CRISPR screen of microRNAs that regulate the regenertive potential of hematopoietic stem cells

摘要 造血干细胞(HSCs)具有自我更新和多向分化的能力 终生生产所有血细胞。然而，HSCs的再生能力随着年龄的增长而下降，并且随着年龄的增长 造血干细胞表现出向髓系偏向分化。这些与年龄相关的变化是 导致免疫力下降，并增加贫血和髓系恶性肿瘤的倾向 老年人。为了深入了解与年龄相关的HSC功能下降的分子机制，我们 建议研究microRNAs(MiRNAs)在HSC自我更新、分化和衰老中的作用。 最近的研究发现，HSCs中存在一种不同于HSCs的miRNA表达模式 分化的造血系，表明一组独特的miRNAs调节 造血干细胞。然而，在HSC中表达的大多数miRNAs还没有被研究过它们对 HSC自我更新和分化。此外，关于这些miRNAs是否为 负责HSC老化。我们假设造血干细胞自我更新和分化的能力是 受一组miRNAs调控，这些miRNAs的缺失会导致HSC自我更新和改变 差异化。我们进一步假设，某些负向调节自我更新和 肝星状细胞的重建能力代表“老化”的miRNAs，而关键的“老化”miRNAs的缺失可以 使老的HSC恢复活力，并逆转HSC的衰老。我们将通过以下两个目标来检验这些假设。在……里面 目的1，我们将使用基于CRISPR的无偏见的遗传筛选来识别调节HSC自我调节的miRNAs 更新和差异化。以1003个单引导RNA(SgRNAs)为靶标的慢病毒CRISPR文库 在小鼠HSCs中表达的230 miRNA将用于感染表达Cas9的幼鼠HSCs。 携带不同miRNA靶向sgRNAs的感染的HSCs将被注射到干细胞耗尽的受者体内 在连续的移植中，小鼠相互竞争地重新繁殖。改变的HSC自我更新 和功能将导致HSCs的浓缩或枯竭。通过分析SgRNAs在 移植前后HSCs和分化的B、T或髓系的深度测序， 我们将确定相应的miRNAs，它们积极或消极地调节HSC的自我更新和 差异化。在目标2中，我们将测试是否有可能通过CRISPR目标4来恢复旧的HSC 已被证明负调控HSC的miRNAs(miR-126、miR-132、miR-193B和miR-212) 幼鼠的自我更新(2-4)。此外，我们将确定并选择下列候选miRNA 目标1根据其在屏幕上的显著浓缩或消耗进行验证。我们将决定 受这些miRNAs影响的过程是否涉及HSC归巢、增殖、静止或 差异化。一旦得到验证，将研究选定的miRNA在衰老和 使老的造血干细胞恢复活力的能力。拟议的研究将提供对潜在原因的新见解 HSC的自我更新、分化和老化机制，并建立翻译范式 HSC返老还童在改善年龄相关疾病和促进健康寿命方面的潜力。

Abstract Hematopoietic stem cells (HSCs) are capable of self-renewal and multi-lineage differentiation to maintain lifelong production of all blood cells. However, the regenerative capacity of HSCs declines with age, and old HSCs exhibit a skewed differentiation towards myeloid lineages. These age-associated changes are responsible for decreased immunity and increased propensity for anemia and myeloid malignancies in the elderly. To gain insight into the molecular mechanisms of age-associated decline in HSC function, we propose to investigate the roles of microRNAs (miRNAs) in HSC self-renewal, differentiation and aging. Recent studies have found a miRNA expression pattern in HSCs that is different from those in the differentiated hematopoietic lineages, suggesting that a distinctive set of miRNAs regulate the function of HSCs. However, the majority of miRNAs expressed in HSCs have not been studied for their regulation of HSC self-renewal and differentiation. Furthermore, little is known about whether these miRNAs are responsible for HSC aging. We hypothesize that the ability of HSCs to self-renew and differentiate is regulated by a set of miRNAs, and deletion of these miRNAs leads to altered HSC self-renewal and differentiation. We further hypothesize that certain miRNAs that negatively regulate the self-renewal and reconstitution capacity of HSCs represent “aging” miRNAs, and deletion of the critical “aging” miRNAs can rejuvenate old HSCs and reverse HSC aging. We will test these hypotheses with the following two Aims. In Aim 1, we will use an unbiased CRISPR-based genetic screen to identify miRNAs that regulate HSC self- renewal and differentiation. A lentiviral CRISPR library with 1003 single-guide RNAs (sgRNAs) that target 230 miRNA expressed in mouse HSCs will be used to infect HSCs from young mice expressing Cas9. Infected HSCs that carry different miRNA-targeting sgRNAs will be injected into stem cell-depleted recipient mice to repopulate competitively with each other in successive transplantations. Altered HSC self-renewal and function will lead to either enrichment or depletion of HSCs. By analyzing the abundance of sgRNAs in HSCs and differentiated B, T or myeloid lineages using deep sequencing before and after transplantations, we will identify the corresponding miRNAs that positively or negatively regulate HSC self-renewal and differentiation. In Aim 2, we will test whether it is possible to rejuvenate old HSCs by CRISPR targeting of 4 miRNAs (miR-126, miR-132, miR-193b and miR-212) that have been shown to negatively regulate HSC self-renewal in young mice (2-4). Furthermore, we will prioritize and select candidate miRNAs identified in Aim 1 for validation based on their significant enrichment or depletion in the screen. We will determine whether the processes affected by these miRNAs involve HSC homing, proliferation, quiescence or differentiation. Once being validated, selected miRNA will be studied for their expression during aging and abilities to rejuvenate old HSCs. The proposed studies will provide novel insights into the underlying mechanisms of HSC self-renewal, differentiation and aging, and establish a paradigm for the translational potential of HSC rejuvenation to ameliorate age-associated diseases and promote healthspan.