Department of Anatomy and Embryology, Leiden University Medical Center, 2300RC Leiden, the Netherlands.
Sequencing Analysis Support Core, Leiden University Medical Center, 2333RC Leiden, the Netherlands.
Cell Rep Methods. 2022 Sep 22;2(10):100300. doi: 10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24.
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.
将大的 DNA 有效负载 (>10 kb) 插入哺乳动物细胞的特定基因组位点仍然具有挑战性。从合成生物学到评估与疾病相关变体的致病性以用于精准医疗计划的应用，如果有能够促进这一过程的工具，将会受益匪浅。在这里，我们融合了不同类别的位点特异性重组酶的优势，并将其与 CRISPR-Cas9 介导的同源重组相结合，开发了一种在人类诱导多能干细胞（hiPSCs）中至少 50 kb 大小的基因组片段进行严格的位点特异性替换的策略。我们通过（1）将各种荧光报告基因组合插入 hiPSCs 中，以评估衍生的心肌细胞中的兴奋-收缩偶联级联，以及（2）同时靶向与遗传性心律失常疾病相关的多个变体，证明了 STRAIGHT-IN（用于高通量研究的丝氨酸和酪氨酸重组酶辅助基因整合）的多功能性。STRAIGHT-IN 提供了一种精确的方法，可以高效且经济有效地生成遗传匹配的 hiPSC 系面板。
