Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPR-Cas9 barcodes by scGESTALT.
Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPR-Cas9 barcodes by scGESTALT.
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DOI：
10.1038/s41596-018-0058-x
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发表时间：
2018-11
期刊：
影响因子：
14.8
通讯作者：
Schier AF
中科院分区：
文献类型：
--
作者：
Raj B;Gagnon JA;Schier AF
在发育过程中产生的大量异质细胞类型之间的谱系关系难以以高通量方式重建。我们最近建立了一种方法scGESTALT，该方法将CRISPR-Cas9对谱系条形码阵列的累积编辑与使用基于液滴的单细胞RNA测序的大规模转录谱分析相结合。该技术使用在早期和晚期斑马鱼胚胎发育期间引入的Cas9和单向导RNA池在多个时间点上在条形码阵列中生成编辑，这将其与类似的Cas9谱系追踪方法区分开来。记录的谱系与数千个细胞转录组一起被捕获沿着，以构建具有数百个分支的谱系树，这些分支表示谱细胞类型之间的关系。在这里，我们提供了（i）生成转基因斑马鱼;（ii）进行多时间点条形码编辑;（iii）从脑组织构建单细胞RNA-seq文库;以及（iv）从捕获的单细胞同时扩增谱系条形码的细节。生成转基因系需要6个月的时间，而进行条形码编辑和生成单细胞文库需要7天的动手时间。scGESTALT提供了一个可扩展的平台，可以在任何允许在发育、再生或疾病期间进行基因组编辑的系统中映射细胞类型之间的谱系关系。该方案描述了如何生成表达条形码阵列的转基因斑马鱼，该条形码阵列可以在多个发育阶段通过CRISPR/Cas9编辑。编辑的条形码和细胞转录组的单细胞RNA测序允许重建谱系关系。
Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR-Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing. The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage tracing methods. The recorded lineages are captured along with thousands of cellular transcriptomes to build lineage trees with hundreds of branches representing relationships among profiled cell types. Here we provide details for (i) generating transgenic zebrafish; (ii) performing multi-timepoint barcode editing; (iii) building single-cell RNA-seq libraries from brain tissue; and (iv) concurrently amplifying lineage barcodes from captured single cells. Generating transgenic lines takes 6 months while performing barcode editing and generating single-cell libraries involve 7 days of hands-on time. scGESTALT provides a scalable platform to map lineage relationships between cell types in any system that permits genome editing during development, regeneration or disease. This protocol describes how to generate transgenic zebrafish expressing a barcode array that can be edited by CRISPR/Cas9 at multiple developmental stages. Single cell RNA sequencing of edited barcodes and cellular transcriptomes allows reconstruction of lineage relationships.
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影响因子：
64.8
作者：
Halpern KB;Shenhav R;Matcovitch-Natan O;Toth B;Lemze D;Golan M;Massasa EE;Baydatch S;Landen S;Moor AE;Brandis A;Giladi A;Avihail AS;David E;Amit I;Itzkovitz S
通讯作者：
Itzkovitz S
影响因子：
9.3
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Baron M;Veres A;Wolock SL;Faust AL;Gaujoux R;Vetere A;Ryu JH;Wagner BK;Shen-Orr SS;Klein AM;Melton DA;Yanai I
通讯作者：
Yanai I
影响因子：
25
作者：
Hrvatin S;Hochbaum DR;Nagy MA;Cicconet M;Robertson K;Cheadle L;Zilionis R;Ratner A;Borges-Monroy R;Klein AM;Sabatini BL;Greenberg ME
通讯作者：
Greenberg ME
影响因子：
64.5
作者：
Dixit, Atray;Pamas, Oren;Li, Biyu;Chen, Jenny;Fulco, Charles P.;Jerby-Amon, Livnat;Marjanovic, Nemanja D.;Dionne, Danielle;Burks, Tyler;Raychowdhury, Raktima;Adamson, Britt;Norman, Thomas M.;Lander, Eric S.;Weissman, Jonathan S.;Friedman, Nir;Regev, Aviv
通讯作者：
Regev, Aviv
影响因子：
48
作者：
Habib N;Avraham-Davidi I;Basu A;Burks T;Shekhar K;Hofree M;Choudhury SR;Aguet F;Gelfand E;Ardlie K;Weitz DA;Rozenblatt-Rosen O;Zhang F;Regev A
通讯作者：
Regev A