Table_1_Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum.XLSX

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, Plasmodium falciparum. 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 P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum 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，由于疟原虫基因组编辑技术的局限性，对分子机制及其相关基因功能的研究受到制约。近期，一种简单而有效的基因组编辑技术——CRISPR/Cas系统（成簇规律间隔的短回文重复序列/与CRISPR相关的蛋白），在包括疟原虫在内的多种生物中极大地推动了相关研究。然而，鉴于疟原虫特殊的基因组特征，该系统在病原体中的操作和基因编辑效率亟待提升，尤其是在人类疟原虫——恶性疟原虫（Plasmodium falciparum）中。本研究基于CRISPR/Cas9系统，开发了一种整合策略，以构建Cas9i系统，显著缩短了在P. falciparum中构建转基因菌株的时间。此外，利用该Cas9i系统，我们成功实现了通过单轮转染实现的多重基因组编辑（包括突变或标记）。更有意义的是，我们首次将AsCpf1（一种酸杆菌属Cpf1蛋白，可作为Cas9的替代品）应用于P. falciparum寄生虫，并对其基因编辑能力进行了检验。这些对CRISPR/Cas系统的优化将进一步促进疟原虫分子机制的研究，并为未来消除疟疾做出贡献。