A CRISPR-based method for testing the essentiality of a gene

Abstract The CRISPR/Cas9 system is a powerful method of editing genes by randomly introducing errors into the target sites. Here, we describe a CRISPR-based test for gene essentiality (CRISPR-E test) that allows the identification of essential genes. Specifically, we use sgRNA-mediated CRISPR/Cas9 to target the open reading frame of a gene in the genome and analyze the in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations in the targeted region of the gene in surviving cells. If the gene is non-essential, the cells would carry both in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations. In contrast, the cells would carry only in-frame (3n) mutations if the targeted gene is essential, and this selective elimination of frameshift (3n + 1 and 3n + 2) mutations of the gene indicate its essentiality. As a proof of concept, we have used this CRISPR-E test in the model organism Dictyostelium discoideum to demonstrate that Dync1li1 is an essential gene while KIF1A and fAR1 are not. We further propose a simple method for quantifying the essentiality of a gene using the CRISPR-E test.

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A CRISPR-based method for measuring the essentiality of a gene

10.1101/736512 ◽

2019 ◽

Author(s):

Yan You ◽

Sharmila G. Ramachandra ◽

Tian Jin

Keyword(s):

Essential Gene ◽

Model Organism ◽

Open Reading Frame ◽

Proof Of Concept ◽

Powerful Method ◽

Simple Method ◽

Target Region ◽

Reading Frame ◽

Target Sites ◽

Selective Elimination

AbstractThe CRISPR/Cas9 system is a powerful method of editing genes by randomly introducing errors into the target sites. Here, we describe a CRISPR-based test for gene essentiality (CRISPR-E test) that allows the identification of essential genes. Specifically, we use sgRNA-mediated CRISPR/Cas9 to target the open reading frame of a gene in the genome and analyze the in-frame (3n) and frameshift (3n+1 and 3n+2) mutations on the target region of the gene in surviving cells. If the gene is non-essential, the cells would carry both in-frame (3n) and frameshift (3n+1 and 3n+2) mutations. In contrast, the cells would carry only in-frame (3n) mutations if the targeted gene is essential, and this selective elimination of frameshift (3n+1 and 3n+2) mutations of the gene indicate its essentiality. As a proof of concept, we have used this CRISPR-E test in the model organism Dictyostelium discoideum to demonstrate that Dync1li1 is an essential gene while KIF1A and fAR1 are not. We further propose a simple method for quantifying the essentiality of a gene using the CRISPR-E test.One Sentence SummaryCRISPR-E measures a gene’s essentiality

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Saccharomyces cerevisiae GPI10, the functional homologue of human PIG-B, is required for glycosylphosphatidylinositol-anchor synthesis

Biochemical Journal ◽

10.1042/bj3320153 ◽

1998 ◽

Vol 332 (1) ◽

pp. 153-159 ◽

Cited By ~ 62

Author(s):

Christine SÜTTERLIN ◽

M. Victoria ESCRIBANO ◽

Peter GEROLD ◽

Yusuke MAEDA ◽

Maria J. MAZON ◽

...

Keyword(s):

Partial Resistance ◽

Essential Gene ◽

Core Structure ◽

Open Reading Frame ◽

Synthesis Inhibitor ◽

Reading Frame ◽

Glycosylphosphatidylinositol Anchor ◽

The Third ◽

Mutant Cells ◽

Functional Homologues

An increasing number of plasma membrane proteins have been shown to be attached to the membrane via a glycosylphosphatidylinositol (GPI) moiety. All eukaryotes share a highly conserved GPI-core structure EthN-P-Man3-GlcN-PI, where EthN is ethanolamine. We have identified a protein encoded by the yeast open reading frame YGL142C that shares 33% identity with the human Pig-B protein. Deletion of this essential gene leads to a block in GPI anchor biosynthesis. We therefore named the gene GPI10. Gpi10p and Pig-B are functional homologues and the lethal deletion of GPI10 can be rescued by expression of the PIG-BcDNA. As found for PIG-B mutant cells, gpi10 deletant cells cannot attach the third mannose in an α-1,2 linkage to the GPI core-structure intermediate. Overexpression of GPI10 gives partial resistance to the GPI-synthesis inhibitor YW3548, suggesting that this gene product may affect the target of the inhibitor.

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Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles

Journal of Cell Science ◽

10.1242/jcs.108.10.3331 ◽

1995 ◽

Vol 108 (10) ◽

pp. 3331-3337 ◽

Cited By ~ 5

Author(s):

C. Adessi ◽

A. Chapel ◽

M. Vincon ◽

T. Rabilloud ◽

G. Klein ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

Cysteine Proteinase ◽

Open Reading Frame ◽

Major Protein ◽

Amino Acid Sequencing ◽

Iron Oxide Particles ◽

Reading Frame ◽

Sds Page ◽

Oxide Particles ◽

Endocytic Vesicles

Magnetic isolation of endocytic vesicles from Dictyostelium discoideum was accomplished after feeding the amoebae with iron oxide particles. Proteins associated with the endocytic vesicles were resolved by SDS-PAGE and digested ‘in-gel’ with endoproteinase Lys-C or Asp-N to generate peptides for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles: namely, the A, B, D, E and 110 kDa subunits of a vacuolar type H(+)-ATPase, actin, a Rab 7-like GTPase, a p34 protein corresponding to a new cysteine proteinase and the 25 kDa product of a recently sequenced D. discoideum open reading frame.

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