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Scientists Create CRISPR/Cas9 Knock-In Mutations in Human T Cells
Jul 27, 2015
| a GenomeWeb staff reporter
NEW YORK (GenomeWeb) – Scientists have found a way to add genes to and delete genes from T cells using CRIPSR/Cas9 genome editing, according to a new study published today in the Proceedings of the National Academy of Sciences.
Led by Kathrin Schumann and Alexander Marson from the University of California, San Francisco, and Steven Lin and Jennifer Doudna from the University of California, Berkeley, the scientists were able to generate knock-in mutations in human T cells using Cas9 ribonucleoproteins (RNPs).
The RNPs are preassembled structures consisting of a Cas9 nuclease and the guide RNA (gRNA). Rather than introduce plasmids encoding Cas9 and the gRNAs into the cell using a viral vector, the scientists used electroporation to introduce the RNPs into the cell.
"We tried for a long time to introduce Cas9 with plasmids or lentiviruses, and then to express separately the single-guide RNA in the cell," Schumann, a postdoc in Marson's laboratory, said in a statement. "Using RNPs made outside the cell, so that the cell is responsible for as little of the process as possible, has made a big difference."
The simplest way to perform CRISPR/Cas9 genome editing is to make a double-stranded break and allow the crude repair mechanism of non-homologous end joining to create a knockout. Alternatively, researchers can also leverage the homology-directed repair (HDR) pathway to create a knock in if a repair template is provided. But getting genome editing to work in human T cells — where there is great potential for immunotherapies for cancer and HIV/AIDS, among other areas — has been "a notable challenge," Marson said, adding he and his colleagues have spent the past year-and-a-half "trying to optimize editing in functional T cells."
The researchers showed that the Cas9 RNPs could not only edit cells' genomes to remove CXCR4 and PD-1 receptors, but also perform the HDR necessary for knock-in editing, reaching knock-in efficiencies of about 20 percent using Cas9 RNPs. HIV uses CXCR4 receptors to infiltrate T cells and PD-1 is an immune checkpoint exploited by cancers to evade apoptosis. The researchers said that the specific pairing of programmed Cas9 RNPs and corresponding HDR templates are required to selectively edit T cell genomes at the nucleotide level. They also added that fluorescence-activated cell sorting (FACS) had the potential to be a useful tool in T cell editing because it could enrich the population of edited cells.
On the research side, the authors suggested that T cell editing could help scientists with experiments on transcription factors, cis-regulatory elements, and genes implicated in T cell function. Of course, there are potential therapeutic applications for T cell editing and the authors said their findings supported that notion.
"There's increasing clinical infrastructure that we could potentially piggyback on as we work out more details of genome editing," Marson said. "I think CRISPR-edited T cells will eventually go into patients, and it would be wrong not to think about the steps we need to take to get there safely and effectively."
科学家创建CRISPR / Cas9敲入突变的人类T细胞
2015年7月27日
|一个GenomeWeb本刊记者
纽约（GenomeWeb） - 科学家发现了一种基因添加到和删除基因使用CRIPSR / Cas9基因组编辑的T细胞，根据今天在美国国家科学院的论文集出版的一项新的研究。
由凯萨琳·舒曼和美国加州大学旧金山分校亚历山大马森，和史蒂芬林和珍妮弗Doudna来自加州大学伯克利分校的领导下，科学家们能够生成敲入使用Cas9核糖人类T细胞突变（体RNP） 。
的的RNP预组装结构组成的Cas9核酸和指导RNA（gRNA）的。而不是引入质粒编码Cas9和gRNAs成使用病毒载体的细胞，科学家使用电穿孔引入的RNP进入细胞。
“我们尝试了很长的时间来与质粒或慢病毒引入Cas9，然后分别表达了单导的RNA在细胞中，”舒曼，在马森的实验室做博士后，在一份声明中说。 “使用电池以外所作的RNP，从而使细胞负责尽可能少的过程成为可能，已经取得了很大的不同。”
执行CRISPR / Cas9基因组编辑的最简单的方法是使一个双链断裂和允许的非同源末端连接以创建一个敲除粗修复机制。可替代地，研究人员还可以利用同源定向修复（HDR）途径如果提供了修复模板中创建一个爆震。但得到的基因组编辑在人类T细胞工作 - 那里是免疫治疗癌症和艾滋病病毒/艾滋病，以及其他领域的巨大潜力 - 一直以“一个巨大的挑战，”马森说，加入他和他的同事们在过去的一年 - 和一个半“，试图在功能性T细胞的优化编辑。”
研究人员表明Cas9的RNP不仅可以编辑细胞的基因组中以除去CXCR4和PD-1受体，但也执行HDR必要敲入编辑，达到敲除中的大约20％使用Cas9的RNP效率。 HIV使用CXCR4受体渗透到T细胞和PD-1是一种免疫检查点由癌症利用来逃避凋亡。研究人员说，编程Cas9的RNP及相应的HDR模板配对的具体要求在核苷酸水平有选择性地编辑T细胞的基因组。他们还增加了荧光激活细胞分选（FACS）必须是在T细胞的编辑一个有用的工具，因为它能够丰富编辑细胞群的潜力。
在研究方面，笔者认为，T细胞的编辑可以帮助科学家对转录因子，顺式调控元件，并参与了T细胞功能的基因实验。当然，也有潜在的治疗应用的T细胞的编辑和作者说，他们的发现支持了这种说法。
“有越来越多的临床基础设施，我们有可能搭载在为我们制定出基因组编辑的详细信息，”马森说。 “我认为CRISPR-编辑的T细胞将最终进入患者，这将是考虑我们需要到那里安全和有效的步骤没有错。 |
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楼主: StephenW
发表于 2015-6-3 20:34
