STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells.
STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells.
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DOI：
10.1016/j.crmeth.2022.100300
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发表时间：
2022-10-24
期刊：
影响因子：
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将大的DNA有效载荷(>；10kb)插入哺乳动物细胞的特定基因组位置仍然具有挑战性。从合成生物学到评估疾病相关变异的致病性，精准医学倡议的应用将极大地受益于促进这一过程的工具。在这里，我们融合了不同类型的位点特异性重组酶的优势，并将这些重组酶与CRISPR-Cas9介导的同源重组结合起来，开发了一种在人诱导多能干细胞(HiPSCs)中对至少50kb的基因组片段进行严格的位点特异性替换的策略。我们展示了STECT-IN(丝氨酸和酪氨酸重组酶辅助的用于高通量研究的基因整合)的多功能性，方法是(1)将各种荧光报告程序组合插入HiPSCs中，以评估衍生心肌细胞中的兴奋-收缩耦合级联反应；(2)同时将与遗传性心律失常相关的多个变种靶向HiPSCs池。Straight-IN提供了一种精确的方法来高效且经济高效地产生遗传匹配的HiPSC系面板。无大小限制的DNA整合到基因组中的高效和快速平台切除步骤在修改的位置留下最小的疤痕后，该程序技术加快了多参数报告HIPSC的建立和评估它也促进了同时产生疾病变异的HIPSC在任何哺乳动物细胞，包括HIPSC中，将大于∼3kb的DNA片段插入到特定的基因组位置是有问题的。为了解决这一问题，我们开发了一种平台，它促进了这种特定位置插入或替换基因组片段的效率，从而允许同时靶向多个构建体。与其他方法相比，Straight-IN显然对可以插入的DNA大小没有限制，同时保持对基因组修改的精确控制；因此，它可以显著提高转基因细胞系的产量。Branch-Askino等人。提出了一种将大DNA有效载荷整合到hPSCs特定基因组位置的方法。这一程序提供了一种精确和快速的方法来生成hPSC系的遗传匹配面板，并应用于疾病建模和合成生物学等研究领域。
Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively. Efficient and rapid platform to integrate DNA without size limits into the genome Excision step leaves minimal scarring at the modified locus after the procedure Technique expedites generating and evaluating multi-parameter reporter hiPSC lines It also facilitates the simultaneous generation of panels of disease variant hiPSCs It is problematic to insert DNA fragments larger than ∼3 kb into a specific genomic locus in any mammalian cell, including hiPSCs. To address this, we have developed a platform that facilitates such site-specific insertion or replacement of genomic fragments with efficiencies that permit targeting of multiple constructs simultaneously. Compared with other approaches, STRAIGHT-IN demonstrably has virtually no restriction on the size of the DNA that can be inserted while maintaining precise control over modifications to the genome; it can thus significantly improve throughput in generating genetically modified cell lines. Blanch-Asensio et al. present a method to integrate large DNA payloads into specific genomic sites of hPSCs. This procedure offers a precise and rapid approach to generate genetically matched panels of hPSC lines with applications in research areas such as disease modeling and synthetic biology.
DOI：
10.1007/7651_2021_368
发表时间：
2022
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
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作者：
Brandão KO;Grandela C;Yiangou L;Mummery CL;Davis RP
通讯作者：
Davis RP
影响因子：
5.2
作者：
Du, Zhong-Wei;Hu, Bao-Yang;Ayala, Melvin;Sauer, Brian;Zhang, Su-Chun
通讯作者：
Zhang, Su-Chun