image.png

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

老規(guī)矩铣减，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

專業(yè)名詞：

Cellular barcoding：細(xì)胞條形碼她君。通過對細(xì)胞進(jìn)行打碼谷朝，從而可以對細(xì)胞譜系進(jìn)行追蹤和溯源糊探。

1. Principles and methods of cellular barcoding

（1）Labelling cells with nucleic acid sequences. 通過特定的核酸序列，形成細(xì)胞標(biāo)記的條形碼杀迹。假設(shè)barcode的長度是10個bp劣针，那么就會有4¹⁰次方種不同的barcode校镐。

情況一： 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.

情況二：barcode還可以是半隨機(jī)或者形成層疊，雖然在barcode多樣性上有些折扣捺典，但卻更容易糾錯和體內(nèi)barcoding鸟廓。

情況三：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. 通過結(jié)合CRISPR-Cas9技術(shù)，隨機(jī)打靶敲除產(chǎn)生barcode襟己。

(2) Methods of barcode delivery

就是不想寫了

image.png

問題：體外barcode效率很高引谜，但體內(nèi)不太行，比較好的解決辦法就是上面提到的:

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

image.png

CRISPR-based in vivo barcoding

上面都是用重組酶（基因編輯工具之一）來完成的barcoding擎浴，那么CRISPR作為新興基因編輯工具员咽，肯定也會被重點(diǎn)研究。只能用優(yōu)秀來形容了贮预。

優(yōu)秀

image.png

一覽表

image.png

(3) Methods of barcode readout.

barcode都想方設(shè)法裝上去了贝室，那么怎么讀么？

對于體外單層細(xì)胞實(shí)驗(yàn)：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.
從PCR擴(kuò)增仿吞，分子量大小鑒定到芯片檢測滑频，桑格測序和高通量測序。舉個例子來說茫藏，單細(xì)胞RNA測序（scRNA-seq）可以幫助讀取單個細(xì)胞的barcode误趴。結(jié)合cellular barcoding和scRNA-seq，可以達(dá)到在基因組篩選务傲，細(xì)胞譜系追蹤和神經(jīng)解剖學(xué)層面的研究凉当。

對于體內(nèi)實(shí)驗(yàn)： 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

每一種方法都要自己的缺點(diǎn)

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. 在細(xì)胞譜系追蹤過程中，一個細(xì)胞的發(fā)育歷史將會被解析售葡，并且形成發(fā)育樹看杭，而在命運(yùn)圖譜中，細(xì)胞起源于特定的組細(xì)胞是重要的鑒定內(nèi)容挟伙，而中間的過程可能并沒有太多明確的要求楼雹。
Lineage tracing requires that cells in intermediate states receive distinct labels. Note that in the literature, “l(fā)ineage tracing” is also used as an umbrella term encompassing all methods that interrogate cellular lineage, and as such often includes fate-mapping approaches. 細(xì)胞譜系追求要求細(xì)胞擁有明確的特定標(biāo)簽，在文章中，“譜系追蹤”也被當(dāng)做一個大的命題來描述贮缅，其中包括細(xì)胞發(fā)育譜系和細(xì)胞命運(yùn)譜圖榨咐。

(2) High-throughput screens.

(3) Mapping the brain with barcodes.

(4) Barcodes as molecular recording devices.

另外推薦一篇文獻(xiàn)：Kalhor R, Kalhor K, Mejia L, et al. Developmental barcoding of whole mouse via homing CRISPR[J]. Science, 2018, 361(6405): eaat9804.