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
<ol><li>
设计 sgRNAs 的目标基因的兴趣使用免费的在线工具。
	<ol>
<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>
</li>
		<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
<ol><li>
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。
</li>
	</ol>
</li>
	<li>
生成反向寡聚
	<ol>
<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>
	<li>
退火和化寡
	<ol>
<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>
</li>
</ol>3. lentiGuide-普洛矢量的消解13
<ol><li>
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>
</li>
	<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>
	<li>
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>
</li>
</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。
<ol><li>
染 HEK-293T 细胞
	<ol>
<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>
</li>
	<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>
</li>
</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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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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

舌细胞外基质的制备与舌鳞癌体外重建

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

基于 crispr-cas9 的竞争检测对遗传依赖性的调查

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

使用 CRISPR/Cas9 在 CAR-T 电池中敲除 GM-CSF

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

细胞毒性细胞因子诱导的杀伤性T细胞的分离和扩张，用于癌症治疗

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

使用转录组学分析绘制无序致癌转录因子的结构-功能关系

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.