Supplemental Information for CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.
Supplementary Figure S1. (Related to Figure 1). Analysis of wildtype or genome edited cells. (a) CXCR4 mRNA expression in HeLa (black bar) or HEK293 (grey bar) cells and (b) ACKR3 mRNA expression in HeLa (black bar) or HEK293 (grey bar) cells. (c-f) Genome-editing resulted in clonal cell lines heterozygous for the insert. PCR amplification using target specific primers of DNA from: (c) wildtype (WT) HEK293 cells, CXCR4/LgBiT clones or cells edited to express both CXCR4/LgBiT and ARRB2/SmBiT (dual), (d) wildtype (WT) HEK293 cells, HiBiT/CXCR4 or Nluc/CXCR4 clones as well as WT HeLa cells and NLuc/CXCR4 HeLa clones, (e) wildtype (WT) HeLa cells and NLuc/ACKR3 clones and (f) wildtype (WT) HEK293 cells, ARRB2/SmBiT clones or clones edited to express both ARRB2/SmBiT and CXCR4/LgBiT (dual). Wildtype PCR product at 200-300bp. In arrows indicate PCR product of inserted tag. Bars represent mean ± s.e.m. of three experiments.
Supplementary Figure S2. (Related to Figure 2). NanoBRET competition ligand binding in a non-clonal pool of HEK293 cells some of which are expressing genome-edited Nluc/CXCR4 (a)Displacement of 12.5 nM CXCL12-AF47 binding by AMD3100 (10 pM – 10 μM) in non-clonal pool of cells transfected with plasmids encoding sgRNAs targeted to the CXCR4 N-terminus, Cas9 and
NLuc/CXCR4 donor repair template (closed circles) or in non-clonal pool of cells transfected with plasmids encoding non-targeted sgRNAs, Cas9 and NLuc/CXCR4 donor repair template (open circles). (b) Luminescence generated from non-clonal pools of cells transfected either with plasmids encoding sgRNAs targeted to the CXCR4 N-terminus (closed bar) or untargeted sgRNA (open bar)
in addition to Cas9 and NLuc/CXCR4 donor repair template. Bars or points represent mean ± s.e.m. of five individual experiments performed in triplicate.
Supplementary Figure S3. (Related to Figure 4). Inhibition of CXCL12-mediated recruitment of genome-edited β-arrestin2/SmBiT recruitment to CXCR4/LgBiT by over-expression of β-arrestin2. HEK293 cells expressing (a) both genome-edited CXCR4/LgBiT and β-arrestin2/SmBiT (Dual CRISPR) or genome-edited β-arrestin2/SmBiT transiently transfected with CXCR4/LgBiT
(CRISPR β-arrestin2/SmBiT) were transfected with 500 ng per well of a
6 well plate of β-arrestin2/Halotag (unlabelled). At time zero CXCL12 (100 nM) or HBSS was applied to the cells and luminescence changes in luminescence over the baseline was measured. Points represent mean ± s.e.m. of four(a) or three (b) individual experiments performed with replicates of 4-6. Baseline-corrected luminescence calculated as described in Methods.
Supplementary Figure S4 (Related to Figure 4). Assessment of the effect of
assay configuration on observations. Basal luminescence observed following application of furimazine (10 μM) in HEK293 cells expressing gene-edited β-arrestin2/SmBiT transiently transfected with CXCR4/LgBiT (CRISPR β-arr2/SmBiT, closed bar), genome-edited CXCR4/LgBiT transiently transfected with β-arrestin2/SmBiT (CRISPR CXCR4/LgBiT, open bar),both genome-edited CXCR4/LgBiT and gene-edited β-arrestin2/SmBiT (Dual CRISPR, grey bar), orHEK293 cells expressing transiently transfected CXCR4/LgBiT and β-arrestin2/SmBiT (Dual exogenous, hatched bar). Points or bars represent mean ± s.e.m. of six (CRISPR CXCR4/LgBiT and Dual exogenous), seven (CRISPR β-arrestin2/SmBiT) or eight (Dual CRISPR) individual experiments performed in triplicate.
Supplementary Figure S5. (Related to Figure 5). (a Analysis of
CXCL12 mRNA expression in wildtype (black bars) or genome-edited CXCL12 knockout HEK293 cells (grey bars). Bars represent mean ± S.D. of mRNA analysis from a single clone performed in triplicate. (b) Determination of the
effect of AMD3100 in the absence of endogenous CXCL12. Change in luminescence in live wildtype HEK293 cells (black bar) or genome-edited CXCL12-KO HEK293 cells (grey bar) transiently transfected with HiBiT/CXCR4 in the absence (basal) or presence of CXCL12 (100 nM) or AMD3100 (1 μM). Luminescence generated by the addition of purified LgBiT (10 nM) and furimazine. Bars represent mean ± s.e.m. of four experiments performed in triplicate.
Supplementary Figure S6. (Related to Figure 5). Effect of ligand addition to HiBiT-tagged receptors. (a) HEK293 cells exogenously expressing HiBiT/ACKR3 were incubated in the absence or presence of increasing concentrations of CXCL12 (closed circles), CXCL11 (open squares) or XAC (closed squares). (b) HEK293 cells expressing genome-edited HiBiT/β2-adrenoceptors were incubated in the absence or presence of increasing concentrations of isoprenaline (closed circles) or propranolol (clos
ed squares). Ligands were incubated for 1 hour at 37 °C before luminescence was generated by the addition of furimazine (10 μM) and purified LgBiT (10 nM). Points represent mean ± s.e.m. luminescence or % baseline luminescence of five experiments performed in triplicate.
Supplementary Table 1: Primers used for in this study (Related to STAR Methods)
Supplementary Table 2: Repair template sequences used in this study (Related to STAR Methods)
|Date made available||12 Feb 2020|