNA 设计师网页工具也可用于筛选不相干效果 (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design)。<ol>
<li>请注意, 麻省理工学院 sgRNA 分析工具的评分每 sgRNA 1-100, 其中得分100表示更高的特异度。分数大于70是理想的, 代表一个 sgRNA 的最低不相干效果。这个网站还会生成一个不相干点击列表, 其中有他们在基因组中的位置, 以及他们与候选指南有多少不匹配。 注意: 四不匹配或更大的非目标命中被视为安全14。如果低于四的不匹配, 则可能会有问题, 如果它们属于显14。</li>
		</ol>
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		<li>选择 2-3 sgRNAs, 目标基因感兴趣的区域内的不同位置, 并具有最高配对的劈裂效率和不相干分数 (参见表 1 , 用于针对Calr的外显子9的 sgRNA 序列。 注: 根据实验目标的不同, 可以更加强调从所述工具中获得的各种值。</li>
	</ol>
</li>
</ol>2. 克隆 sgRNA 寡13
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生成正向寡
	<ol>
<li>创建没有 PAM 站点的 sgRNA 序列的列表。</li>
		<li>如果 sgRNA 序列的5结尾不是 g, 则将 g 添加到序列的5的末尾。 注意: 这 G 是必要的最大化 U6-driven sgRNA 转录水平。添加 G 对于 m1 sgRNA (表 1) 是必需的。</li>
		<li>将序列 "CACC" 添加到 G 优化引导序列的5端 (没有 PAM) (表 2), 以便生成将退火的寡结扎到 BsmBI消化 lentiGuide 向量 (参见步骤 3)所需的悬垂。
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	</ol>
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生成反向寡聚
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<li>反向补全 G 优化引导序列 (无 PAM)。</li>
		<li>将序列 "AAAC" 添加到反向补充的 G 优化指南序列的5端 (表 2)。</li>
		<li>从底漆合成公司订购设计的寡。</li>
	</ol>
</li>
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退火和化寡
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<li>添加1µL 的正向寡核苷酸 (100 µM), 1 µL 的反向寡糖 (100 µM), 1 µL 的 10x T4 结扎缓冲, 0.5 µL 的 T4 核苷酸激酶 (PNK) 和6.5 µL 的 H2O 到 PCR 管 (总 = 10 µL)。</li>
		<li>运行以下程序在 thermocycler:37 ˚C 为30分钟, 95 ˚C 为 5 min, 舷梯从95到25在0.1 ° c/s。</li>
		<li>在 H2O 中, 将反应产物稀释在1:250。</li>
	</ol>
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</ol>3. lentiGuide-普洛矢量的消解13
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文摘 lentiGuide-普洛向量
	<ol>
<li>用以下成分组装50µL 消化反应: 5 µg 的循环质粒, 2 µL (30 单位) BsmBI 限制性酶, 5 µL 限制酶缓冲和高达50µL 的 H2O。</li>
		<li>在55° c 下运行2小时的反应。在第一个小时之后, 添加1µL 更多的 BsmBI 限制性酶。</li>
	</ol>
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	<li>
Dephosphorylate 切割骨干
	<ol>
<li>添加7µL 磷酸酯酶反应缓冲剂和2µL 磷酸腺酵素对消化载体。</li>
		<li>孵育30分钟, 在37° c。</li>
	</ol>
</li>
	<li>PCR 纯化切割和化骨干使用商业套件。 注: 用 miniprep 套件蓝色的列替换提供的粉红色的列, 因为它们可以更好地容纳大质粒大小。在洗脱之前, 通过在90˚C 热块中加热洗脱缓冲剂, 通过在末端两次从柱上洗脱 dna (从第一次洗脱中运行洗 dna) 可以提高产量。</li>
</ol>4. 结扎 ofAnnealed 寡成消化骨干13
<ol><li>添加 50 ng 消化骨干, 1 µL 退火寡稀释, 1 µL 的 10x T4 结扎缓冲, 1 µL T4 连接和高达10µL 的 H2O 到 PCR 管。室温孵育1小时</li>
	<li>用2µL 的结扎反应对 Stbl3 细胞进行转化。用100µg/毫升氨苄西林在性汤 (LB) 琼脂板上, 在37° c 的夜间孵育。</li>
	<li>选择3殖民地和接种到一个 mini-prep 的文化。</li>
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验证正确的寡聚插入
	<ol>
<li>使用商业套件从 U6 启动子的底漆开始, 对每种文化和序列进行 miniprep。</li>
		<li>执行一个 midi 准备或最大的顺序验证的文化准备。</li>
	</ol>
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</ol>5. 细胞 lLines 稳定表达 SpCas9 的生成
注: 本协议涉及慢感染 pLX_TRC311-Cas9 质粒的交付。本协议详细描述了小鼠 interleukin-3 (mIL-3) 依赖亲 B (Ba/F3) 细胞, 悬浮细胞线, 并可以适应其他细胞类型使用的首选培养条件为每个细胞类型。Ba/F3 细胞培养基由 RPMI 补充10% 胎牛血清、1% 青霉素/链霉素/l-谷氨酰胺和10小鼠白细胞介素3。
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染 HEK-293T 细胞
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<li>种子 3 x 106 HEK-293T 细胞每10厘米组织培养皿。在转染前一夜保留种子细胞。 注: HEK-293T 细胞是附着的细胞系, 通常用于病毒生产。细胞维持在 DMEM 补充10% 胎牛血清, 1% 青霉素/链霉素/l-谷氨酰胺。细胞在转染时应80% 汇合。</li>
		<li>前温热还原的血清培养基 (优化) 和生长培养基。</li>
		<li>添加500µL 的减少血清培养基, 7 µg pLX_TRC311-Cas9 结构, 4 µg pCMV-VSV-慢包装质粒和4µg psPAX2 慢包装质粒成管 注: 每 10 cm 293T 细胞的总 DNA 是15µg。</li>
		<li>混合 DNA, 加入45µL 的转染试剂。轻轻地混合, 让它站立20分钟。 注意: 由于血清会抑制转染试剂和质粒 DNA 之间的复杂形成, 所以使用降低血清培养基是很重要的。</li>
		<li>在293T 板中吸取旧培养基, 加入5毫升的新培养基。</li>
		<li>添加 DNA 和转染试剂混合在一个下降明智的方式, 以293T 板。轻轻地旋转, 在37° c 和 5% CO2下孵育24小时。</li>
		<li>收获病毒清在24和 48 h 后转染。通过一个0.22 µm 过滤器和分到一个 cryovial 管 (1.6 毫升/管和1.5 毫升将用于感染)。</li>
		<li>在-80 ° c 贮存病毒上清液。 注: 慢上清液可在6月内使用。如果可能, spinfection transductions 应该用新鲜的病毒来执行。</li>
	</ol>
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	<li>
Ba/F3 细胞的慢感染
	<ol>
<li>冰冻时, 将病毒上清液解冻。</li>
		<li>离心机细胞在 300 x g 为4分钟。</li>
		<li>吸气上清和重细胞的浓度为 3 x 106细胞/毫升。</li>
		<li>添加500µL 的悬浮细胞, 1.5 毫升的病毒上清, 4 µL 的凝聚 (股票: 2 毫克/毫升) 和 10 ng/毫升 m-IL3 在6良好的组织培养板。确保包括一个未感染的良好与1.5 毫升的媒体, 而不是病毒上清作为一个控制。 注: 增加的病毒上清液的数量应对应于多种感染 (莫伊) < 1。</li>
		<li>离心板在 440 x g 为 120 min 在37° c。</li>
		<li>从离心机中取出盘子, 在37° c 和 5% CO2孵化器中放置, 并允许在一夜之间生长。</li>
		<li>在24小时后旋转下来的细胞, 重与5毫升的新鲜温暖的媒体在一个6井板。</li>
	</ol>
</li>
	<li>
Cas9 感染细胞的选择
	<ol>
<li>旋转下来的细胞和重与新鲜温暖的媒体补充5µg/毫升的 blasticidin, 48 小时后 spinfection。</li>
		<li>选择9天与 blasticidin 或直到未感染 (阴性) 控制细胞死亡的细胞。</li>
		<li>一旦选择完成, 将耐药细胞转移到低浓度 blasticidin 的培养基中。</li>
	</ol>
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</ol>6. 记者检测 Cas9 活动15
<ol><li>传感器父母细胞和细胞稳定表达 Cas9 与 pXPR-011 的慢感染 (见步骤 5.1-5.2)。 注: 此矢量包含 gfp 和以 gfp 为靶的指南。含有活性 Cas9 的细胞会导致 GFP 的减少 (图 2)。</li>
	<li>选择3天的细胞与2µg/毫升的嘌呤, 48 小时后 spinfection。</li>
	<li>用流式细胞仪分析 GFP 表达的样品。 注意: GFP 减少50% 或以上代表最佳 Cas9 活动。如果观察到少于50% 的减少, 可以使用更高浓度的 blasticidin 选择来增加 Cas9 活性。并非所有的单元格线都能容忍 blasticidin 选择。在这种情况下, 使用 Cas9 向量与其他选择磁带可能是必要的。此外, 并非所有细胞系都能耐受 Cas9 的稳定表达。在这种情况下, 可以使用瞬态转染 Cas9。</li>
</ol>7. sgRNAs Spinfection Cas9 表达细胞的研究
<ol><li>按照步骤 5.1-5.2 的慢感染的 sgRNAs 成细胞的利益。 注意: 在步骤5.1.3 中, 将 pLX_TRC311-Cas9 构造替换为从4节中验证的 lentiGuide-普洛构造。</li>
	<li>48 h 后 spinfection, 选择3天的细胞与2µg/毫升的嘌呤。</li>
	<li>将耐药细胞转移到较低浓度抗生素的培养基中, 再继续选择4天, 以便进行足够的编辑。</li>
</ol>8. 利用 m-IL3 提取 Ba/F3 细胞转化和阳性选择
注: 本法用于 mIL-3 依赖的 Ba/F3 细胞, 但可应用于任何细胞因子依赖的干细胞系。
<ol><li>Ba/F3 细胞 ectopically 表达 Cas9 和 sgRNA 的兴趣。</li>
	<li>吸入上清液, 用5毫升磷酸缓冲盐水 (PBS) 冲洗细胞。</li>
	<li>旋转下来的细胞和重复洗涤步骤在8.2 四次 (这一步确保媒体是免费的 IL-3)。</li>
	<li>吸入 PBS 和重5毫升的新鲜媒体没有 IL-3 的细胞。</li>
	<li>使用例或单元生存能力分析器对单元格进行计数。</li>
	<li>在6个组织培养板中, 以 1 x 105细胞/毫升的浓度, 在2毫升的新鲜培养基的总体积内, 在没有 IL-3 的情况下, 将细胞种在一式三份。</li>
	<li>每2天监测和计数细胞, 总共8天。 注: Ba/F3 细胞缺乏 IL-3 通常死亡2天后 IL-3 饥饿。重要的是要在分析中包括一个负控制 (通常是一个互不指南被用作一个控制)。</li>
</ol>9. CRISPR 的目标编辑筛选
<ol><li>sgRNA 劈裂部位上游和下游 CRISPR 筛选底漆的设计 注: 从预测的劈裂部位使用至少 100 bp, 以确保检测不会受到 sgRNA 目标站点上的大插入和/或删除 (indel) 的影响。</li>
	<li>分离基因组 DNA (gDNA) 从被转换的细胞 (在成长曲线的末端) 和从细胞 pre-cytokine 撤退。 注意: 始终包括表达互不指南中的单元格作为基因编辑的控制。将 gDNA 从细胞中分离出来之前和之后的转变将会发现那些积极选择并具有增殖优势的克隆。</li>
	<li>组装50µL pcr 与以下组成部分:25 µL 的 2x pcr 混合, 1 µL 前引物 (10 µM), 1 µL 的反向底漆 (10 µM), 50-100 ng gDNA, 和 H2O 高达50µL。 注: 许多高保真聚合可在步骤9.3 中使用。</li>
	<li>使用以下参数在 thermocycler 中运行示例:95 1 分钟, 30 周期 (94 ° c 为 1 min, 52 ° c 为三十年代, 72 ° c 为三十年代), 和72° c 为 10 min。此 PCR 针对表 3中列出的Calr引物进行了优化。在 gDNA 试验的基础上对9.1 步设计的引物对 PCR 条件进行优化。</li>
	<li>使用1x 醋酸三乙酸-EDTA (泰) 缓冲液在10伏/cm 上运行2% 琼脂糖凝胶样品的5µL。检查样本的存在/缺席的放大带相应大小的基因感兴趣。</li>
	<li>使用 pcr 反应的其余部分 (45 µL) 进行 pcr 纯化。</li>
	<li>克隆增与 PCR 克隆试剂盒成质粒载体。例如, 通常使用 pGEM T 容易向量。</li>
	<li>将质粒转化为 Stbl3 细胞或其它相容的细胞, 并用相关的抗生素将其放入 LB 琼脂板上。选择10-20 殖民地, mini-prep 每一个, 并服从每一个克隆到桑格排序的特点 indels 创建的 CRISPR/Cas9 编辑。 注意: 如果需要, 可以在批量编辑的种群上执行单细胞荧光活化细胞分类 (资产管制), 以将克隆与特定的 indel 分离。此外, 下一代测序 (NGS) 方法可用于更有力地量化的目标编辑使用 PCR 增跨越 sgRNA 目标区域的深度排序。例如, 可以使用分解或潮汐跟踪 Indels, 而不是亚精确地确定在一个单元格池 (https://tide.nki.nl/#about)16中生成的目标突变的频谱和频率。</li>
</ol>

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S., 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/Cas9-mediated+Gene-knockout+screens+and+target+identification+via+Whole+genome+sequencing+uncover+host+genes+required+for+picornavirus+infection.">CRISPR/Cas9-mediated Gene-knockout screens and target identification via Whole genome sequencing uncover host genes required for picornavirus infection.</a> J Biol Chem. 10, 1074 (2017).</li><li>Heckl, D., 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=Generation+of+mouse+models+of+myeloid+malignancies+with+combinatorial+genetic+lesions+using+CRISPR-Cas9+genome+editing.">Generation of mouse models of myeloid malignancies with combinatorial genetic lesions using CRISPR-Cas9 genome editing.</a> Nat Biotechnol. 32 (9), 941-946 (2014).</li><li>Daley, G. Q., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+an+interleukin+3-dependent+hematopoietic+cell+line+by+the+chronic+myelogenous+leukemia-specific+bcr/abl+protein.">Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific bcr/abl protein.</a> Proc Natl Acad Sci USA. 85, 9312-9316 (1988).</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> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>Haeussler, 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=Evaluation+of+off-target+and+on-target+scoring+algorithms+and+integration+into+the+guide+RNA+selection+tool+CRISPOR.">Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.</a> Genome Biol. 17, 148 (2016).</li><li>Doench, J. 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=Rational+design+of+highly+active+sgRNAs+for+CRISPR-Cas9-mediated+gene+inactivation.">Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.</a> Nat Biotechnol. 32, 1262-1267 (2014).</li><li>Brinkman, E. K., 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=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucl Acid Res. 42, e168 (2014).</li><li>Klampfl, T., 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=Somatic+mutations+of+calreticulin+in+myeloproliferative+neoplasms.">Somatic mutations of calreticulin in myeloproliferative neoplasms.</a> N Engl J Med. 369, 2379-2390 (2013).</li><li>Nangalia, J., 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=Somatic+CALR+mutations+in+myeloproliferative+neoplasms+with+nonmutated+JAK2.">Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.</a> N Engl J Med. 369, 2391-2405 (2013).</li><li>Elf, S., 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=Mutant+Calreticulin+requires+both+its+mutant+C-terminus+and+the+thrombopoietin+receptor+for+oncogenic+transformation.">Mutant Calreticulin requires both its mutant C-terminus and the thrombopoietin receptor for oncogenic transformation.</a> Cancer Discovery. 6 (4), 368-381 (2016).</li><li>Bauer, D. E., Canver, M. C., Orkin, 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=Generation+of+Genomic+Deletions+in+Mammalian+Cell+lines+via+CRISPR/Cas9.">Generation of Genomic Deletions in Mammalian Cell lines via CRISPR/Cas9.</a> J. Vis. Exp. (95), e52118 (2015).</li><li>Watanabe-Smith, K., 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=Analysis+of+acquired+mutations+in+transgenes+arising+in+Ba/F3+transformation+assays:+findings+and+recommendations.">Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations.</a> Oncotarget. 8, 12596-12606 (2017).</li><li>Fu, Y., 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=Improving+CRISPR-Cas+nuclease+specificity+using+truncated+guide+RNAs.">Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.</a> NatBiotechnol. 32, 279-284 (2014).</li></ol>

我们没有与本报告有关的利益冲突。

在这里, 我们展示了使用 CRISPR/Cas9 基因编辑研究的生物学功能的CALR突变的造血细胞。该协议的成功与否高度依赖于多个因素。首先, 重要的是要知道什么类型的突变可能是负责的表型, 是需要的。在本协议中, 读数是 Ba/F3 细胞向 mIL-3 独立的转化, 而突变的类型在CALR的外显子9中 indels。但是, 如果所需的突变是单个基对替代, 那么 HDR 介导的修复是首选方法, 因为它可以引入精确点突变或...

CRISPR/Cas9 技术最近在活细胞和生物体中彻底改变了目标基因组的编辑。它已成为生物医学研究的一个非常强大的工具, 目前正被用作治疗遗传性疾病的潜在途径1。所有基因组编辑工具的基础是建立一个核酸诱导的 DNA 双链断裂 (争端) 在基因组所在地要修改。DSBs 可以通过同源结束连接 (NHEJ) 或同源定向修复 (HDR)2,3进行修复。Cas9 核酸优于其他基因组工程核酸, 如锌指核酸 (ZFNs) 和转录激活剂样效应核酸 (TALENs) 的优势是它依赖于 RNA 的目标核酸到一个理想的 DNA 序列, 比较到 ZFNs 和 TALENs 中发现的蛋白质-DNA 相互作用2,3。
在原核细胞 CRISPR/Cas9 核酸通路作为自适应免疫系统的发现后4,5, 许多努力已经适应了哺乳动物细胞系和模型生物体的使用途径2,3. 作为基因编辑的工具, CRISPR/Cas9 通路利用两个主要组成部分:链球菌化脓(Sp) 衍生 Cas9 核酸和 sgRNAs 的目标基因的兴趣2,3。sgRNA 由20核苷酸组成, 通过 RNA-DNA 基对互补2,3, 直接 Cas9 到基因组上的特定站点。sgRNA 的目标位置必须位于相邻的 protospacer 相邻主题 (PAM) 的位置, 其形式为 5 "NGG, 这是 SpCas9 核酸所认可的。有了这些工具, Cas9 可以通过设计 sgRNAs 的目标区域来引导到任何 DNA 序列。除了 Sp 派生的 Cas9, 还有其他的变体, Cas9 具有不同的功能, 这取决于特定的应用程序。例如, 有 Cas9 的变种具有更高的特异性为在目标编辑或单分裂容量为脱氧核糖核酸偷6,7。此外, 催化的非活动 Cas9 最近已经发展为转录调节8。科学家们现在已经使用 CRISPR/Cas9 系统的各种应用, 如基因敲除和击倒研究的生物学功能的基因9, 功能和功能库屏幕10和遗传模型有机体的工程学11。
在本协议中, 我们将 CRISPR/Cas9 方法与 Ba/F3 细胞转化法结合起来, 以了解CALR突变的生物学功能。Ba/F3 细胞是一种小鼠 IL-3 依赖的造血细胞系, 可以 IL-3 独立于某些致癌基因的表达, 如 BCR 的12。为了了解突变体钙是否能将 Ba/F3 细胞转化为细胞因子的独立生长, 我们针对内生的Calr轨迹的外显子9使用 CRISPR/Cas9 引入 indel 突变, 然后从细胞中撤回 IL-3, 以应用积极的选择压力, 与目标综述功能CALR突变发现 MPN 患者。该协议包括 sgRNAs 的设计、克隆和交付、稳定 Cas9 表达细胞的开发和 CRISPR 靶基因编辑的筛选。该协议可以应用于不同的基因和各种细胞因子相关的兴趣, 在模型和研究癌症相关基因的生物学功能方面特别有价值。

<table><tbody><tr><td>BsmBI</td><td>New England Biolabs</td><td>R0580L</td><td></td></tr><tr><td>Blasticidin</td><td>Sigma Aldrich</td><td>15025</td><td></td></tr><tr><td>Puromycin</td><td>Life Technologies</td><td>A1113803</td><td></td></tr><tr><td>Stbl3 cells</td><td>Life Technologies</td><td>C737303</td><td></td></tr><tr><td>TransIT-LT1</td><td>Thermo Fisher Scientific</td><td>MIR2300</td><td></td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>51985034</td><td></td></tr><tr><td>RPMI</td><td>Thermo Fisher Scientific</td><td>MT10040CV</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>10-017-CV</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Omega Scientific</td><td>FB-11</td><td></td></tr><tr><td>Penicillin/streptomycin/L-glutamine</td><td>Life Technologies</td><td>10378016</td><td></td></tr><tr><td>mIL-3</td><td>Peprotech</td><td>213-13</td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>N/A</td><td></td></tr><tr><td>pCMV-VSV-G</td><td>Addgene</td><td>N/A</td><td></td></tr><tr><td>pLX_TRC311-Cas9</td><td>Addgene</td><td>N/A</td><td></td></tr><tr><td>polybrene</td><td>Sigma Aldrich</td><td>H9268</td><td></td></tr><tr><td>pXPR-011</td><td>Addgene</td><td>N/A</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Genessee Scientific</td><td>25-507</td><td></td></tr><tr><td>TAE buffer</td><td>Thermo Fisher Scientific</td><td>FERB49</td><td></td></tr><tr><td>LentiGuide-Puro</td><td>Addgene</td><td>Plasmid #52963</td><td></td></tr><tr><td>PNK</td><td>New England Biolabs</td><td>M0201S</td><td></td></tr><tr><td>T4 ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Miniprep kit</td><td>Qiagen</td><td>27104</td><td></td></tr></tbody></table>

using crispr/cas9 gene editing to investigate the oncogenic activity of mutant calreticulin in cytokine dependent hematopoietic cells

聚类定期 interspaced 短复发重复 (CRISPR) 是一种自适应免疫系统的原, 已被科学家用来产生 RNA 引导核酸, 如 CRISPR 相关 (Cas) 9 site-specific 真核基因组编辑.Cas9 的基因组工程被用来有效、容易和有力地改变许多生物相关的哺乳动物细胞系和生物体内源基因。在这里, 我们展示了一个如何利用 CRISPR/Cas9 方法来理解特定基因突变的生物学功能的例子。我们模型钙 (CALR) 突变的小鼠 interleukin-3 (mIL-3) 依赖亲 B (Ba/F3) 细胞通过交付单一指南 rna (sgRNAs) 的目标内源性CALR所在地在特定地区的插入和/或删除 (indel) CALR突变发生在骨髓肿瘤 (MPN), 一种类型的血液癌症患者。sgRNAs 在目标区域创建双链断裂 (DSBs), 由同源端连接 (NHEJ) 修复, 以提供各种尺寸的 indels。然后, 我们采用标准 Ba/F3 细胞转化法, 以了解生理水平表达的Calr突变对造血细胞转化的影响。这种方法可应用于其他基因, 以研究其生物学功能的各种哺乳动物细胞系。

采用本文所概述的方法, 本实验的目的是研究在造血细胞转化过程中引入 indel 突变对内源性Calr轨迹的功能效应。CRISPR/Cas9 系统被用作在 Ba/F3 细胞中创建内源性的Calr突变的工具。选择了两个 sgRNAs 来针对Calr (图 1) 的外显子 9, 在该区域中, 插入和/或删除 (indel) 突变通常发生在Calr变种 MPN 患者17、<sup ...

Watch this Scientific Journal Video about Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells at JoVE.com

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

有针对性的基因编辑使用 CRISPR/Cas9 极大地促进了对基因的生物功能的理解。在这里, 我们利用 CRISPR/Cas9 的方法, 模型钙突变的细胞因子依赖性造血细胞, 以研究其致癌活动。

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

using-crisprcas9-gene-editing-to-investigate-oncogenic-activity

Research

JoVE Journal

Cancer Research

13.0K 次观看.  Brigham and Women's Hospital, Harvard Medical School. 有针对性的基因编辑使用 CRISPR/Cas9 极大地促进了对基因的生物功能的理解。在这里, 我们利用 CRISPR/Cas9 的方法, 模型钙突变的细胞因子依赖性造血细胞, 以研究其致癌活动。

视频: Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

科研

遗传学

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

癌症研究

Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.

Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.

将 CRISPR/Cas9 技术与细胞计数相结合评估造血干细胞和祖细胞生长相关基因的异常

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. ...

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

<meta charset="utf8"></head><body>向导 RNA 的逆转录病毒转导用于 Th17 分化中的基因编辑

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

<meta charset="utf8"></head><body>逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

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此视频中的章节

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增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

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CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

将 CRISPR/Cas9 技术与细胞计数相结合评估造血干细胞和祖细胞生长相关基因的异常

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

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化