Introducing Large Genomic Deletions in Human Pluripotent Stem Cells Using CRISPR-Cas3.
Introducing Large Genomic Deletions in Human Pluripotent Stem Cells Using CRISPR-Cas3.
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DOI：
10.1002/cpz1.361
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发表时间：
2022-03
期刊：
影响因子：
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通讯作者：
Zhang Y
中科院分区：
文献类型：
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作者：
Hou Z;Hu C;Ke A;Zhang Y
CRISPR-CAS系统为研究人员提供了真核基因组编辑工具和治疗平台，允许靶向躯体器官中的疾病突变。这些工具中的大多数使用II型(例如Cas9)或V型(例如Cas12a)CRISPR酶在基因组中创建RNA引导的精确双链断裂。然而，这类技术在进行有针对性的大规模删除方面的能力有限。最近，I型CRISPR系统在微生物中普遍存在，并显示出独特的酶特性，已被利用来有效地在人类细胞中造成大量染色体缺失。I型CRISPR首先使用一个名为Cascade的多亚基核糖核蛋白(RNP)复合体来寻找其引导互补的靶部位，然后招募解旋酶核酸酶Cas3，以高处理速度远距离切割靶DNA。当作为纯化的RNPs被引入人类细胞时，CRISPR-Cas3复合体可以有效地诱导CRISPR靶点上不同长度(1到100 kb)的大量基因组缺失。由于这种独特的编辑结果，CRISPR-Cas3在移除整合的病毒基因组和询问影响基因功能和人类疾病的结构变异等任务方面具有很大的前景。在这里，我们提供了使用CRISPR-Cas3引入大片段缺失的详细协议。我们描述了从青海热双歧中提纯I-E型CRISPR蛋白Cascade和Cas3的步骤，将RNPs电穿孔到人类细胞中，并使用PCR和测序鉴定DNA缺失。由于人类多能干细胞的临床潜力，我们在这里重点关注它们，但这些方案将广泛用于其他细胞系和模式生物的应用，包括大基因组缺失、全基因或染色体移除、非编码元件的CRISPR筛查等。人多能干细胞TfuCas3蛋白的表达和纯化电穿孔技术将TFU级联RNP和Cas3蛋白导入人多能干细胞基因组DNA损伤的长程聚合酶链式反应、Topo克隆和Sanger测序基于TN5的NGS单细胞克隆综合分析基因组损伤
CRISPR-Cas systems provide researchers with eukaryotic genome editing tools and therapeutic platforms that allow targeting disease mutations in somatic organs. Most of these tools employ Type II (e.g Cas9) or Type V (e.g. Cas12a) CRISPR enzymes to create RNA-guided precise double-strand breaks in the genome. However, such technologies are limited in their capacity to make targeted large deletions. Recently, the Type I CRISPR system, which is prevalent in microbes and displays unique enzymatic features, has been harnessed to effectively create large chromosomal deletions in human cells. Type I CRISPR first uses a multi-subunit ribonucleoprotein (RNP) complex called Cascade to find its guide-complementary target site, and then recruits a helicase-nuclease enzyme, Cas3, to travel along and shred the target DNA over a long distance with high processivity. When introduced into human cells as purified RNPs, the CRISPR-Cas3 complex can efficiently induce large genomic deletions of varying lengths (1 to 100 kb) from the CRISPR-targeted site. Because of this unique editing outcome, CRISPR-Cas3 holds great promise for tasks such as removal of integrated viral genomes, and interrogation of structural variants impacting gene function and human disease. Here, we provide detailed protocols for introducing large deletions using CRISPR-Cas3. We describe step by step procedures for purifying the Type I-E CRISPR proteins Cascade and Cas3 from Thermobifida fusca, electroporating RNPs into human cells, and characterizing DNA deletions using PCR and sequencing. We focus here on human pluripotent stem cells due to their clinical potential, but these protocols will be broadly useful for other cell lines and model organisms for applications including large genomic deletion, full gene or chromosome removal, CRISPR screening for non-coding elements, and others. Expression and purification of TfuCascade RNP Expression and purification of TfuCas3 protein Culturing of human pluripotent stem cells (hPSCs) Introduction of Tfu Cascade RNP and Cas3 proteins into hPSCs via electroporation Characterization of genomic DNA lesions using long-range PCR, TOPO cloning, and Sanger sequencing Comprehensive analysis of genomic lesions by Tn5-based NGS Single cell clonal isolation