Efficient labeling and imaging of protein-coding genes in living cells using CRISPR-Tag

The lack of efficient tools to image non-repetitive genes in living cells has limited our ability to explore the functional impact of the spatiotemporal dynamics of such genes. Here, we addressed this issue by developing a CRISPR-Tag system using one to four highly active sgRNAs to specifically label protein-coding genes with a high signal-to-noise ratio for visualization by wide-field fluorescence microscopy. Our approach involves assembling a CRISPR-Tag within the intron region of a fluorescent protein and then integrating this cassette to N- or C-terminus of a specific gene, which enables simultaneous real-time imaging of protein and DNA of human protein-coding genes, such as HIST2H2BE, LMNA and HSPA8 in living cells. This CRISPR-Tag system, with a minimal size of ~250 bp DNA tag, represents an easily and broadly applicable technique to study the spatiotemporal organization of genomic elements in living cells.

(a) Schematic of fluorescence signal amplification by tandem GFP11. (b) Representative images demonstrate MUC4 and 5S rDNA labeling using dCas9-EGFP or dCas9-GFP 14X . All images are maximum intensity projections from z stacks. Scale bars: 10 µm. (c) Quantifications of signal-to-noise ratio to compare labeling efficacies. Each dot represents a single cell. Green line indicates the median value, n ≥ 119 cells.

Supplementary Figure 3
Labeling MUC4 gene in the non-repetitive or repetitive DNA region (related to Fig. 1).
(a) Labeling of MUC4 intron 1 and exon 1 by CRISPR imaging. Non-repetitive intron 1 was labeled by using 36 sgRNAs, whereas repetitive exon 1 was labeled by using only one sgRNA. 2 to 3 spots (arrows) can be detected. All images are maximum intensity projections from z stacks. Scale bars: 5 µm. (b) Labeling efficiency of non-repetitive and repetitive region in MUC4 gene was compared by measuring the signal-to-noise ratio. Dots represent individual cells. Green line denotes the median value, n = 34 cells. (c) Labeling efficiency of MUC4 loci was determined by counting the number of spots in each cell. Non-repetitive DNA, n = 265; Repetitive DNA, n = 289.

Supplementary Figure 4
Experimental work flow of CRISPR-Tag based DNA tagging (Related to Fig. 1, 2 and 3).
First, CRISPR-Tag together with mCherry was inserted into a specific locus by CRISPR-Cas9 mediated knock-in. The knock-in experiments were carried out by electroporation of Cas9/sgRNA ribonucleoprotein (RNP)/HDR donor into HeLa cells. Left homology arm, CRISPR-Tag, mCherry and right homology arm were assembled as a donor plasmid harboring two Cas9 cleavage sites as indicated. Four days after nucleofection, mCherry + cells, which represent knock-in + cells were sorted out by FACS. Genotyping was then performed to confirm the successful knock-in. The final step was to transfect sgRNAs into the cells for CRISPR labeling.

Supplementary Figure 5
Quantitative analysis of HDR efficiency (Related to Fig. 1).
(a-d) FACS analysis was carried out four days after RNP/donor electroporation. BFP serves as an irrelevant channel. The percentage of mCherry+ cells represents the HDR-mediated knock-in efficiency. (a) mCherry was integrated to C-terminus of H2B locus by using conventional plasmid donor or double-cut plasmid donor in HeLa cells. Cells were first gated for the intact cells by FSC/SSC plot and then gated for single cells based on FSC-A/FSC-W. mCherry positive cells were established by first analyzing cells negative for mCherry and setting the gate for mCherry positive cells. (b) mCherry was inserted to C-terminus of H2B locus by using double-cut plasmid donor in 293T cells. (c) Left: mCherry was integrated to the C-terminus of H2B locus; Right: mCherry plus CRISPR-Tag were inserted to the C-terminus of H2B locus. (d) Left: mCherry was integrated to the N-terminus of LMNA locus; Right: CRISPR-Tag plus mCherry were inserted to the N-terminus of H2B locus. (e-f) mCherry + cells isolated by FACS were imaged to examine the subcellular localization of H2B-mCherry (e) and mCherry-LMNA (f). All images are maximum intensity projections from z stacks. Scale bars: 5 µm.
(a, c) Schematic of primer designs to validate mCherry (a) or mCherry-CRISPR-Tag (c) insertion at the C-terminus of H2B. The size of each PCR fragment is indicated for every pair of primers. (b, d) Genomic DNA from each sample was purified and used as the PCR template. Representative gels of PCR products are shown to indicate the correct insertions. The size of all PCR products is correct as expected. (e) Genomic DNA PCR was performed for Clone 9, 12 and 14 by using indicated primers. Arrows indicated the expected PCR products and the star highlighted an extra band in Clone 9. Sequencing results suggested that Clone 9 may contain two CRISPR-Tag modified alleles. One allele harbors a full sequence of mCherry-CRISPR-Tag, while another allele only contains part of CRISPR-Tag sequence, which might be resulted in genomic rearrangement in HeLa cells.

Supplementary Figure 7
Labeling of H2B loci in mix pool cells and clonal cell lines (Related to Fig. 1).
Large field of images to show the labeling efficiency of H2B loci in mix pool and clonal cells, including Clone 9 and Clone 14. The sgRNA vector co-expressing four sgRNAs was transfected to label H2B loci. Images were acquired on wide-field fluorescent microscopy using 100x NA 1.40 PlanApo oil immersion objective. Arrows indicate the spots which represent H2B loci. All images are maximum intensity projections from z stacks. Scale bars: 10 µm.

Supplementary Figure 8
Application of CRISPR-Tag to label human HSPA8 gene (Related to Fig. 1).
(a) Human HSPA8 gene codes a member of the heat shock protein 70 family. mCherry was integrated to the C-terminus of HSPA8. HSPA8-mCherry is mainly localized to the cytoplasm as shown. Transfection of sgRNAs did not result in specific fluorescent spots. (b) mCherry-CRISPR-Tag was inserted at the C-terminus of HSPA8. HSPA8 loci could be observed by co-expressing sgTS1 and sgTS3. All images are maximum intensity projections from z stacks. Scale bars: 10 µm.

Supplementary Figure 9
Insertion of CRISPR-Tag in the UTR region down regulates protein expression in HeLa cells (Related to Fig. 2).
(a) Schematic of mCherry or CRISPR-Tag_v1 insertion to a genomic locus of interest. The CRISPR knock-in fragments are highlighted in yellow. (b) Quantitative analysis of FACS indicates mCherry intensity of individual HeLa cells for three conditions, control, mCherry and CRISPR-Tag-mCherry integration at the C-terminus of HSPA8. (c) Quantitative analysis of FACS to report H2B-mCherry expression in 293T cells for three conditions, control, mCherry and mCherry-CRISPR-Tag integration at the C-terminus of H2B. For all FACS plots, dots denote single cells. BFP serves as an irrelevant channel.
(a-d) Representative images of mCherry expression driven by a strong promoter at four different conditions: mCherry without introns (a), mCherry with one artificial intron (b), mCherry with one intron harboring CRISPR-Tag_v1 (c) and mCherry with one intron carrying CRISPR-Tag_v2 (d). Overall expression of mCherry was dramatically decreased when CRISPR-Tag_v1 was included in the intron region, whereas CRISPR-Tag_v2 included in the intron did not affect mCherry expression. All images in are from a single focal plane. Scale bars: 10 µm.

Supplementary Figure 11
Protein expression remains normal when CRISPR-Tag_v2 is embedded in the intron region (Related to Fig. 2).
(a) Schematic of mCherry or CRISPR-Tag_v2 insertion to a genomic locus of interest. (b) FACS quantitative analysis reports mCherry intensity of individual HeLa cells for the two conditions, integrations of mCherry, mCherry with intron plus CRISPR-Tag_v2 containing 4 repeats to the C-terminus of H2B, respectively. (c) Quantitative analysis of FACS was performed for the following conditions, integrations of mCherry, mCherry with intron plus CRISPR-Tag_v2 containing containing 4 repeats to the N-terminus of LMNA, respectively. Dots denote single cells. BFP serves as an irrelevant channel.

Supplementary Figure 12
The insertions of CRISPR-Tag have no significant effect on gene transcription (Related to Fig. 2).
Quantitative PCR measurement of transcript abundance of H2B (Left) and LMNA (right). The y axis shows transcript abundance values that are normalized to control cells (dCas9-GFP 14X stable cell line), which does not have genomic modifications of target gene. RNA abundance is normalized to UBC. The data is displayed as means ± SEM for three technical replicates. One-way ANOVA analysis using PRISM does not show significant difference in relative RNA expression for all the comparisons.