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快速进化的归巢CRISPR条形码。

Rapidly evolving homing CRISPR barcodes.

作者信息

Kalhor Reza, Mali Prashant, Church George M

机构信息

Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

Department of Bioengineering, University of California San Diego, La Jolla, California, USA.

出版信息

Nat Methods. 2017 Feb;14(2):195-200. doi: 10.1038/nmeth.4108. Epub 2016 Dec 5.

Abstract

We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9-hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.

摘要

我们提出了一种在活细胞中构建不断进化的DNA条形码的方法。一种归巢引导RNA(hgRNA)支架将Cas9-hgRNA复合物引导至hgRNA自身的DNA位点。我们表明,这种归巢CRISPR-Cas9系统作为一种表达的遗传条形码,其序列会多样化,并且在培养细胞中可以控制多样化的速率。我们进一步在细胞群体中评估了这些条形码,并表明它们可用于记录谱系历史,并且条形码RNA可以原位扩增,这是原位测序的一个先决条件。这种综合方法将有广泛的应用,如在深度谱系追踪、细胞条形码、分子记录、剖析癌症生物学和连接体图谱绘制等方面。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce6f/5322472/61b40db8a352/nihms831612f1.jpg

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