Large DNA fragment knock-in and sequential gene editing in Plasmodium falciparum: a preliminary study using suicide-rescue-based CRISPR/Cas9 system

CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce marker-free genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.

Automated summary

The application of CRISPR/Cas9 technology to Plasmodium falciparum has the potential to greatly improve gene editing efficiency, particularly in regard to large DNA fragment knock-ins and sequential gene editing. By modifying the suicide-rescue-based system for conventional gene editing, we have achieved a major advance in addressing these challenges. Our improved approach has been confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, generate marker-free genetically engineered parasites, and show potential for sequential gene editing.