Zhao Yuemeng, Wang Fei, Wang Changhong, Zhang Xiaobai, Jiang Cizhong, Ding Feng, Shen Li, Zhang Qingfeng
Research Center for Translational Medicine, Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China.
Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, Tongji University, Shanghai, China.
Front Microbiol. 2021 Jan 8;11:625862. doi: 10.3389/fmicb.2020.625862. eCollection 2020.
Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, . Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in . Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in . In addition, we for the first time adapted AsCpf1 ( sp. Cpf1), an alternative to Cas9, into parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.
长期以来，疟原虫分子机制及相关基因功能的研究一直受到其有限的基因组编辑技术的限制。最近，一种简单有效的基因组编辑技术——CRISPR/Cas（成簇规律间隔短回文重复序列/CRISPR相关蛋白）系统，极大地促进了在包括疟原虫在内的许多生物体中的此类研究。然而，由于疟原虫特殊的基因组特征，CRISPR/Cas系统在这种病原体中的操作和基因编辑效率有待提高，特别是在人类疟原虫中。在此，基于CRISPR/Cas9系统，我们开发了一种整合策略来生成Cas9i系统，这显著缩短了在……中产生转基因菌株的时间。此外，利用这种Cas9i系统，我们成功地通过单次转染在……中实现了多重基因组编辑（突变或标记）。此外，我们首次将Cas9的替代物AsCpf1（……属Cpf1）应用于疟原虫，并对其进行基因编辑检测。CRISPR/Cas系统的这些优化将进一步促进疟原虫的机制研究，并为未来消除疟疾做出贡献。
