Name: silencing the spark: crispr/cas9 genome editing in weakly electric fish
Uploaded: 2019-10-27T14:00:00
Description: Watch this Scientific Journal Video about Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish at JoVE.com

作者感谢莫妮卡·卢卡斯、凯瑟琳·肖、瑞安·泰勒、贾里德·汤普森、妮可·罗比乔和霍普·希利在鱼类养殖、数据收集和早期协议开发方面所付出的英勇努力。我们还要感谢三位审评员对手稿的建议。我们相信，在发表他们的意见后，最终产品的质量会更好。这项工作由美国国家科学基金会#1644965和#1455405资助，并资助了自然科学和工程研究理事会向VLS提供DG赠款。

此处描述的所有方法均已获得密歇根州立大学机构动物护理和使用委员会 （IACUC） 的批准。
1. 选择sgRNA靶点
注：在步骤 1.1 中为 sgRNA 的手动设计提供了一个协议。这被用于scn4aa目标选择。提供了一个附加协议，以方便此过程（步骤 1.2）使用 EFISHGENOMICS Web 门户。建议用户选择协议 1.2，它具有几个自动"检查"功能，以确保成功为自定义目?...

弱电鱼的表型丰富性，加上最近基因组学资源的扩散，激发了弱电鱼模型中对功能基因组工具的强烈需求。该系统特别有吸引力，因为鱼类平行谱系中许多型特征的收敛进化，这些特征很容易保存在实验室中。
此处描述的协议演示了 CRISPR/Cas9 技术在同时进化电成和电接收的弱电鱼谱系中的有效性，因此代表了该模型承诺解决的一个重要步骤表型进化的比较基因组学的未来工?...

在脊椎动物的进化史上，电接收和电发生已经发生了变化。两个谱系的远发鱼，骨状和硅状，平行地进化电接收，和五个谱系的远程（金体素，莫米里德，和属星体，马拉普特鲁鲁斯，和西诺多特斯）并联进化电发生。这些独立衍生的表型具有惊人的收敛性，它们共享着一个共同的遗传结构1，2，3。
这也许最好的例证是，健身房和莫米里德的众多收敛特征，这两个物种丰富的电镀层，产生和检测弱电场，被称为弱电鱼。在发现弱电鱼利用电来感知周围环境和交流的50年里，越来越多的科学家对发展的演变有了巨大的了解。 ，6， 系统和电路神经科学7，8，9，10，细胞生理学11，12，生态学和能量13，14，15，16，17，行为18，19，和宏观进化3，20，21.
最近，电鱼1、22、23、24、25、26的基因组、转录组和蛋白酶资源激增。 27，28.这些资源的使用已经产生了关于基因型和表型在这些物种1，2，3，28，29之间的联系的重要见解 ， 30.将基因组学数据与弱电鱼表型数据整合的一个主要障碍是目前缺乏功能基因组学工具31。
其中一个工具是最近开发的聚类定期间隔短帕林德罗次，与Cas9内分酶（CRISPR/Cas9，CRISPR）技术配对。CRISPR/Cas9是一种基因组编辑工具，在模型32、33、34和非模型生物体35、36、37中都广泛使用。CRISPR/Cas9技术已经发展到一个点，一个实验室能够基本分子生物学可以很容易地产生基因特异性的探针称为短导RNA（sgRNA），使用非克隆方法38低成本。CRISPR 比其他敲除/敲除策略具有优势，例如变形39、40、转录激活器样效应素核酸酶（TALEN）和锌指核酸酶（ZFN），这些策略成本高昂，为每个目标基因生成非常耗时。
CRISPR/Cas9 系统通过针对基因组的特定区域（由 sgRNA 序列引导）创建基因敲除，并造成双链断裂。细胞检测到双链断裂，并优先使用非同源端连接（NHEJ）路径触发内源性DNA修复机制。此通路极易出错：在修复过程中，DNA 分子通常会在双链断裂部位插入或删除（indels）。这些印子可能导致功能丧失，原因包括 （1） 打开阅读框架中的偏移，（2） 插入过早停止的芯子，或 （3） 基因产物的关键初级结构的偏移。在此协议中，我们使用CRISPR/Cas9编辑，在弱电鱼种中使用NHEJ来靶向目标基因的点突变。虽然比其他技术更简单、更有效，但这种诱变方法预计在F0中会产生一系列表型分裂，这归因于遗传马赛克41、42、43 ， 44.
有机物的选择 为了便于今后研究弱电鱼的比较基因组学，需要选择具有代表性的品种，用于植物体和摩尔米里德的礼仪开发。在乌拉圭蒙得维的亚举行的2016年电鱼会议上，经过讨论，社区达成共识，利用已在实验室中培育且有基因组资源的物种。健身房形式布拉奇希波姆高德里奥和莫米里德布里诺米鲁斯小球被选为符合这些标准的物种。在这两个物种中，诱导和维持繁殖条件的自然线索在圈养中很容易模仿。B. gauderio，一种来自南美洲的健身房品种，具有低饲养要求的优势：鱼可以在相对较小的（4L）水箱中保持相对较高的密度。在俘虏条件下，高德里奥也有快速的代际周转。在实验室条件下，B.高德里奥可以在4个月内从卵子发育到成人。
B. 小鱼，一种来自中西部非洲的莫米里德鱼，在圈养中繁殖容易。B. 近距离比乌斯通过水族馆贸易很容易获得，已被广泛用于许多研究，现在有一些基因组资源可用。其生命周期为 1⁄3 年，具体取决于实验室条件。对于该物种，养殖需求较为密集，由于在繁殖过程中受到攻击，需要中等大小的水箱（50–100 L）。
研究其他种类的电鱼的实验室应该能够轻松地适应这一协议，只要该物种可以繁殖，并且单细胞胚胎可以收集和饲养到成年。住房、幼虫饲养和体外受精（IVF）率可能会随着其他物种的变化而变化;然而，该协议可以作为其他弱电鱼繁殖尝试的起点。
概念验证的理想基因靶点：scn4aa 弱电虫和健美鱼通过释放一种称为电器官的专用器官产生电场（电发生）。电器官放电（EODs）是电细胞中称为电细胞同时产生作用电位的结果。EOD被皮肤中的电受体阵列检测到，以产生鱼类周围环境的高分辨率电图像45。弱电鱼还能够检测其共分物的EOD波形18及其放电速率46的特征，使EOD能够另外作为类似于鸟鸣或青蛙的社会通信信号发挥作用发声47.
在莫米里德和健力酸弱电鱼的电细胞中，作用电势生成的主要成分是电压门钠通道NaV1.42。非电电电表达两个副基因副本，scn4aa和scn4ab，编码为电压门钠通道NaV1.430。在健身房和莫米里德弱电鱼系中，scn4aaa发展迅速，经历了许多氨基酸替代，影响其动力学特性48。最重要的是，scn4aaa在电器官2、3的两个谱系中都变得分门别类。scn4aa对电器官的表达相对受限，在EOD的产生中起着关键作用，使其成为CRISPR/Cas9敲除实验的理想目标，因为它具有最小的有害性多效效应。由于弱电鱼在受精后6-8天开始排出幼虫电器官（DPF），因此，scn4aa的靶向非常适合在胚胎显注后快速表示。

<table>
	<tbody>
		<tr>
			<td>20 mg/mL RNA grade Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
			<td></td>
		</tr>
		<tr>
			<td>50 bp DNA ladder</td>
			<td>NEB</td>
			<td>N3236L</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillary with filament</td>
			<td>Sutter Instrument</td>
			<td>BF100-58-10</td>
			<td>(O.D. 1.0mm, I.D. 0.58 mm, 10 cm length)</td>
		</tr>
		<tr>
			<td>Cas9 protein with NLS; 1 mg/mL</td>
			<td>PNA Biology</td>
			<td>CP01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dneasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf FemptoJet 4i Microinjector</td>
			<td>Fisher Scientific</td>
			<td>E5252000021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader Pipette Tips</td>
			<td>Fisher Scientific</td>
			<td>10289651</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Fisher Scientific</td>
			<td>14-824-654</td>
			<td>referred to as &#34;precision glass syringe&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td>referred to as &#34;delicate task wipe&#34; in the protocol</td>
		</tr>
		<tr>
			<td>MEGAscript T7 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 3</td>
			<td>NEB</td>
			<td>B7003S</td>
			<td>used for in vitro cleavage assay</td>
		</tr>
		<tr>
			<td>OneTaq DNA kit</td>
			<td>NEB</td>
			<td>M0480L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovaprim</td>
			<td>Syndel USA</td>
			<td>https://www.syndel.com/ovaprim-ovammmlu010.html</td>
			<td>referred to as &#34;spawning agent&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>S37440</td>
			<td>referred to as &#34;thermoplastic&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>WPI</td>
			<td>SU-P97</td>
			<td>sutter brand</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable needle- requires customization</td>
			<td>Fisher Scientific</td>
			<td>7803-02</td>
			<td>Customize to 0.7 inches long; point style 4 and angle 25</td>
		</tr>
		<tr>
			<td>T4 DNA polymerase</td>
			<td>NEB</td>
			<td>M0203L</td>
			<td>Use with the 10X NEB buffer that is included</td>
		</tr>
		<tr>
			<td>Teflon coated tools</td>
			<td>bonefolder.com</td>
			<td>T-SPATULA4PIECE</td>
			<td>referred to as &#34;polytetrafluoroethene&#34; in the protocol</td>
		</tr>
	</tbody>
</table>

silencing the spark: crispr/cas9 genome editing in weakly electric fish

在脊椎动物的进化史上，电接收和电发生已经发生了变化。这些独立衍生的表型具有惊人的收敛性，它们有着共同的遗传结构。这也许最好的例证是健身房和莫米里德的众多收敛特征，这两种物种丰富的电镀层能够产生和检测弱电场，被称为弱电鱼。自从发现弱电鱼用电来感知周围环境和交流以来的50年里，越来越多的科学家对发展、系统和电路神经科学的演变有了巨大的见解，细胞生理学、生态学、进化生物学和行为学。最近，电鱼的基因组资源激增。这些资源的使用已经促进了对这些物种基因型和表型之间联系的重要见解。将基因组学数据与弱电鱼表型数据集成的一个主要障碍是目前缺乏功能基因组学工具。我们在这里报告一个完整的协议，执行CRISPR/Cas9诱变，利用内源性DNA修复机制在弱电鱼。我们证明，该协议在莫米里德物种布诺米鲁斯小虫和健身房，通过使用CRISPR/Cas9来靶向第一个外显子中的印贝和点突变，同样有效。钠通道基因scn4aa。使用该协议，从两个物种获得胚胎和基因型，以确认在钠通道scn4aa的第一个外向的预测突变存在。与未注射大小匹配的对照组相比，记录显示电器官放电振幅降低，证实了敲除成功的表型。

sgRNA靶点位于B.gauderio和B.b.brachyisus的scn4aa的外量1内，如第1节所述。sgRNA 的生成情况如下，如第 2 节所述。在成功选择和合成sgRNA（图1）后，对体外裂解进行了测试（图2）。然后选择在体外切割的sgRNA进行单细胞显微注射。
成年鱼被以繁殖为条件（第4.1节），然后注射产卵剂（第4.2节），然后挤压（B.gauderio?...

Watch this Scientific Journal Video about Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish at JoVE.com

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

在这里，提出了一个协议，以生产和后CRISPR/Cas9基因组敲除电鱼。详细列出了运动和生物形态所需的分子生物学、育种和饲养要求，以及生产 Cas9 诱导 Indel F0幼虫的注射技术。

沉默的火花：CRISPR/Cas9基因组编辑在弱电鱼

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

CRISPR/Cas9基因编辑在弱电鱼，特别是在比较的背景下，将使研究人员能够测试假设，具体基因和基因差异如何促成表型变异。CRISPR/Cas9是产生突变F-零胚胎的高效方法。该协议允许研究人员对两种融合进化的弱电鱼进行CRISPR-Cas9操作。将这种方法与其他电鱼或其他鱼类物种进行一些修改时使用的关键，是能否获得转录或基因组资源来制作 sgRNA。在 2D 平面内的 3D 位置执行微注射和可视化单元格可能很困难。获得一个小斑马鱼群，供鸡蛋练习一般的微注射技术。与萨瓦斯·康斯坦丁努一起演示手术的将是贾里德·汤普森，一个来自我实验室的技术人员。在适当的繁殖条件下，在饲养感兴趣的鱼类品种后，确定出现格雷维德的雌性鱼。在B.gauderio，女性会肿胀的性腺只是考达尔到通风口。在B.腹皮，女性会有肿胀的肚子，并出现深体。麻醉后，添加产卵剂到四倍的PCR管的DPCS体积与彻底混合。解决方案将变得多云。在将溶液绘制到装有 28 量表 19 至 25 毫升长度斜面针的精密玻璃注射器中之前，使用移液器测量计算出的剂量，将稀释剂分配到一块热塑性塑料中。以平滑的速度将溶液注入背部躯干肌肉，让针头在去除前坐两到四秒钟。然后，立即将鱼放入淡水中进行回收。对于B.gauderio精子的收集，24小时后，选择一只大型雄性鱼，并在重新加水后，彻底干燥鱼，特别是头部和通风口周围。将干鱼的腹侧向上，在适当的支架左侧，用 MS222 浸泡的湿纸巾覆盖，头部与桌子尽可能平行。立即施加压力，在孔达尔到旋转方向，光挤压立即对风向向通风口，并使用带尖端的微管，小心收集精子，因为它被挤压从男性在50微升增量。将等分直接添加到500微升的精子扩展器溶液上。收集后，立即将鱼放入粘液涂层保护产品处理的新鲜系统水。对于 B.gauderio 卵子收集，干燥麻醉的 B.gauderio 雌鱼后，立即将鱼放在其一侧，头部朝向非多米诺手，并在鱼尾中施加压力，向栖息方向施加压力，用光将局部挤压到通风口。使用聚四氟乙烯工具，小心收集鸡蛋，因为它们被挤压从氯卡，并迅速将鸡蛋放在一个小的覆盖培养皿。如果挤压后独自工作，触摸鸡蛋质量到小培养皿的基础，因为他们被逐出女性。收集鸡蛋后，立即将鱼放在粘液涂层保护产品处理的新鲜系统水中。对于B.gauderio体外受精，直接在卵质量上加入100微升的混合精子SES溶液，然后加入1毫升的新鲜系统水。将游戏液完全混合30至60秒，然后向细胞中再加入1至2毫升0.22微米孔过滤系统水。用体外受精时间标记菜，将菜放入29摄氏度的培养箱中，设置50分钟的定时器，以检查开发进度。在施肥期间，使用微载重移液器尖端将感兴趣的注射溶液的两微升回装到微注射针中，并尽可能将液体排出靠近尖端。当单个细胞在至少一个细胞的动物极形成时，在解剖显微镜下转移盘子，并使用一对细钳子以斜角断开针尖，朝针的锥度开始表现出刚性的位置。针的孔应保持尽可能小，同时保持刚性锥度。在显微镜下识别正在发育的酶，并使用带切尖的塑料转移移液器，将 10 到 20 个鸡蛋放在尽可能少的水中，放在 Petri 盘倒置顶部的玻璃显微镜幻灯片的边缘。使用精细的任务擦拭，轻轻按压幻灯片以去除多余的水，使鸡蛋牢牢地粘在幻灯片的边缘。水在滑梯下会很邪恶，把鸡蛋拉到边缘。应该有足够的水来保持鸡蛋的湿润，同时保持很少的站立水分，以避免鸡蛋在注射过程中移动。将鸡蛋垂直地与滑梯对齐，垂直于接近的针头，并使用微操纵器将针头对角。然后，以大约 45 度角握住针头，先将尖端插入胆小球，然后插入单个细胞，如果可能，通过蛋黄注射。在注射过程中用细钳取出任何破鸡蛋。当所有鸡蛋都注射后，使用喷瓶0.22微米孔过滤系统水，轻轻地将鸡蛋从注射阶段冲洗成新的100毫米直径的培养皿。在成功的短指南RNA选择和合成后，体外裂解可以测试单细胞微注射的选择。经过PCR清理和克隆，在这个代表性的实验中，CRISPR-Cas9诱导突变在单个B.gauderio和B.b.B.b.9诱导突变中被识别出来，这些克隆具有很强的电器官放电幅度减小，而未诱导的对子只显示参考基因型。确认的CRISPR突变体与未释放的对控器之间的电器官放电振幅的可视化表明，SCN-4 AA突变体B.b.brachyistius和B.gauderio胚胎的放电幅度都明显低于对联。进行微注射时，尽可能快速和刻意地移动，以最大限度地增加存活的单细胞注射胚胎的数量。不要浪费时间在困难鸡蛋。一旦成功突变体被识别出来，传统的育种方法就可以创建稳定的突变线，从而消除CRSPR相关的马赛克问题。该协议将允许研究人员进行比较基因组研究，在两个融合进化的弱电鱼物种的功能验证。

silencing-the-spark-crisprcas9-genome-editing-in-weakly-electric-fish

Research

JoVE Journal

Biology

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

科研

生物学

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

的弱散射主题内嵌数字全息显微镜（DIHM）

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

基因组编辑在<em&gt; Astyanax mexicanus</em&gt;使用转录因子样效应核酸酶（TALENS）

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

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 ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&amp;#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

TGF-β介质到中微过渡（末端MT）和使用CRISPR/Cas9基因编辑对末端效应器的功能评估

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

基于CRISPR/Cas9技术构建迁徙蝗虫纯合突变体

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...