Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in .

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.