Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in

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.

CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes

“…We have reported changes in expression following genome editing that depends on the tag sequence (Khan et al, 2019); however, here this appears unlikely since the two HEK293 cell lines expressing genome-edited CXCR4/LgBiT did not show comparable effects, and analysis of ARRB2 expression showed no observable differences following editing. An alternative explanation is that changes in expression are due to on/off-target effects of the editing (Zhang et al, 2015), or due to the subsequent cloning procedure resulting in amplification of a founder cell with acquired changes to the cellular proteome.…”

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

“…The multiple applications of CRISPR/Cas9 system are described in a nature video [93]. Khan et al [94] introduced endogenously expressed tags for advanced Single Molecule Localization Microscopy (such as halotags), suggesting that the CRISPR mediated labeling (CRISPR-PALM) could have quantitative benefits. In another work, this technology has been used to study the induction and repair of DNA lesions occurring in specified genome sites, by targeting locations of the Cas9 nuclease [95].…”

Ligand-directed covalent labelling of a GPCR with a fluorescent tag

“…Each of these technologies has their limitations and are not readily transferrable to endogenously expressing systems where GPCRs are expressed at low levels. Gene editing techniques, such as CRISPR/Cas9 2 , have allowed labelling of proteins 3 , including GPCRs 4,5 , at endogenous levels but the fluorophore used can dramatically alter expression levels 6 . An orthogonal approach to protein labelling uses ligand-directed chemistry whereby connecting a fluorophore via a highly reactive, electrophilic linker to a ligand that binds to the protein of interest.…”