Expanding the Coverage of CRISPR-Gene Targets in Plasmodium Falciparum Using Cpf1 Endonucleases
Mammalian Synthetic Biology Workshop
2018
Fifth International Mammalian Synthetic Biology Workshop
Poster Session
Poster Session
Saturday, May 5, 2018 - 5:30pm to 6:30pm
CRISPR/Cas class II systems are powerful and efficient DNA editing tools that are finding broad applications in medicine and biology, and in diverse organisms. We have been particularly interested in editing the genome of P. falciparum, the causative agent of the most severe form of human malaria for drug discovery applications. While Cas9-based approaches are being used quite successfully to perform genome-editing studies in P. falciparum, the AT-richness of the parasiteâs genome (~80-90%) constrains the number of suitable Cas9-NGG motifs required for DNA cleavage. Cpf1, a novel class II CRISPR endonuclease, was recently shown to cleave DNA through recognizing a TTTN motif, opening the possibility of leveraging this system to increase the range of target sites in the P. falciparum genome that can be manipulated. Here we describe the use of Cpf1 as an additional genome-editing tool to manipulate different native gene loci in P. falciparum asexual blood stages. We use these approaches to perform various manipulations, including epitope tagging and installation of genetically encoded elements for protein expression regulation, which are critical for enabling various downstream functional genetics studies. Through bioinformatics analyses, we show on a genome-wide scale that, compared to Cas9, Cpf1 has significantly more high specificity binding sites in the P. falciparum genome . Altogether, Cpf1 and Cas9 are highly complementary genome editing technologies for maximizing access to diverse loci in the challengingly AT-rich P. falciparum genome, and extend our ability to more flexibly perform genome manipulation studies in this organism.