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

Abdullah O. Khan,Alexandre Slater,Jeremy A. Pike,Natalie S. Poulter,Steven G. Thomas,Carl W. White,Stephen J. Hill,Jack Yule,Neil V. Morgan

doi:10.1038/s41598-019-50733-9

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.

Highlights

Super-resolution imaging techniques offer unique insights into cellular structures and organisations on a nanoscale inaccessible through conventional light microscopy
HDR donor templates for TubA1B were generated carrying a codon optimised mEos 3.2 and both the original and a codon optimised version of mEos 4b (Fig. S1) reported by Paez-Segala et al.[26]. These donors were co-transfected with a TubA1B targeting guide and Cas[9] expressing plasmid into Hel 92.1.7, a cell line which we previously demonstrated expresses high levels of TubA1B but still exhibits significantly less mEos 3.2 tagged TubA1B when compared to endogenously labelled mEGFP at the same locus[15]
When compared to our previously reported mEos 3.2 clones, no significant difference in expression is observed in clones carrying a codon optimised variant of the original mEos 3.2 (Fig. 2B). mEos 4b shows a significant decrease in expression (p < 0.0001), the codon optimised version of this sequence demonstrates a significant increase in expression when compared to the mEos 3.2 clone (p = 0.0005, 0.0244, and 0.022 respectively) (Fig. 2B). This Western blot data was further supported by quantitative Real Time PCR amplifying the edited gene using tag specific forward primers, and a reverse primer anchored to the TUBA1B sequence (Fig. S4)

Summary

Introduction

Super-resolution imaging techniques offer unique insights into cellular structures and organisations on a nanoscale inaccessible through conventional light microscopy. Cells expressing a genomically encoded tag will provide a large sample with defined expression profiles and label distributions, minimising sample to sample variation which is a major concern in nanoscale measurements[15,16]. This approach has been used very successfully in prokaryotic cells, with a number of excellent studies highlighting the utility of endogenous labels in applying SMLM to interrogate nanoscale cellular architecture[17,18,19]. Hansen et al effectively used their HaloTag knock-ins, and while the data is not shown, report the generation of homozygous knock-ins with no effect of insertion on the target’s expression level compared to wild type protein[23,24]

Methods

Results

Discussion

Conclusion