Papaioannou Nikoletta Y, Patsali Petros, Naiisseh Basma, Papasavva Panayiota L, Koniali Lola, Kurita Ryo, Nakamura Yukio, Christou Soteroula, Sitarou Maria, Mussolino Claudio, Cathomen Toni, Kleanthous Marina, Lederer Carsten W
Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
Research and Development Department, Central Blood Institute, Blood Service Headquarters Japanese Red Cross Society, Tokyo, Japan.
Front Genome Ed. 2023 Mar 8;5:1141618. doi: 10.3389/fgeed.2023.1141618. eCollection 2023.
Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34 cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for manipulation. Moreover, editing of CD34 cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Here, we detail an transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34 cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal delivery of genome editing tools. Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.
基因组编辑工具，如CRISPR/Cas、转录激活样效应因子核酸酶（TALE nucleases）以及最近出现的双链断裂非依赖型编辑器，已成功用于基因治疗和反向遗传学研究。在该领域的诸多挑战中，将编辑器以可耐受且高效的方式递送至靶细胞和靶点，以及摆脱对商业工具的依赖以实现新编辑技术的灵活快速应用，是最为紧迫的问题。对于许多造血研究应用而言，原代CD34细胞和人脐带血来源的祖红细胞系2（HUDEP-2）是极具信息价值的底物，且易于进行操作。此外，对CD34细胞进行编辑具有直接的治疗意义。这两种细胞类型对标准转染程序和试剂（如用质粒DNA进行脂质转染）都很敏感，因此需要更合适的方法来实现所选编辑器编辑的高效率和耐受性。这些挑战可以通过RNA递送解决，对于基于CRISPR/Cas的系统，可作为向导RNA和mRNA的混合物进行递送；对于TALENs，则作为mRNA的混合物进行递送。与核糖核蛋白或蛋白质相比，RNA作为载体具有灵活性，无需依赖新型编辑器蛋白质的商业可得性或繁琐的内部制备。与DNA相比，RNA毒性更小，且通过避免mRNA的核转录和输出，具有更快的动力学和更高的编辑效率。在此，我们详细介绍一种基于质粒DNA模板的转录方案，该方案使用T7 RNA聚合酶添加抗逆转录帽类似物（ARCA），并使用聚腺苷酸聚合酶进行聚腺苷酸化尾，同时结合对HUDEP-2细胞和患者来源的CD34细胞进行核转染。我们基于RNA的递送方案使用广泛可得的试剂和设备，并且能够轻松用于基因组编辑工具的通用递送。借鉴一个常见的应用案例，我们采用该方案靶向β-珠蛋白突变并重新激活γ-珠蛋白表达，作为β-血红蛋白病的两种潜在治疗方法，随后进行红细胞分化和功能分析。我们的方案可实现高编辑效率，且细胞活力和分化不受影响，具有可扩展性、适用于编辑结果的功能评估以及在应用于不同编辑器时具有高度灵活性。
