Herrera Curbelo, A. | bioRxiv Science

bioRxiv2024

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei

Many Plasmodium genes remain uncharacterised due to low genetic tractability. Previous large scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called PbHiT, which uses a single cloning step to generate targeting vectors with 100 bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide vector designs and sequences to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasites biology at scale.

molecular biology↗

bioRxiv2022

Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression

Mitochondria are central to numerous anabolic and catabolic pathways whereby mitochondrial dysfunction has a profound impact on metabolism and can manifest in disease. The consequences of mitochondrial dysfunction can be ameliorated by adaptive responses that rely on mito-cellular crosstalk to communicate mitochondrial distress to the rest of the cell. Such mito-cellular signaling slows cell cycle progression in mitochondrial-DNA deficient ({rho}0) Saccharomyces cerevisiae cells, but the initial trigger and the pathway mediating the response has remained unknown. Here, we show that decreased mitochondrial membrane potential ({Delta}{Psi}m) acts as the initial signal of mitochondrial stress that delays G1-to-S phase transition in both {rho}0 and control cells. Accordingly, experimentally increasing {Delta}{Psi}m was sufficient to restore timely cell cycle progression in {rho}0 cells. Neither the RTG retrograde pathway nor central DNA damage checkpoint kinases were involved in mediating this form of mito-cellular communication. The identification of {Delta}{Psi}m as a novel regulator of cell cycle progression may have implications for disease states involving mitochondrial dysfunction.

cell biology↗