The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells.
The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells.
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DOI：
10.1038/s41467-020-20792-y
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发表时间：
2021-01-20
影响因子：
16.6
通讯作者：
Majeti R
中科院分区：
文献类型：
--
作者：
Sharma R;Dever DP;Lee CM;Azizi A;Pan Y;Camarena J;Köhnke T;Bao G;Porteus MH;Majeti R
造血干细胞和祖细胞（HSPC）中致病突变的靶向DNA校正可以治疗血液和免疫系统的遗传疾病。现在可以通过将CRISPR/Cas9与同源DNA供体组合来校正HSPC中的高频率突变。由于基因校正的精确性，这些方法排除了基因靶向HSPC的克隆追踪。在这里，我们描述了使用测序（TRACE-Seq）跟踪克隆移植中的扩增等位基因，这是一种利用条形码AAV6供体模板文库的方法，携带编码区外的框内沉默突变或半随机核苷酸，以跟踪基因靶向HSPC克隆的体内谱系贡献。通过用由约20，000个可能的独特外显子1框内沉默突变组成的AAV6供体模板文库靶向HBB基因，我们在小鼠中追踪了HBB靶向的骨髓偏斜、淋巴偏斜和平衡的多谱系重建人HSPC克隆的造血重建。我们预计这种方法可能用于在翻译和基础研究环境中对Cas9 RNP和AAV6介导的基因靶向结果进行HSPC克隆跟踪。遗传条形码已用于跟踪细胞的克隆动力学。在这里，作者开发了一种使用测序（TRACE-Seq）的克隆移植中的跟踪扩增等位基因，通过引入沉默突变或编码区外部来条形码修复等位基因，以显示移植后编辑的CD34+细胞的克隆复杂性。
Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings. Genetic barcoding has been used to track clonal dynamics of cells. Here, the authors develop a Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), to barcode repaired alleles by introducing silent mutations or outside of coding regions, to show clonal complexity of edited CD34 + cells following engraftment.
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影响因子：
64.8
作者：
Barzel, A.;Paulk, N. K.;Shi, Y.;Huang, Y.;Chu, K.;Zhang, F.;Valdmanis, P. N.;Spector, L. P.;Porteus, M. H.;Gaensler, K. M.;Kay, M. A.
通讯作者：
Kay, M. A.
影响因子：
64.8
作者：
Genovese, Pietro;Schiroli, Giulia;Escobar, Giulia;Di Tomaso, Tiziano;Firrito, Claudia;Calabria, Andrea;Moi, Davide;Mazzieri, Roberta;Bonini, Chiara;Holmes, Michael C.;Gregory, Philip D.;van der Burg, Mirjam;Gentner, Bernhard;Montini, Eugenio;Lombardo, Angelo;Naldini, Luigi
通讯作者：
Naldini, Luigi
影响因子：
14.8
作者：
Bak RO;Dever DP;Porteus MH
通讯作者：
Porteus MH
影响因子：
64.8
作者：
Eyquem J;Mansilla-Soto J;Giavridis T;van der Stegen SJ;Hamieh M;Cunanan KM;Odak A;Gönen M;Sadelain M
通讯作者：
Sadelain M
影响因子：
46.9
作者：
Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
通讯作者：
Porteus MH