Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells

Xia, Baolong; Viswanatha, Raghuvir; Hu, Yanhui; Mohr, Stephanie E.; Perrimon, Norbert

doi:10.7554/elife.85542

Cited by 2 publications

(2 citation statements)

References 46 publications

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Section: Discussionmentioning

confidence: 99%

“…Specifically, CRISPRa might be considered a valuable tool for orthogonally validating immunity and fitness studies in I. scapularis. By overexpressing genes of interest in I. scapularis cells, one may complement RNAi-based technologies and develop small-or large-scale studies, such as commonly reported genomewide pooled CRISPRa screens in mammals and Drosophila (28,30). Collectively, the development of genetic tools for tick research offers unique avenues to identify crucial genes related to growth, physiology, immune signaling and detection of microbes.…”

Section: Discussionmentioning

confidence: 99%

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Genetic manipulation of anIxodes scapulariscell line

Singh,

Rolandelli,

O’Neal

et al. 2023

Preprint

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Although genetic manipulation is one of the hallmarks in model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the blacklegged tickIxodes scapularis,an arthropod that serves as a vector of several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology dependent recombination. Targeting the E3 ubiquitin ligase X-linked inhibitor of apoptosis (xiap) and its substratep47led to alteration in molecular signaling within the immune deficiency (IMD) network and increased infection of the rickettsial agentAnaplasma phagocytophiluminI. scapularisISE6 cells. Collectively, our findings complement techniques for genetic engineering of ticksin vivoand aid in circumventing the long-life cycle ofI. scapularis,of which limits efficient and scalable molecular genetic screens.ImportanceGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology dependent recombination. We ablated the expression ofxiapandp47, two signaling molecules present in the immune deficiency (IMD) pathway ofI. scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agentA. phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the blacklegged tickIxodes scapularis.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Genetic manipulation of anIxodes scapulariscell line

Singh,

Rolandelli,

O’Neal

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells

Xia

Viswanatha

et al. 2023

eLife

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Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In Drosophila cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in Drosophila cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (CG8468), a member of the lipocalin protein family (CG5399), and a zinc finger C2H2 transcription factor (CG9932). Mechanistically, we demonstrate that CG5399 overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by CG5399 requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in Drosophila cells.

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Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells

Cited by 2 publications

References 46 publications

Genetic manipulation of anIxodes scapulariscell line

Genetic manipulation of anIxodes scapulariscell line

Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells

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