DOI: 10.1101/482596Nov 30, 2018Paper

Optimised CRISPR-Cas9 mediated single molecule imaging for accurate quantification through endogenous expression.

BioRxiv : the Preprint Server for Biology
Abdullah O KhanNeil V Morgan

Abstract

The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR knock-in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4, and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

Related Concepts

Complication
Ligands
Penicillin-Binding Proteins
CXCR4 Receptors
G-Protein-Coupled Receptors
Protein Expression
Knock-in
Protein Function
TUBA1B gene
Monomeric IgA Immunoglobulin Complex

Related Feeds

CRISPR for Genome Editing

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here is the latest research on the use of CRISPR-Cas system in gene editing.

BioRxiv & MedRxiv Preprints

BioRxiv and MedRxiv are the preprint servers for biology and health sciences respectively, operated by Cold Spring Harbor Laboratory. Here are the latest preprint articles (which are not peer-reviewed) from BioRxiv and MedRxiv.

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

CRISPR Genome Editing & Therapy (Preprints)

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on the application of this system for gene editing and therapy in human diseases.

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.

CRISPR for Genome Editing (Preprints)

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here are the latest preprints on the use of CRISPR-Cas system in gene editing.

Related Papers

Journal of Genetics and Genomics = Yi Chuan Xue Bao
Renjie Jiao, Caixia Gao
Nature Chemical Biology
Paul S-H Park, Krzysztof Palczewski
© 2021 Meta ULC. All rights reserved