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DNA条形码新技术实现原位标记
作者:小柯机器人 发布时间:2019/11/19 15:14:21

美国加州理工学院Michael B. Elowitz研究小组利用体外转录实现了在原位读出DNA条形码并进行单基编辑的新技术。2019年11月18日《自然—生物技术》在线发表了该成果。

研究人员报道了一种基于图像的短(20个碱基对)DNA条形码读取系统。在这个称为Zombie的系统中,噬菌体RNA聚合酶在固定细胞中转录工程条形码。随后通过荧光原位杂交检测所得的RNA。使用竞争的匹配和不匹配探针,Zombie可以准确区分条形码中的单核苷酸差异。这种方法无需活细胞中的条形码表达即可在原位读出由CRISPR碱基编辑器产生的密集组合条形码文库和单碱基突变。Zombie在各种环境中起作用,包括细胞培养、雏鸡胚胎和成年小鼠脑组织。通过成像灵敏地读出紧凑而多样的DNA条形码的能力将促进广泛的条形码和基因组记录策略。

据了解,条形码识别的局限性阻碍了唯一识别单个细胞的分子条形码技术。通过测序读出不能保留组织中细胞的空间组织,而成像方法可以保留空间结构,但对条形码序列不那么敏感。

附:英文原文

Title: In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription

Author: Amjad Askary, Luis Sanchez-Guardado, James M. Linton, Duncan M. Chadly, Mark W. Budde, Long Cai, Carlos Lois, Michael B. Elowitz

Issue&Volume: 2019-11-18

Abstract: Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

DOI: 10.1038/s41587-019-0299-4

Source: https://www.nature.com/articles/s41587-019-0299-4

期刊信息

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:31.864
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex