Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may usher in a new class of medicines to treat genetic diseases of the blood and immune system. With state-of-the-art methodologies, it is now possible to correct disease-causing mutations at high frequencies in HSPCs by combining ribonucleoprotein (RNP) delivery of Cas9 and chemically modified sgRNAs with homologous DNA donors via recombinant adeno-associated viral vector serotype six (AAV6). However, because of the precise nucleotide-resolution nature of gene correction, these current approaches do not allow for clonal tracking of gene targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a novel methodology that utilizes barcoded AAV6 donor template libraries, carrying either in-frame silent mutations or semi-randomized nucleotide sequences 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 immunodeficient mice. We anticipate that this methodology has the potential to be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

中文翻译：

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TRACE-Seq揭示了基因靶向人类造血干细胞的克隆重建动力学。

造血干细胞和祖细胞（HSPC）中致病突变的靶向DNA校正可能会引入一类新的药物来治疗血液和免疫系统的遗传性疾病。使用最新的方法，现在可以通过重组腺相关病毒载体血清型将Cas9的核糖核蛋白（RNP）递送和化学修饰的sgRNA与同源DNA供体结合起来，从而在HSPC中以高频率纠正引起疾病的突变六（AAV6）。但是，由于基因校正具有精确的核苷酸分辨率特性，因此这些当前方法无法进行基因靶向HSPC的克隆追踪。在这里，我们描述了使用测序（TRACE-Seq）（一种利用条形码AAV6供体模板库的新颖方法）在克隆移植中追踪重组等位基因，在编码区外携带框内沉默突变或半随机核苷酸序列，以追踪靶向基因的HSPC克隆的体内谱系贡献。通过将ABB6供体模板库（包括约20,000个独特的外显子1框内沉默突变）靶向HBB基因，我们追踪了HBB靶向的髓样偏斜，淋巴样偏斜以及平衡的多谱系人类HSPC克隆的造血重建。在免疫缺陷小鼠中。我们预计，该方法有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。通过将ABB6供体模板库（包括约20,000个独特的外显子1框内沉默突变）靶向HBB基因，我们追踪了HBB靶向的髓样偏斜，淋巴样偏斜以及平衡的多谱系人类HSPC克隆的造血重建。在免疫缺陷小鼠中。我们预计，该方法有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。通过将ABB6供体模板库（包括约20,000个独特的外显子1框内沉默突变）靶向HBB基因，我们追踪了HBB靶向的髓样偏斜，淋巴样偏斜以及平衡的多谱系人类HSPC克隆的造血重建。在免疫缺陷小鼠中。我们预计，该方法有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。