Direct Visualization of Single-Nucleotide Variation in mtDNA Using a

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Direct Visualization of Single-Nucleotide Variation in mtDNA using a CRISPR/Cas9-Mediated Proximity Ligation Assay (CasPLA Kaixiang Zhang, Ruijie Deng, Xucong Teng, Yue Li, Yupeng Sun, Xiaojun Ren, and Jinghong Li J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05309 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 20, 2018

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Journal of the American Chemical Society

Direct Visualization of Single-Nucleotide Variation in mtDNA using a CRISPR/Cas9-Mediated Proximity Ligation Assay (CasPLA) Kaixiang Zhang, Ruijie Deng, Xucong Teng, Yue Li, Yupeng Sun, Xiaojun Ren, Jinghong Li* Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China. ABSTRACT: The accumulation of mitochondrial DNA (mtDNA) mutations in cells is strongly related to aging‐associated diseases. Imaging of single‐nucleotide variation (SNV) in mtDNA is crucial for understanding the heteroplasmy of mtDNAs that harbor pathogenic changes. Herein, we designed a CRISPR/Cas9‐mediated proximity ligation assay (CasPLA) for direct visualization of the ND4 and ND5 genes in the mtDNAs of single cells. Taking advantage of the high specificity of CRISPR/Cas9, CasPLA can be used to image SNV in the ND4 gene at single‐molecule resolution. Using CasPLA, we observed a mtDNA transferring process between different cells through a tunneling nanotube, which may account for the spreading of mtDNA heteroplasmy. Moreover, we demonstrated that CasPLA strategy can be applied for imaging of single copy ge‐ nomic loci (KRAS gene) in nuclear genome. Our results establish CasPLA as a tool to study SNV in situ in single cells for basic research and genetic diagnosis.

INTRODUCTION Mitochondrial DNA (mtDNA) encodes multiple tRNAs, rRNAs and proteins necessary for oxidative phosphoryla‐ tion, which generates energy in eukaryotic cells.1‐4. Each eukaryotic cell holds hundreds of mitochondria and each mitochondrion contains multiple mtDNA‐protein com‐ plexes known as nucleoids 3,5. Since mtDNA suffers from a high mutation rate and has a limited repair capacity, cells often contain mtDNAs with different genotypes, a phe‐ nomenon termed heteroplasmy 6,7. The heteroplasmy of mtDNA, which arises in somatic tissues and accumulates throughout life, is thought to contribute to diseases of ag‐ ing, including neurodegeneration, metabolic disorders, cancer, heart disease and sarcopenia 8‐11. Therefore, the ability to visualize single‐nucleotide variation (SNV) in mtDNA in single cells is important for resolving the com‐ plexity and heterogeneity of diseases related to mutations in mtDNA 12. Conventional mtDNA imaging methods include fluo‐ rescence in situ hybridization (FISH) and target‐primed rolling‐circle amplification (tpRCA) 13‐17. However, both of these methods require denaturation or digestion of mtDNA, which leads to lower detection efficiency and af‐ fects the structural and organizational integrity of the mi‐ tochondrial genome (Table S3) 18,19. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)‐ associated protein 9 (Cas9) system, a revolutionary tool for genome editing, has been utilized to visualize genomic lo‐ cus in cells without global DNA denaturation 20‐22. However, the existing Cas9‐based imaging methods are mostly appli‐ cable for highly repetitive genomic loci, such as telomeres,

major satellites or the MUC4 gene.23‐26 Imaging of non‐re‐ petitive loci requires a minimum of 4 separate sgRNAs and advanced imaging technology (such as lattice light sheet microscopy, LLSM) to achieve sufficient signal 27‐30. As far as we know, imaging of SNV remains as an unmet chal‐ lenge for Cas9 based imaging method, since single Cas9 binding event cannot provide a sufficient signal to distin‐ guish the target molecule from background. To address this challenge, we designed a CRISPR/Cas9‐ mediated proximity ligation assay (CasPLA) to image SNV in mtDNA at single‐molecule resolution. This method uses two Cas9 probes to target a specific mtDNA sequence, fol‐ lowed by proximity ligation and in situ rolling circle ampli‐ fication (RCA) to reveal the spatial localization of individ‐ ual wild‐type and mutated mtDNAs in single cells. The mechanism of CasPLA is shown in Scheme 1. Cas9/sgRNA probes, consisting of a Cas9 enzyme molecule bound to a sgRNA molecule, efficiently identify and bind to the target mtDNA by CRISPR‐based programmable recognition31. The sgRNA sequence includes a spacer of 20 nucleotides (nt), which is complementary to the target se‐ quence (termed the protospacer) in mtDNA. The target se‐ quence must be near a NGG sequence termed protospacer‐ adjacent motif (PAM). Cas9 recognizes the PAM and facil‐ itates complementary base pairing between the sgRNA spacer and the protospacer. Base pairing is sensitive to the presence of a mutation, so the Cas9/sgRNA probe can dis‐ tinguish SNVs when binding to the mtDNA. For each tar‐ get sequence, a pair of Cas9/sgRNA probes, termed the CasPLA probe, are specifically designed, which recognize sequences that are nearby in the genome. When the paired CasPLA probes bind in close proximity to each other, they

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Scheme 1. Schematic diagram of CRISPR/Cas9‐mediated proximity ligation (CasPLA) for in situ imaging of single‐nucleotide variation (SNV) in mitochondrial DNAs (mtDNAs). Step 1: Pairs of in vitro assembled Cas9/sgRNA probes specifically bind to the mutant (MUT; red) mtDNA. Each pair consists of two probes. One probe has an sgRNA spacer that is complementary to the mutant (red) target sequence. The other probe (blue) binds to a sequence nearby and facilitates proximity ligation. Step 2: Hybridization of the proximity probes to the Cas9/sgRNA probes, followed by proximity ligation to form a circular DNA structure. Step 3: Rolling circle amplification (RCA) is initiated by Phi29 DNA polymerase from the ligated circular DNA to generate a long RCA product, which is then visualized through hybridization of fluorescence‐labeled oligonucleotides. can then guide the subsequently added linear oligonucleo‐ tides to form a circular structure, which can be covalently joined by enzymatic DNA ligation. The circularized DNA is amplified through RCA, and the in situ‐synthesized RCA product then hybridizes with fluorescently labeled probes, which enable the amplified product to be distinguished from background. Since RCA is a localized isothermal am‐ plification, it can provide information about the localiza‐ tion of target mtDNAs with single‐molecule resolution 32‐ 34 . In previous Cas9‐based in situ imaging studies (CasFISH), imaging of non‐repetitive loci required synthe‐ sis of 73 unique sgRNAs to achieve a sufficient signal‐to‐ noise ratio for spot detection. 20 Herein, using CasPLA, we can successfully visualize individual mtDNAs using only two sgRNAs. Taking advantage of the high specificity of CRISPR/Cas9, CasPLA can be used to image SNV in mtDNA at single‐molecule resolution.

may remain bound to the cleaved site after the cleavage re‐ action. Therefore, we reason that wild‐type Cas9 proteins may be sufficient to assemble the CasPLA probes for in situ imaging.

RESULTS AND DISCUSSION Development of CasPLA for detection of mtDNA. To construct the sgRNA within the CasPLA probe, we added a proximity probe binding site to the stem‐loop of the sgRNA to recruit the DNA proximity probe (Scheme 1). We first tested the activity of the modified sgRNA and found that the added stem‐loop did not affect the binding or cleavage activity of the Cas9/sgRNA complex (Figure S1), which is consistent with a previous report23. It has also been demonstrated that the Cas9/sgRNA complex does not dissociate from the cleaved DNA, except under extremely harsh conditions 35,36, which indicates that Cas9/sgRNA

Figure 1. CasPLA for detection of mtDNA. a) Schematic di‐ agram of CasPLA for the detection of mtDNAs using the poly‐ merase chain reaction (PCR). The proximity probes are hy‐ bridized to the stem‐loop structure of the sgRNA. A short strand of DNA (the bridge oligo) hybridizes with both of the proximity probes to create a double‐stranded segment which can be joined by DNA ligase. The resultant ligated DNA is then detected by standard qPCR methods. b) Real‐time fluores‐ cence intensity of qPCR reactions in the presence of increasing


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amounts of purified mtDNA. c) Linear relationship between ΔCt and the mtDNA concentration. Error bars are based on triplicate experiments.

To test this possibility, we first used a PCR‐based prox‐ imity ligation assay to detect a purified mtDNA sequence (Figure 1a) 37,38. Briefly, a 110 nt‐long single‐strand DNA (ssDNA) was separated into two 55 nt‐long ssDNAs which were attached to CasPLA probes made with a commercially available Cas9 enzyme. Then, a short (20 nt) bridge oligo was added to hybridize to the two ssDNA strands (Figure 1a), thus creating a short stretch of dsDNA, which enabled DNA ligase to join the two fragments to form the 110 nt‐ long DNA strand. The DNA produced in this way was quantified with high sensitivity by qPCR. According to the real‐time fluorescence intensity of qPCR (Figure 1b) and the linear relationship between ΔCt and the mtDNA con‐ centration (Figure 1c), a strong dose‐dependent signal was observed, suggesting that the target mtDNA sequence was driving proximity ligation and the enzymatically active Cas9 nuclease can be used for CasPLA. CasPLA for in situ imaging of mtDNA in single cells. To investigate whether the CasPLA process can be used for in situ mtDNA imaging, a pair of CasPLA probes were de‐ signed to target the 13703G site of the ND5 gene in MCF‐7 cells. The ND5 (NADH dehydrogenase 5) gene encodes a subunit of NADH dehydrogenase, which plays an im‐ portant role in the electron transport chain for generation of ATP (the location of the ND5 gene in mtDNA is shown in Diagram S1). Mutations of the ND5 gene are related with distant metastasis of colon cancer 39 and reduced mito‐ chondrial Ca2+ transients 40. As shown in Figure 2, the green fluorescent signals from the RCA amplicons were predom‐ inantly present in the cytoplasm. The number of amplicons was 231.7/cell, which is around 25% of total mtDNA accord‐ ing to the estimated mtDNA copy number, 41 and the de‐ tection efficiency is around 2.5 times higher than tpRCA.14

CasPLA probe resulted in no obvious signal, which con‐ firms the requirement for binding of both probes. We also compared CasPLA pairs in which the two probes were sep‐ arated by 10 nt or 195 nt. As shown in Figure 2, no obvious signal was observed when the probes were 195 nt apart, while the 10 nt pair gave a strong signal, suggesting that CasPLA requires adjacent binding of the two probes. The influence of Cas9 probe orientation was also tested (Figure S3). In agreement with theoretical assumptions concerning the distance between the ends of the probes, the head‐to‐ head probe configuration resulted in a stronger signal than the head‐to‐tail configuration, and the tail‐to‐tail probe configuration gave no obvious signal. Recently, dCas9 pro‐ tein becomes commercial available, and we also performed direct comparison between Cas9 protein and dCas9 pro‐ tein for mtDNA imaging (Figure S4). By counting the aver‐ age amplicons/cell, we found that, comparing with Cas9 protein, dCas9 showed around 1.2‐fold increase in detec‐ tion efficiency. We think it’s because the dCas9 protein without cleavage activity may stay more firmly binding to the target dsDNA. CasPLA for in situ imaging of SNV in mtDNA. After verifying that CasPLA can be used for mtDNA imaging, we then explored the potential of CasPLA for SNV detection. Specifically, 6 sgRNAs were synthesized by in vitro tran‐ scription (Figure 3a). One contained the wild‐type spacer sequence from the ND5 gene and 5 had SNVs at different positions relative to the PAM. The specificity of the sgR‐ NAs was analyzed by in vitro cleavage assay with a PCR‐ amplified mtDNA sequence (Figure 3b), followed by PCR‐ based CasPLA test. As shown in Figure 3c, 3d, the ΔCt value was highly related to the distance between mismatch site and PAM sequence, indicating that PCR‐based CasPLA strategy may serve as a new way for analyzing SNV in vitro.

Figure 2. CasPLA for in situ imaging of mtDNA in MCF‐7 cells. To ascertain that the CasPLA reaction depends on proximity binding of both probes, two pairs of CasPLA probes were de‐ signed and used for mtDNA imaging. The distance between the probes in the first pair was 10 nt and the distance between the probes in the second pair was 195 nt. The cell nuclei are shown in blue (DAPI), and the RCA amplicons appear as green spots (Alexa Flour 488). Scale bar represents 25 μm.

To ascertain that the CasPLA reaction depends on sim‐ ultaneous binding of both probes, we replaced one CasPLA probe at a time with the corresponding concentration of free oligonucleotide. Figure S2 shows that omission of one



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NADH dehydrogenase and the C12084T mutation has been demonstrated to induce mitochondrial respiration defects and overproduction of reactive oxygen species (ROS) which may account for the ability of tumor cells to develop metastatic potential 10. The sequence data available in the GenBank databases identified that the MDA‐MB‐231 cell line (high metastatic potential) had the C12084T mutation (AB626609), while the MCF‐7 cell line did not have the mutation (AB626610). MDA‐MB‐231 and MCF‐7 cells have completely different morphologies, which allow them to be easily distinguished from each other. MDA‐MB‐231 cells are spindle‐shaped and relatively small in size, while MCF‐ 7 cells aggregate and are relatively large in size.

Figure 3. CasPLA for imaging of SNV in mtDNA. a) The sgRNA target sequences used in the experiment. Red rectan‐ gles represent single‐nucleotide changes. b) Gel electrophore‐ sis to analyze the recognition and cleavage activity of different sgRNAs. The substrate is a PCR‐amplified mtDNA sequence (3 kb) which is cleaved by Cas9 to give two products of 1.7 kb and 1.3 kb. c), d) qPCR analysis of the efficiency of CasPLA with different sgRNAs. e) In situ imaging of mtDNA in MCF‐7 cells using CasPLA with sgRNAs with or without SNVs. The cell nu‐ clei are shown in blue, and the RCA amplicons appear as green spots. Frequency histograms of RCA amplicons per cell are shown to the right of the fluorescence images (50 cells were analyzed for each experiment).

According to the in vitro data, the efficiency of CasPLA was much lower when the SNV was near the PAM sequence, suggesting it is possible to image SNVs using CasPLA. To test this possibility, three pairs of CasPLA probes were de‐ signed to cover the same sequence of the ND5 gene (Figure 3e). In one pair, the sgRNA was designed to match the wild‐type sequence, while the other two contained SNVs at different positions. The in situ imaging data are shown in Figure 3e. With the wild‐type sgRNA, 208.3 RCA amplicons were detected per cell, and this was reduced dramatically to 0.3/cell when the sgRNA contained a SNV 3 nt away from the PAM. When the SNV was 10 nt away from the PAM, there were around 71.1 RCA amplicons per cell, which is evidence of non‐specific amplification. According to the results, CasPLA can recognize SNV in mtDNA near the PAM sequence, which can be utilized for SNV imaging. To use CasPLA for practical in situ mtDNA mutation analysis, we designed a pair of CasPLA probes to genotype the mitochondrial C12084T mutation in the ND4 gene in MDA‐MB‐231 cell. The ND4 gene encodes a subunit of

Figure 4. In situ imaging of the A11002G point mutation in mtDNA in MCF‐7 and MDA‐MB‐231 cells. A pair of CasPLA probes were designed to image the mutated 11002G site of mtDNA in separately cultured and mixed MCF‐7 and MDA‐ MB‐231 cells. The cell nuclei are shown in blue and the RCA amplicons appear as red spots. Frequency histograms of RCA amplicons per cell are shown to the right of the fluorescence images (50 cells were analyzed for each experiment).

Therefore, we first applied the CasPLA probes to geno‐ type the C12084T point mutation in the MCF‐7 and MDA‐ MB‐231 cells. The separately cultured and mixed cells were fixed on glass slides and then treated with the CasPLA probes, followed by proximity ligation and RCA. The RCA products were detected by hybridization of two fluorescent oligonucleotide probes (green for wild‐type mtDNA, red for mutated mtDNA). Surprisingly, when we performed the imaging experiment to detect the mutated mtDNA (12084T) in MDA‐MB‐231 cells, no obvious signal was ob‐ served (Figure S5). But, when we used wild‐type probes to detect 12084C in mtDNA, 392.4 and 262.1 amplicons per cell were found in MCF‐7 and MDA‐MB‐231, respectively (Figure S6). The in situ imaging data indicated that the MCF‐7 and MDA‐MB‐231 cells used in this experiment only contained wild‐type mtDNAs, which is inconsistent with the GenBank databases. To verify this result, we isolated the mtDNAs from both cell lines for sequencing. As shown in Figure S7, the sequencing result indicates that neither of the cells contained the C12084T mutation, which is con‐ sistent with the CasPLA imaging data but not with the GenBank database. This result further illustrates that mtDNA mutation is highly complex and that methods to


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visualize mtDNA mutations in situ in single cells are of high importance. By analyzing sequencing data, we identified a new point mutation, A11002G, in MDA‐MB‐231 cells (Figure S8). A11002G was also located in the ND4 gene (Diagram S1). This mutation does not appear to have been studied previ‐ ously. We designed a new pair of CasPLA probes to image the A11002G mutation and the imaging data are shown in Figure 4. The single‐nucleotide difference between the mtDNAs in the two cell lines was clearly visualized, with MDA‐MB‐231 cells showing predominantly red signals (203.6 per cell) and the MCF‐7 cells showing no obvious signal (3.1 per cell), consistent with the sequencing result. Moreover, the cell lines could clearly be distinguished from each other in the mixed cell culture experiment. Interest‐ ingly, we noticed an obvious intercellular mtDNA transport phenomenon between cells in the co‐culture ex‐ periment (Figure S9). MtDNA was clearly in a transferring process in a tunneling nanotube, which is a structure that facilitates selective transfer of membrane vesicles and or‐ ganelles between cells, which can be an important cause for the spreading of mtDNA heteroplasmy 42. The applica‐ tion of CasPLA for SNV imaging has some limitations in choosing target sites. Considering the availability of appro‐ priate paired CRISPR target sites, Streptococcus pyogenes Cas9 (SpCas9) needs NGG and CCN in proximity for Cas‐ PLA imaging. However, there are some other CRISPR en‐ zymes such as Staphylococcus aureus Cas9 or Cpf1 devel‐ oped as alternative for SpCas9, which can offer additional target sites on the genome43. Moreover, a mutagenesis screen process has been used to increase the targeting range of Cpf1, and the structural basis for the altered PAM recognition by engineered CRISPR‐Cpf1 has been studied44. We think that, in near future, the CRISPR system will not be restricted to conventional NGG PAM sequence, and more potential target will be available for CasPLA based analysis. Simultaneous imaging of wild‐type and mutated mtDNA for studying mtDNA heteroplasmy. To explore the possibility of using CasPLA for simultaneous detection of sequence variants, we performed an imaging experiment with CasPLA probes to detect the wild‐type (A) and mu‐ tant (G) sequences at position 11002 in mtDNA. Specifically, MCF‐7 cells and MDA‐MB‐231 cells were co‐cultured for 2 days and three probes were used in the experiment. One was specific for the wild‐type sequence, one was specific for the mutant sequence, and one was able to pair with ei‐ ther of the other two to create two distinct circularized DNAs for RCA (Figure 5a). RCA from the wild‐type probe gave a green signal, while RCA from the mutant probe gave a red signal (Figure 5b). In the simultaneous imaging ex‐ periment, the number of amplicons per cell was signifi‐ cantly decreased comparing to the imaging performed with a single pair of probes (Figure 4). It is because the two SNV recognition probes (Green and Red in Figure 5a) were designed to pair with the same locating probe (Blue in Fig‐ ure 5a) 32, and the backbone probes were with same bind‐ ing region, which will interfere the probe binding and de‐ crease the detection efficiency. Specifically, for MCF‐7, the

average numbers of green and red amplicons are 47.9/cell and 8.21/cell, respectively. For MDA‐MB‐231, the average numbers of green and red amplicons are 16.5/cell and 25.6/cell, respectively. By plotting the number of red and green amplicons per cell, mtDNA heteroplasmy was clear in the cell co‐culture experiment (Figure 5c, 5d), which may because of the mtDNA transferring process.

Figure 5. Simultaneous imaging of wild‐type (A) and mutant (G) mtDNAs at position 11002 in MCF‐7 and MDA‐MB‐231 cells. a) Detailed mechanism of CasPLA for simultaneous de‐ tection of wild‐type and mutated mtDNA. Three probes were used in the experiment, colored blue, green and red in the fig‐ ure. The blue probe can pair with either of the other two to create two distinct circularized DNAs for the rolling circle am‐ plification. b) Fluorescence imaging of mtDNA in MCF‐7 and MDA‐MB‐231 cells by CasPLA. The green spots represent RCA amplicons hybridized with Alexa488‐labeled detection probes, and the red spots represent RCA amplicons hybridized with Cy5‐labeled detection probes. c) The number of red and green amplicons in each individual MCF‐7 and MDA‐MB‐231 cell. The cell types were distinguished by their morphologies. d) Quantification of the average number of red and green ampli‐ cons per cell in MCF‐7 and MDA‐MB‐231 cells (50 cells were analyzed for each experiment).

CasPLA imaging on a tissue section. Identification of somatic mtDNA alterations is important for genetic diag‐ nosis45. However, conventional methods, such as sequenc‐ ing of bulk tissue samples, cannot accurately determine the spatial distribution of mutated mtDNA and may ignore the small portion of mutated ones in the majority of wild‐type mtDNAs46. Since CasPLA has been proved for imaging of SNV in mtDNA at single‐cell level, we further tested whether CasPLA can be applied for mtDNA analysis in tis‐ sue section. Specifically, we bought a formalin‐fixed paraf‐ fin‐embedded MCF‐7 xenograft tumor tissue sections from Servicebio and proceeded with the CasPLA assay to detect the A>G SNV at 11002 in mtDNA. As shown in Figure 6, the wild‐type CasPLA probe penetrated the tumor sections and efficiently labeled their targets (green), while the mu‐ tant CasPLA probe showed no obvious signal (red). This



indicates that the mtDNA in the tissue section mainly has the wild‐type sequence (A) at position 11002. We also iso‐ lated mtDNA from the tissue section and sequenced the gene to confirm that the mtDNAs were mainly wild‐type (Figure S11). When both CasPLA probes were used, the de‐ tection efficiency was decreased, most likely due to the backbone probe design, as discussed above. Nevertheless, many green fluorescent signals were detected in the simul‐ taneous imaging experiment and no obvious red fluores‐ cent signals were observed. These results suggest that Cas‐ PLA is a powerful tool for visualizing SNV in mtDNA in primary tissue sections for genetic diagnosis applications.

Figure 6. CasPLA imaging of mtDNA at position 11002 in a xenograft tumor tissue section. Two pairs of CasPLA probes (for wild‐type and mutated mtDNA) were designed and used for mtDNA imaging in the tissue section. The cell nuclei are shown in blue and the RCA amplicons appear as green (wild‐ type, A) and red (mutant, G) spots. The xenograft tumor tissue section were expected to contain only the wild‐type target se‐ quence. Scale bar: 200 μm.

CasPLA for imaging single copy loci in nuclear ge‐ nome. Variations in nuclear genome are important indi‐ cators for a number of disease47. Since nuclear genome is packing with histones in a highly condensed nucleosome structure, it’s still challenging to analysis genetic alteration inside chromatin48. Considering Cas9 probes can effi‐ ciently bind to dsDNA in cell nuclei, we further hypothesis that CasPLA may serve as a strategy for in situ imaging of single copy genomic loci in nuclear genome As a proof of concept, we chose KRAS gene as a model system. KRAS gene, which encodes a small G‐protein downstream of EGFR, can acquire activating mutations in exon 2 and isolate the pathway from the effect of EGFR, which make the EGFR inhibitors ineffective49. KRAS gene is mutated in approximately 40% of metastatic colorectal cancer and the most frequent mutations occurring at co‐ dons 12 and13 (G13D) (GCT GGT G G/A C GTA GGC)50. To image KRAS gene in situ in single cells, two pairs of CasPLA probes were designed to target wild‐type and mutated

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KRAS gene at the G13D codon, which only has single‐nu‐ cleotide difference in the sgRNA recognition sequence (GTA GTT GGA GCT GGT G G/A C GT). Since KRAS is a single copy gene located on homo sapiens chromosome 12 and each cell nuclei have one pair of autosomes, we ex‐ pected to see 2 signals per cell. Specifically, MCF‐7 cells were treated with no CasPLA probes/ CasPLA probes tar‐ geting wild‐type KRAS gene/ CasPLA probes targeting mu‐ tated KRAS gene, followed by proximity ligation and RCA. Imaging data are shown in Figure 7 and Figure S12. Using CasPLA probe to target wild‐type KRAS gene, approxi‐ mately 40% of cells showed expected 2 signals per cell, and around 50% cells showed 1 signal per cell. In contrast, the CasPLA probes targeting mutated KRAS gene generated no obvious fluorescent signal. According to the data, CasPLA can be applied for imaging KRAS gene in nuclear genome with high specificity and around 60% detection efficiency.

Figure 7. CasPLA for imaging KRAS gene in nuclear genome. CasPLA probes were designed for targeting wild‐type and mu‐ tated KRAS gene with only single‐nucleotide variation (GCT GGT G G/A C GTA GGC). The DNA amplicons were stained with AF488 probes (Green) and DNA nuclear were stained with DAPI (Blue). RCA amplicons were marked by white ar‐ row. 50 cells were counted in each experiment. Scale bar: 20 μm.

CONCLUSION Imaging of non‐repetitive genomic loci with Cas9‐based methods relies on strategically‐designed signal amplifica‐ tion. In previous technologies, multiple Cas9 probes were used to generate adequate fluorescent signals for imaging. This is problematic for biological applications due to the challenges of delivering multiple sgRNAs into cells and the increased off‐targeting by the large number of sgRNAs 27. In this study, we show that by incorporating a proximity ligation strategy, CasPLA provides strong and discrete flu‐ orescent signals for mtDNA imaging using only two sgR‐ NAs, which allows in situ SNV imaging in single cells. Comparing with previous DNA imaging methods, Cas‐ PLA presents some advanced features (Table S3). First, CasPLA takes advantage of the CRISPR‐based mechanism for rapid and specific DNA hybridization. This enzymatic probe is much more efficient than the nucleic acid probes


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used in FISH and tpRCA. Therefore, CasPLA is able to de‐ tect dsDNA without DNA denaturation with significantly higher detection efficiency. Second, using two probes binding close to each other to initiate proximity ligation and RCA, CasPLA achieves increased specificity due to the necessary of two sequences in the correct spacing and ori‐ entation. The fluorescent signals from RCA amplicons are strong and discrete, allowing the study of mtDNA distribu‐ tion in single cells at single‐molecule resolution. Third, the CRISPR/Cas9 system recognizes target sequences with sin‐ gle‐nucleotide specificity. Binding of the sgRNA spacer se‐ quence to its target is sensitive to single nucleotide changes. Therefore, CasPLA can be utilized for detection and imaging of subtle DNA variations, such as SNVs. We have demonstrated that mtDNAs with a point mutation can be clearly distinguished from the wild‐type mtDNAs by CasPLA imaging. Fourth, the mild conditions of CasPLA can better preserve cell morphology and DNA structure, and thus CasPLA can potentially be used to visualize the interaction of proteins or lncRNA with specific DNA se‐ quences. Moreover, CasPLA is applicable for tissue sec‐ tions mtDNA imaging and even nuclear genome single copy loci imaging, thus offering potential for basic research and genetic diagnosis. Future development of CasPLA will be aimed at live‐cell imaging with a compatible signal am‐ plification method, such as the hybridization chain reac‐ tion (HCR)51,52, and further improving the specificity and efficiency. We expect that the CasPLA imaging method will serve as an important tool for direct visualization of SNVs in genomic loci with high spatiotemporal resolution in sin‐ gle cells.

MATERIALS AND METHODs Materials and apparatus. All oligonucleotide se‐ quences in Table S1 and Table S2 were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). Cas9 Nuclease, S. py‐ ogenes (1,000 nM), EnGen® Spy dCas9 protein, HiScribeTM T7 Quick High Yield RNA Synthesis Kit and dNTP Mix were purchased from NEB (New England Biolabs, Ipswich, MA, USA). T4 DNA ligase, T4 polynucleotide kinase, Phi29 DNA polymerase and RiboLock RNase Inhibitor were pur‐ chased from Life Technologies (Carlsbad, CA, USA). A CFX 96 real‐time PCR detection system (Bio‐Rad) was used for qPCR. A NanoDrop 2000 UV‐Vis Spectrophotometer was used to measure nucleic acid concentration. Leica TCS SP5 laser scanning confocal microscope was used for fluores‐ cent imaging. sgRNA synthesis. The sgRNAs were synthesized in vitro from DNA templates using T7 RNA polymerase. The tem‐ plate DNA was synthesized by PCR reaction using different primers and the produced sgRNAs were purified by Trizol. Primers used in this work are listed in Table S1. The se‐ quence of modified sgRNA1: 5’‐ NNN NNN NNN NNN NNN NNN NNG UUU AAG AGC UAU GCU GGA (AAA AAG AAA AAU GCA AGU GGA AUA CCA AAA AGA AAA A)AA CAG CAU AGC AAG UUU AAA UAA GGC UAG UCC GUU AUC AAC UUG AAA AAG UGG CAC CGA GUC GGU GCU UUU UUU CUU ‐3’. The sequence of modified

sgRNA2: 5’‐ NNN NNN NNN NNN NNN NNN NNG UUU AAG AGC UAU GCU GGA (AAA AAG AAA AAC CUG CUC UAG CAA UGA AAA AGA AAA A)AA CAG CAU AGC AAG UUU AAA UAA GGC UAG UCC GUU AUC AAC UUG AAA AAG UGG CAC CGA GUC GGU GCU UUU UUU CUU ‐3’. Cell culture and tumor section preparation. MCF‐7 and MDA‐MB‐231 cells were cultured in DMEM supple‐ mented with 10% FBS and 1% penicillin/streptomycin at 37 o C in a humidified atmosphere with 5% CO2. The xenograft tumor tissue FFPE (formalin‐fixed paraffin‐embedded) section was purchased from Servicebio and deparaffinized using established protocols (Weibrecht et al., 2013). PCR‐based CasPLA assay. A PCR‐based CasPLA assay was first used to verify the applicability of CasPLA probes. Specifically, 500 pM of paired CasPLA probes were first suspended in Cas9 reaction buffer [20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH=6.5]. Target DNA with different concentrations was then added and incu‐ bated at 37 oC for 40 min. The reaction mix was added to pre‐made ligation mix [40 mM Tris‐HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, 0.025 U/μL T4 DNA ligase, 100 nM bridge oligo, pH=7.5] and then incubated for 15 min at 30 oC. 25 μL of the ligation mix was then added to 25μL 2x PCR Master Mix with 10 nM PLA‐PCR primers and amplified by PCR (95 oC for 10 min, 60 oC for 30 s, 95 oC for 15 s, 13 cycles). The PCR reaction was then diluted 1:20 in ddH2O. 8.5 μL of the diluted PCR samples were added to 10 μL 2x qPCR Mas‐ ter Mix with 1.5 μL PLA‐PCR primer. qPCR was performed with a Bio‐Rad CFX96 with standard thermocycling. CasPLA for mtDNA imaging. Unless indicated, the standard CasPLA protocol is as follows: Cells cultured on collagen‐coated glass were fixed at ‐20 oC for 20 min in a pre‐chilled solution of methanol and acetic acid at a 1:1 ra‐ tio. Samples were washed 3 times (5 min each time) with PBS with gentle shaking, followed by incubation for 2 hours at 37 oC in blocking/reaction buffer [20 mM HEPES (pH 6.5), 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM freshly added DTT, 250 μg/ml BSA, 25 μg/ml sonicated salmon sperm DNA, 0.05% Tween 20]. To assemble Cas‐ PLA probes, Cas9 protein (20 nM) was mixed with sgRNA at a molar ratio of 1:4 in blocking/reaction buffer and was incubated at room temperature for 10 min. The PLA‐RCA probes 1 and 2 were then added to the reaction mix with a final concentration of 200 nM. The assembled CasPLA probes were applied to pre‐blocked cells and incubated for 1 hour at 37 oC. The reaction was terminated by removal of the CasPLA probe solution and washing 3 times with blocking/reaction buffer for 5 min each. To ensure optimal conditions for the ligation reaction, the cells were soaked for 5 min in ligation buffer, prior to addition of the ligation mix [100 nM PLA‐RCA backbone, 100 nM PLA‐RCA insert, 40 mM Tris‐HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, 0.05 U/μL T4 DNA ligase]. The ligation reaction was con‐ ducted at 37 oC for 2 hours. After circularization, the sam‐ ples were washed 3 times with blocking/reaction buffer to remove excess oligonucleotides. The RCA reaction was then performed by adding RCA mix [33 mM Tris‐acetate, 10 mM Mg‐acetate, 66 mM K‐acetate, 0.1%(v/v) Tween‐20, 1 mM DTT, 10 μM dNTPs, 0.1 U/μL Phi 29 DNA polymerase] and incubating for 2 hours at 37 oC. The cells were washed



three times with blocking/reaction buffer for 5 min each, then 10 nM detection probe was added and incubated for 30 min at 37 oC. From this step onwards, the slide was kept in the dark. The samples were washed three times with PBS and the nuclei were stained with DAPI. The slides were mounted with mounting media and pictures were taken with a confocal microscope. CasPLA for nuclear genome imaging. Cells cultured on collagen‐coated glass were fixed at ‐20 oC for 10 min in a pre‐chilled solution of methanol and acetic acid at a 3:1 ratio. The fixation procedure was repeated two times. The fixed cells were treated with 0.01% pepsin in 0.1 M HCl for 90 s at 37 oC, followed by washes in PBS. Cells were then washed with a series of ethanol baths (70%, 90% and 100%) for dehydration. The following CasPLA procedure was the same with CasPLA for mtDNA imaging. Microscopy and image analysis. All CasPLA samples were imaged using a Leica TCS SP5 inverted confocal mi‐ croscope (Leica, Germany). The images were acquired with a 40 x oil‐immersion objective. Ar laser (488 nm) was used as the excitation source for Alexa488‐labeled probes, and a 500‐535 nm bandpass filter was used for fluorescence de‐ tection. The Cy5 dye was excited with a HeNe633 (633 nm) laser and detected with a 650‐750 bandpass filter. Z‐stacks were collected at a step size of 0.2 μm for 20 slices to image the entire cell. The images were processed using ImageJ. Amplicons with a bright fluorescent signal were distin‐ guished from background by adjusting the threshold value to 50. To determine the copy number per cell, the number of bright pixels was counted by particle analysis in ImageJ.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figures S1‐S12, Table S1‐S3.

AUTHOR INFORMATION Corresponding Author * [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was financially supported by National Natural Sci‐ ence Foundation of China (No. 21621003, No. 21235004 and No. 21327806), National Key Research and Development Program of China (No. 2016YFA0203101) and Tsinghua University Initi‐ ative Scientific Research Program.

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Direct Visualization of Single-Nucleotide Variation in mtDNA Using a

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