A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9.
A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9.
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DOI：
10.1007/978-1-0716-1720-5_1
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发表时间：
2022
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人多能干细胞（hPSC）具有自我更新和分化成大多数细胞类型的能力，并且与广泛使用的细胞系相反，其核型正常且未转化。因此，hPSC被认为是疾病建模的黄金标准系统，特别是在再生医学领域。尽管hPSC和诱导多能干细胞（iPSC）的广泛研究使用，但对多能性和谱系分化机制的系统理解仍然不完整。在用遗传筛选解决谱系分化的复杂性之前，关键是要对多能状态下细胞适应性和增殖的一般遗传要求进行编目，并在常用的培养条件下评估其可塑性。我们描述了一种通过在诱导型S.化脓性链球菌Cas9 H1 hPSC系。为了解决上下文依赖的基因的重要性的问题，我们包括筛选hPSC饲养细胞和层粘连蛋白，两种常用的生长基质上培养的协议。该方法建立了hPSC中全基因组筛选的参数，使人类干细胞适合于功能基因组学方法，以促进hPSC生物学的研究。
Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete. Before tackling the complexities of lineage differentiation with genetic screens, it is critical to catalogue the general genetic requirements for cell fitness and proliferation in the pluripotent state and assess their plasticity under commonly used culture conditions. We describe a method to map essential genetic determinants of hPSC fitness and pluripotency, herein defined as cell reproduction, by genome-scale loss-of-function CRISPR screens in an inducible S. pyogenes Cas9 H1 hPSC line. To address questions of context-dependent gene essentiality, we include protocols for screening hPSCs cultured on feeder cells and laminin, two commonly used growth substrates. This method establishes parameters for genome-wide screens in hPSCs, making human stem cells amenable for functional genomics approaches to facilitate investigation of hPSC biology.