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.
| Original language | English |
|---|---|
| Number of pages | 1 |
| Journal | Scientific Reports |
| Volume | 9 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2 Oct 2019 |
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Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in. / Khan, Abdullah O.; White, Carl W.; Pike, Jeremy A.; Yule, Jack; Slater, Alexandre; Hill, Stephen J.; Poulter, Natalie S.; Thomas, Steven G.; Morgan, Neil V.
In: Scientific Reports, Vol. 9, No. 1, 02.10.2019.Research output: Contribution to journal › Article
TY - JOUR
T1 - Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in
AU - Khan, Abdullah O.
AU - White, Carl W.
AU - Pike, Jeremy A.
AU - Yule, Jack
AU - Slater, Alexandre
AU - Hill, Stephen J.
AU - Poulter, Natalie S.
AU - Thomas, Steven G.
AU - Morgan, Neil V.
PY - 2019/10/2
Y1 - 2019/10/2
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85072930516&partnerID=8YFLogxK
U2 - 10.1038/s41598-019-50733-9
DO - 10.1038/s41598-019-50733-9
M3 - Article
VL - 9
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
ER -