Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells
Curation statements for this article:-
Curated by eLife
eLife assessment
This valuable manuscript presents resources for genome-wide genetic perturbation in the fruitfly, Drosophila. The evidence for the usefulness is solid, with the authors demonstrating that they can identify novel genes that affect an important pathway, the mTOR pathway, which plays key roles in cell proliferation and cell death. The genetic resources are significant for their availability to colleagues in the Drosophila community seeking to to identify genes with important cellular functions.
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Abstract
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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eLife assessment
This valuable manuscript presents resources for genome-wide genetic perturbation in the fruitfly, Drosophila. The evidence for the usefulness is solid, with the authors demonstrating that they can identify novel genes that affect an important pathway, the mTOR pathway, which plays key roles in cell proliferation and cell death. The genetic resources are significant for their availability to colleagues in the Drosophila community seeking to to identify genes with important cellular functions.
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Reviewer #1 (Public Review):
The manuscript entitled "Pooled genome-wide CRISPRa screening for rapamycin resistance gene in Drosophila cells" by Xia et al. is a well-structured piece of work with clear objectives and experiments. The authors successfully demonstrated genome-wide gene activation using CRISPRa using a novel sgRNA design, which overcame previous failed attempts to replicate gene activation that worked well in mammalian systems. The study is detailed and highly relevant for the application of CRISPRa in understanding the molecular mechanism of gene candidates.
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Reviewer #2 (Public Review):
In this work Xia et al have generated CRISPR resources for genome-wide gain-of-function genetic perturbation in the Drosophila genome and have used them to identify novel genes that cause Rapamycin resistance in Drosophila cells. To do so, they have used the SAM system, already established to work well in flies (Jia et al., PNAS 2018). 3 of these candidate genes they discovered in the screen, were further characterized to study how they affect the mTOR pathway leading to Rapamycin resistance. Since genome-wide libraries for GOF studies do not exist for Drosophila, these resources will be very useful for a wider Drosophila community.
Strengths
1. GOF CRISPR library does not exist currently to be used in Drosophila and hence this is going to be useful for the wider Drosophila community
2. Authors have used …Reviewer #2 (Public Review):
In this work Xia et al have generated CRISPR resources for genome-wide gain-of-function genetic perturbation in the Drosophila genome and have used them to identify novel genes that cause Rapamycin resistance in Drosophila cells. To do so, they have used the SAM system, already established to work well in flies (Jia et al., PNAS 2018). 3 of these candidate genes they discovered in the screen, were further characterized to study how they affect the mTOR pathway leading to Rapamycin resistance. Since genome-wide libraries for GOF studies do not exist for Drosophila, these resources will be very useful for a wider Drosophila community.
Strengths
1. GOF CRISPR library does not exist currently to be used in Drosophila and hence this is going to be useful for the wider Drosophila community
2. Authors have used already established and currently the most effective SAM system for gene activation for a genome-wide genetic screen.
3. From this screen they have found candidate genes overexpression which leads to Rapamycin resistance. They have validated 3 of these genes by multiple methods and have also tried to elucidate the mechanism by which these genes might regulate mTOR signaling and confer resistance to Rapamycin. The authors have shown the strength and usefulness of the resource that they have generated and this resource will be complementary to loss-of-function screens of similar nature.Weaknesses
1. Authors have taken a number of measures to maintain the integrity of the CRISPRa library, including multiple gRNA targets per gene, 1000 cells per gRNA, and deep sequencing. However, do the authors have an idea of what percentage of the gRNA vectors are functional? Looking at the data they show for the 3 candidate genes, at least half of them are not functional, which could be either because of gRNA location or efficiency. Considering this to be an average situation, there might be a large number of genes for which all gRNAs might not function at all. I understand this might be a caveat for all such studies, but an estimate of some kind in discussion might be useful for anyone who might want to use these resources.
2. As the authors mention that ~32% of genes in Drosophila have transcription start side <1kb apart, off-targeting (neighbouring genes getting activated in addition to the intended gene) will be an issue. To address this, the authors describe one example of genes where although the genes TSS are within one kb of each other, the sgRNA specifically activated only one gene and not the other. However, since following this, authors have generated genome-wide resources keeping 500bp upstream as their benchmark, a large percentage of these 32% genes might have off-targets. It would be useful to know the estimates of off-targeting for such a resource. In addition, have authors looked at the transcripts of genes close to the specific genes they have studied? CG9932 is in close proximity to (although not within the 1 kb range) a few genes including mTOR. -
