细胞谱系示踪条形码，Cellular barcoding: lineage tracing, screening and beyond

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Kebschull J M, Zador A M. Cellular barcoding: lineage tracing, screening and beyond[J]. Nature methods, 2018: 1.
原文链接：https://doi.org/10.1038/s41592-018-0185-x

老规矩，先文章框架：

Title
Abstract
Introduction

1. Principles and methods of cellular barcoding
   (1) Labeling cells with nucleic acid sequences.
   (2) Methods of barcode delivery.
   (3) Methods of barcode readout.
2. Applications of cellular barcoding
   (1) Lineage tracing and fate mapping
   (2) High-throughput screens.
   (3) Mapping the brain with barcodes.
   (4) Barcodes as molecular recording devices.
3. Outlook

专业名词：

Cellular barcoding：细胞条形码。通过对细胞进行打码，从而可以对细胞谱系进行追踪和溯源。

1. Principles and methods of cellular barcoding

（1）Labelling cells with nucleic acid sequences. 通过特定的核酸序列，形成细胞标记的条形码。假设barcode的长度是10个bp，那么就会有4¹⁰次方种不同的barcode。

情况一： A random 10-bp barcode therefore can assume any of 4¹⁰ (~10⁶) different sequences, and a random 30-bp barcode can assume any of 4³⁰ (~10¹⁸) different sequences, each of which can act as a unique label.

情况二：barcode还可以是半随机或者形成层叠，虽然在barcode多样性上有些折扣，但却更容易纠错和体内barcoding。

情况三：Finally, barcodes can be generated via random deletions in known sequences, as is common in CRISPR–Cas9-based methods. Which barcode type is chosen for any given study depends largely on the required barcode diversity and the method used to read out the barcodes. 通过结合CRISPR-Cas9技术，随机打靶敲除产生barcode。

(2) Methods of barcode delivery

就是不想写了

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问题：体外barcode效率很高，但体内不太行，比较好的解决办法就是上面提到的:

As discussed below, implementations of such in vivo barcoding rely on either the shuffling of sequence fragments or the introduction of random insertions or deletions at a specific site. So far, neither approach has been able to generate
sufficient diversity to exhaustively label an adult vertebrate, but the
field is progressing quickly

Recombinase-based in vivo barcoding

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CRISPR-based in vivo barcoding

上面都是用重组酶（基因编辑工具之一）来完成的barcoding，那么CRISPR作为新兴基因编辑工具，肯定也会被重点研究。只能用优秀来形容了。

优秀

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一览表

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(3) Methods of barcode readout.

barcode都想方设法装上去了，那么怎么读么？

对于体外单层细胞实验：The methods used to read out barcodes have varied with the available technology, beginning with PCR amplification and sizing and progressing to microarray detection, Sanger sequencing and high-throughput sequencing. In a paradigm shift, scRNA-seq approaches have recently been applied to dissociated cells for the simultaneous readout of a cell’s barcode and transcriptome. This is an extremely powerful approach, as it combines information about cellular history or anatomy from the barcode with the independently measured high dimensional phenotype of the cell’s transcriptional state and transcriptional cell type. The combination of cellular barcoding with scRNA-seq has been exploited in genome-wide screens, lineage-tracing approaches and neuroanatomy studies.
从PCR扩增，分子量大小鉴定到芯片检测，桑格测序和高通量测序。举个例子来说，单细胞RNA测序（scRNA-seq）可以帮助读取单个细胞的barcode。结合cellular barcoding和scRNA-seq，可以达到在基因组筛选，细胞谱系追踪和神经解剖学层面的研究。

对于体内实验： Recently, the MEMOIR method was used with highly multiplexed fluorescence in situ hybridization (FISH) to read out a combination of in vitro and CRISPR–Cas9-generated barcodes.

In situ sequencing approaches

每一种方法都要自己的缺点

2. Applications of cellular barcoding

（1）Lineage tracing and fate mapping.

Here, we distinguish between the related concepts of lineage tracing and fate mapping. In lineage tracing, the developmental history of a cell is decoded and a tree is produced, whereas in fate mapping, cells originating from a specific progenitor are marked identically, which can obscure information about intermediate steps. 在细胞谱系追踪过程中，一个细胞的发育历史将会被解析，并且形成发育树，而在命运图谱中，细胞起源于特定的组细胞是重要的鉴定内容，而中间的过程可能并没有太多明确的要求。
Lineage tracing requires that cells in intermediate states receive distinct labels. Note that in the literature, “lineage tracing” is also used as an umbrella term encompassing all methods that interrogate cellular lineage, and as such often includes fate-mapping approaches. 细胞谱系追求要求细胞拥有明确的特定标签，在文章中，“谱系追踪”也被当做一个大的命题来描述，其中包括细胞发育谱系和细胞命运谱图。

(2) High-throughput screens.

(3) Mapping the brain with barcodes.

(4) Barcodes as molecular recording devices.

另外推荐一篇文献：Kalhor R, Kalhor K, Mejia L, et al. Developmental barcoding of whole mouse via homing CRISPR[J]. Science, 2018, 361(6405): eaat9804.