Single Genomic Loci Labeling and Manipulation Using SIMBA System

The SIMBA (Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies) system is an innovative CRISPR‐based imaging technique that leverages dCas9‐SunTag and FRB‐mCherry‐HP1α, with scFv‐FKBP acting as a bridge. This powerful system enables simultaneous visualization and manipulation of genomic loci. The dCas9‐SunTag fusion protein allows for precise targeting of specific genomic sites, and the FRB‐mCherry‐HP1α fusion protein facilitates the condensation of chromatin at the targeted loci. The scFv‐FKBP bridge protein links dCas9‐SunTag and FRB‐mCherry‐HP1α, ensuring efficient and specific recruitment of HP1α to the desired genomic loci. This integrated approach allows us to visualize and manipulate genomic regions of interest, opening up new avenues for studying genome organization, gene expression regulation, and chromatin dynamics in living cells. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.


INTRODUCTION
In recent years, significant advancements have been made in the development of various microscopic techniques aimed at visualizing and accurately mapping the localization of chromatin compartments.One widely used approach is the application of DNA fluorescent in situ hybridization (FISH) techniques (Manuelidis, 1985;Price, 1993).By leveraging the power of fluorescently labeled DNA probes, these methods enable researchers to precisely target and visualize specific genomic regions within the nucleus.The traditional FISH design was combined with super-resolution imaging techniques, leading to the development of high-resolution intranuclear imaging methods (Jerkovic & Cavalli, 2021;Wang et al., 2017).These innovative approaches push the boundaries of optical resolution, allowing for the visualization of chromatin compartments at an unprecedented level of detail.
Although DNA FISH has proven to be a valuable tool in chromatin research, it does have certain limitations.One such limitation is its restriction to fixed cell samples, which do not provide temporal information or allow capture of dynamic processes occurring within the cell.This lack of temporal resolution poses challenges when studying timedependent phenomena such as chromatin remodeling, gene expression dynamics, or the spatial reorganization of chromatin during cellular processes.
The emerging usage of the CRISPR/Cas9 system provides us with more opportunity in tracking genome dynamics at the live-cell level (Liu et al., 2017;Wang et al., 2019;Wu et al., 2016).For example, the live-FISH system uses nuclease-defective Cas9 (dCas9) and fluorescent-labeled guide RNA (gRNA) complex (Wang et al., 2019).dCas9/gRNA complex recognizes the target gene sequence, which carries its chromophore to the loci to be visualized by a confocal microscope.Various catalytically inactive CRISPR/Cas9 (dCas9)-based labeling systems have been developed to visualize genomic loci in living cells, aiming to overcome challenges such as low signal-to-noise ratio (SNR) at specific genomic loci, particularly those with non-repetitive sequences (Chen et al., 2013;Cheng et al., 2016;Hong et al., 2018;Ma et al., 2016;Qin et al., 2017;Ye et al., 2017).Strategies to enhance the SNR and amplify signals for locus labeling include bimolecular fluorescence complementation (BiFC) (Chen et al., 2013;Ma et al., 2018), tandem repeats of MS2 stem-loop (Gu et al., 2018;Ye et al., 2017), and multiple repeats of SunTag (Chen et al., 2013;Hong et al., 2018;Ye et al., 2017), typically integrated with the dCas9-single guide RNA (sgRNA) system.However, for non-repetitive loci, where there is only one anchoring site for a dCas9-sgRNA complex, the efficiency of locus-specific labeling using a single sgRNA is generally low.Currently, multiple sgRNAs targeting neighboring regions of the target locus are still required for efficient labeling of non-repetitive loci (Cheng et al., 2016;Gilbert et al., 2013;Mao et al., 2019;Shao et al., 2018;Ye et al., 2017).However, this approach introduces increased complexity, reduced efficiency, and a higher risk of off-target labeling.
Oligomers have demonstrated the capacity to form membrane-less organelles, including condensed biomolecular assemblies (BAs) within the nucleus.These assemblies have the ability to interact with chromatin, thereby influencing genome architecture and gene expression (Brangwynne et al., 2009;Cheutin et al., 2003;Frey et al., 2006;Qi & Zhang, 2021).One key player in this context is heterochromatin protein 1α, often abbreviated as HP1α, which serves as a significant component of constitutive heterochromatin.HP1α plays pivotal roles in shaping chromatin structure, upholding genome stability, and governing gene expression (Larson et al., 2017;Wang et al., 2016).HP1α exhibits the capability to condense both in vitro and in vivo, driven by weak hydrophobic interactions primarily facilitated by an intrinsically disordered region (IDR) situated in the N-terminal extension and hinge domain (Keenen et al., 2021;Strickfaden et al., 2020;Strom et al., 2017).This increased local concentration of HP1α encourages the formation of BAs at genomic loci, involving intricate interactions, both homotypic and heterotypic, with proteins, RNAs, and DNAs (Wang et al., 2020).Furthermore, specific buffer conditions and environmental co-factors present at these genomic regions can further promote the formation of condensed chromatin structures (Strickfaden et al., 2020).
The structural makeup of HP1α encompasses distinct domains, including an Nterminal chromodomain (CD) responsible for binding to K9-trimethylated histone H3 (H3K9me3), a middle positively charged hinge domain (HD) that facilitates interactions with DNA and RNA, and a C-terminal chromo shadow domain (CSD) crucial for Chen et al.

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Current Protocols self-association (Strom et al., 2017).HP1α exhibits direct interactions with histone methyltransferases, such as SUV39H1/2 and SETDB1, thereby enabling selfpropagation and sequential methylation of adjacent nucleosomes and the recruitment of additional HP1α molecules within the local genomic region (Chaudhary et al., 2020;Krouwels et al., 2005;Schotta et al., 2002;Schultz et al., 2002).These unique characteristics of HP1α make it a promising candidate for triggering BA formation for genomic locus labeling.Additionally, the transcription suppression capability of HP1α may allow for localized gene silencing at the labeled loci in living cells.Thus, HP1α is emerging as a promising candidate for initiating the formation of BAs at genomic loci for the purpose of labeling.Additionally, its ability to suppress transcription suggests the potential for localized gene silencing at labeled loci in living cells.
Here, we introduce basic protocols for the CRISPR-based SIMBA (Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies) system (Peng et al., 2022).In this design, dCas9 recognizes specific gene loci, recruiting fluorescent proteinfused HP1α in a rapamycin-dependent manner, which facilitates chromatin condensation through HP1α and SUV39H1-mediated heterochromatin formation.The system could be easily turned on by rapamycin induction, and the condensed chromatin at specific genomic loci (repetitive and non-repetitive) not only enables precise imaging with an enhanced SNR but also alters the local H3K9me3 level, leading to gene silencing.

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Current Protocols 4. Take a small aliquot (100 to 200 μl bacterial culture) and mix with 1 volume of 50% (v/v) glycerol in a 1.5-ml Eppendorf tube or cryovial to prepare glycerol stock.Store stock at −80°C for long-term storage.Spin down the remaining culture for 5 min at 5000 × g.Extract plasmid DNA using GenElute HP Plasmid DNA Miniprep Kit following the manufacturer's protocol.
5. Elute plasmid DNA with 30 to 60 μl molecular biology-grade water.Measure the DNA concentration using a spectrophotometer.
6. Verify the amplified plasmid by Sanger sequencing or restriction enzyme digestion.

Preparation of basic coding cassettes and vector backbone for recombination
7. Design and obtain the appropriate primers for cassette cloning (containing 20-to 25-bp annealing region) with a homologous arm region (16 to 25 bp with Tm ≥ 48°C) for Gibson Assembly.
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10. Recover the linearized vector fragment by agarose gel electrophoresis and gel recovery using the Zymoclean Gel DNA Recovery Kit.
11. Build expression vector for each of the components by Gibson Assembly, including dCas9-24×SunTag, scFv-FKBP, and FRB-mCherry-HP1α, preparing the reaction mix using Gibson Assembly Master Mix following the manufacturer's protocol.
Mix by pipetting, perform a quick spin, and then incubate at 50°C for 15 min in a thermocycler.
12. Amplify and screen for successful clones by transformation of reaction product into Stbl3 competent cells and streak on an ampicillin-selective LB plate.
13. Verify the construct by Sanger sequencing or restriction enzyme digestion.
sgRNA expression vector subcloning 14.Design the 20-nt targeting sequence sgRNA sequence for specific labeling loci or look up from previous publications.
The sgRNA expression vector for dCas9 complex targeting and labeling is another important component.We list some useful sgRNA targeting sequences used in the SIMBA system in Table 2.
17. Incubate the PCR tube containing reaction system in a thermocycler at 95°C for 3 min and then slowly cool to 25°C with a ramp rate of 0.5°C/s.Then, dilute 5 μl annealed oligos with 245 μl water to a final concentration of 200 nM, which is ∼3 ng/μl and ready for use.
19. Incubate the reaction at 37°C for 15 min in a thermocycler or incubator and then transform 2 μl reaction mix into 25 μl Stbl3 competent cells for further amplification to obtain single clones.
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Current Protocols 20.Examine the single clones by Sanger sequencing or restriction enzyme digestion.

SIMBA constructs
21.For SIMBA imaging and further lentivirus packaging, subclone the sgRNA to the pHR vector by Gibson Assembly following the manufacturer's protocol.
CRISPR dCas9-sgRNA complex, which functions as a targeting unit to specific gene loci, could be substituted by other genomic DNA-binding units.Here, we demonstrate this with NFAT1, a transcription factor.The NFAT1 and 8×SunTag fragments were respectively PCR-amplified and then recombined into the pHR vector by Gibson Assembly.
For BA formation, other IDR sequence-containing components besides HP1α could also be used as the reporter unit in the SIMBA system.Here, we demonstrate this by using the N-terminal region of the human FUS (fused in sarcoma) protein (FUS N ) to replace HP1α in the construct FRB-mCherry-HP1α (i.e., FRB-mCherry-FUS N ).
23. Amplify the corresponding plasmids for either experimental usage or storage.

TRANSIENT TRANSFECTION IN MAMMALIAN CELLS AND LIVE-CELL IMAGING
Basic Protocol 1 yields the essential constructs needed for delivery and expression in mammalian cells.To assess the functionality of the SIMBA system, transient transfection is performed in HEK 293T cells (ATCC®, CRL-3216).Upon successful transfection, rapamycin treatment is administered to the transfected cells, initiating the dimerization of FKBP-FRB.Subsequently, images of BAs in the transfected cells are captured following rapamycin treatment, facilitating the visualization of genomic loci (refer to Fig. 2).Specific SIMBA-labeled puncta undergo analysis based on SNR, employing established methods for SNR assessment as detailed in prior studies (Mao et al., 2019).This experimental trajectory serves to validate the potential of the SIMBA system at the livecell level.2. One day before transfection, seed 0.2 × 10 6 HEK 293T cells per well onto 12-well plates to obtain 70% to 90% confluency by the time of transfection (no longer than 24 hr after cell seeding).Culture cells in complete DMEM in a humidified incubator at 37°C supplemented with 5% CO 2 .

Materials
Ensure that the cells have good viability and are without contamination before use.
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Current Protocols 3. Incubate overnight to reach 70% to 90% confluency for optimal transfection performance.Exchange medium with fresh medium before transfection.
4. Transfect the corresponding plasmids using Lipofectamine 3000 transfection reagent following the manufacturer's protocol.For transfection of one well of cells, prepare two 1.5-ml Eppendorf tubes separately: (a) 50 μl Opti-MEM medium with 1.5 μl Lipofectamine 3000 reagent and (b) 50 μl Opti-MEM with plasmid to transfect and P3000 reagent at a ratio of 1:2 (w/v), i.e., 2 μl P3000 reagent per 1 μg plasmid.Mix contents of tube (a) with contents of tube (b) and incubate for 10 min at room temperature.Then, add the mixture to the culture well.
Note that the sgRNA sequence varies when targeting different intended gene loci for imaging.Representative sgRNA sequences with their target genes are shown in Table 3.
5. Seed the transfected cells onto glass-bottom dishes (one well per dish in complete DMEM) coated with fibronectin at 24 hr after transfection.
6. Add 100 μM rapamycin stock to medium to a final concentration of 100 nM at 2 hr before imaging.Add the same amount of DMSO to the cells of control group.At 36 to 48 hr after transfection, switch medium to live-cell imaging solution without phenol red for best imaging quality and image the cells using a confocal microscope.
7. Use MetaFluor software and/or ImageJ for imaging data analysis.Spot SIMBA puncta manually to determine their average intensity.Quantify background signals from regions within the nucleus outside of the puncta area and the nucleolus, allowing measurement of the average background intensity and standard deviation.
8. Determine the SNRs by dividing the corrected SIMBA puncta signal (average SIMBA puncta intensity subtracted mean background intensity) by the standard deviation of the background signal.Utilize the designated SNR value of 10 as a threshold for identifying specific SIMBA puncta in response to rapamycin induction.

GENERATION OF SIMBA-EXPRESSING STABLE CELL LINES
Generating a stable cell line expressing the desired components can provide stable expression and induction levels for further imaging and analysis.In this protocol, we demonstrate that human U2OS cells can be transduced by lentiviral vectors encoding the SIMBA components to generate stable cell lines, which can be verified by fluorescence microscopy imaging and/or flow cytometry.
Ensure that the cell is in good condition, with >90% viability, and without contamination.
A sufficient amount of plasmids is vital for successful lentivirus production.Expand plasmid copies according to Basic Protocol 1 and perform maxi prep using a HiSpeed Plasmid Maxi Kit (Qiagen, 12662) following the manufacturer's protocol.The suggested plasmid concentration is >1 μg/μl for lentivirus packaging.
3. Day 3: Prepare for lentivirus collection at 48 hr after transfection by collecting supernatant containing lentivirus and filtering through a 0.45-μm low-binding filter to remove any residue cell debris.Concentrate the virus using Clontech Lenti-X Concentrator following the manufacturer's protocol.Immediately store the concentrated virus at −80°C in single-use aliquots in 1.5-ml Eppendorf tubes (∼10 μl/aliquot).

Cell line generation
4. Seed U2OS cells in complete McCoy's 5A (Modified) Medium into 10-cm culture dishes (2 × 10 6 cells in 10 ml medium per dish) or 12-well plates (0.2 × 10 6 cells in 1 ml medium per well) and culture in humidified incubator at 37°C with 5% CO 2 to an optimal confluence (about 50% to 70%) for infection.Exchange medium with fresh medium before virus infection.
The U2OS cell line is used for lentivirus infection, generating a stable cell line for further imaging and manipulation.
5. For lentivirus infection, pre-mix virus with 4 μg/ml polybrene (from 10 mg/ml stock).Then, dilute 10 μl SIMBA virus from step 3 10-fold in complete DMEM and add to cells.Infect the cells for 24 hr.
6. Sort the SIMBA-transduced cells (mCherry-positive) using a FACS sorter 3 days postinfection.Passage the stable cell lines at least six times after sorting before rapamycin induction for experiments (see step 7).
To passage the cells, aspirate the culture medium containing fetal bovine serum (FBS) completely.Wash once with pre-warmed phosphate-buffered saline (PBS) to remove any residual medium and aspirate completely.Then, add one volume of 0.25% trypsin solution to cover the cells and digest at room temperature or 37°C till most of the cells detach from the culture plate surface.Add more than 2 volumes of complete cell culture medium to terminate digestion.Gently pipet the cells off the plate and collect the cells for 5 min at Chen et al.

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Current Protocols 500 × g.Discard the supernatant with trypsin and resuspend cell pellet in 1 ml complete culture medium.Seed ∼200 μl cells for sub-culture.
Imaging using SIMBA stable cell lines 7. Seed the stable cell line in 1 ml medium in a glass-bottom dish coated with fibronectin and prepare for imaging with 100 nM rapamycin induction 6 hr in advance.Add the same amount of DMSO to the cells of control group.
The U2OS cell line expressing SIMBA system works well as a tool for long-term and stable imaging at specific genomic loci.
8. Image cells using a confocal microscope and analyze as in Basic Protocol 2, steps 7 and 8.

MANIPULATION OF GENOMIC LOCI USING SIMBA
The SIMBA system allows genomic loci labeling and manipulation via the BAs.At its core, the system comprises two fundamental units: the dCas9-sgRNA complex as the targeting unit and HP1α as the functional unit.By custom-designing for specific genomic loci, the system allows for straightforward substitution of sgRNA, thereby achieving precise labeling for different gene loci.Further characterization of target gene transcription, often carried out through quantitative PCR (qPCR), unveils the transcriptional status of the labeled gene within its genomic context.
For the simultaneous labeling of multiple genomic loci, the SIMBA system offers strategies such as parallel labeling at multiple sites by mixing sgRNA that targets different loci.Moreover, the system is useful to directly manipulate transcription at specific sites, leveraging downstream genes affected by transcription factors, which facilitates comprehensive analysis of transcription factor impact on gene regulation across the entire genome, offering profound insights into the intricacies of gene regulatory networks.
Another key feature of the system is the capability to replace HP1α with other IDR sequences, thereby achieving phase separation effects without necessitating chromatin condensation.This could enhance our understanding of gene distribution and interactions within three-dimensional chromatin space.Therefore, the SIMBA system stands as a versatile tool empowering researchers in gene tracing and manipulation studies.

24-well plates
Additional reagents and equipment for qPCR and RNA-seq

Part 1: Gene manipulation at specific loci on chromosome
1. Seed HEK 293T cells with good viability in 12-well plates at 0.2 × 10 6 cells in 1 ml medium per well.Incubate until about 70% to 80% confluent and then exchange medium with fresh medium for transfection preparation.
For chromatin manipulation at specific gene loci, appropriate targeting sgRNAexpressing vector must be prepared along with essential elements for SIMBA system.
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A group transfected with the same dCas9 construct without HP1α can serve as the negative control.
3. Incubate the cells in humidified cell culture incubator at 37°C supplemented with 5% CO 2 for 36 to 48 hr.
4. Collect the cell sample, sort out mCherry-positive cells by FACS, and re-seed them in 500 μl culture medium in 24-well plates.
5. Incubate the mCherry-positive cells to optical confluence and then treat with 100 nM rapamycin to induce BA formation.
6. Six hours after rapamycin induction, extract total RNA from cell sample using RNeasy Mini Kit following the manufacturer's protocol.
7. Once fresh total RNA sample is acquired, perform reverse transcription PCR using SuperScript IV Reverse Transcriptase following the manufacturer's protocol.
8. Design and obtain qPCR primers for the gene targets based on previous publications or database.Perform qPCR on cDNA samples using SYBR Green Mix.Use betaactin (ACTB) as an internal control for qPCR analysis.Sample preparation is critical for a successful qPCR reaction.Preparation of a master mix is recommended, followed by splitting into at least three parallel samples for a single reaction, which is good for statistical correction.
9. Analyze qPCR results and evaluate the manipulation efficiency of SIMBA system.
The 2 -Ct method is recommended for qPCR analysis.Correct the Ct value of the target gene of each group to that of a housekeeper gene (ACTB, for example).Then, correct the values of the experimental groups to those of controls and calculate the fold change in target gene expression.A biological repeat of at least three is required.
11. Incubate the cells in humidified culture incubator at 37°C with 5% CO 2 for 36 to 48 hr.
12. Collect the cell sample and sort out mCherry-positive cells with FACS sorter.
13. Re-seed the sorted cells into a 24-well plate with 500 μl growth medium.
14. Incubate the mCherry-positive cells to optimal confluency and treat cells with 100 nM (final concentration) rapamycin to induce BA formation.15.Harvest the cells at 6 hr after rapamycin induction.Extract total RNA from cell sample using RNeasy Mini Kit by following the manufacturer's protocol.
16. Send collected RNA samples for RNA-seq (including library preparation and nextgeneration sequencing).

COMMENTARY Background Information
Emerging CRISPR-Cas9 systems The advent of CRISPR-Cas9 technology has sparked a revolution in gene editing, providing researchers with a powerful tool for precise genetic modifications (Doudna & Charpentier, 2014).CRISPR-Cas9, initially harnessed from the bacterial immune system (Jinek et al., 2012), has been ingeniously repurposed to facilitate targeted DNA cleavage and subsequent repair processes (Cofsky et al., 2022;Jinek et al., 2014;O'Connell et al., 2014).This revolutionary technique enables the manipulation of specific genetic sequences, holding immense promise for therapeutic applications, disease modeling, and fundamental biological research.
In parallel, the emergence of the catalytically inactive Cas9 mutant, known as dCas9, has expanded the toolkit of genetic manipulation strategies (Gilbert et al., 2013;Lebar et al., 2020;Zalatan et al., 2015).Unlike its active counterpart, dCas9 lacks endonuclease activity, rendering it incapable of inducing DNA double-strand breaks (Gilbert et al., 2013).However, this deficiency has proven to be its strength, as dCas9 can be tailored for locus-specific targeting without altering the DNA sequence.By utilizing dCas9 fused with effector domains or reporter molecules, the system allows for precise localization and dynamic monitoring of genomic loci (Lebar et al., 2020;Zalatan et al., 2015).The synergy of dCas9 with these functional moieties has laid the foundation for the SIMBA system, which combines precise targeting with realtime visualization, opening up new avenues for exploring gene function within living cells.

Phase separation and biomolecule assembly in cells and their functions
Recent advancements in cell biology have unveiled the phenomenon of phase separation and BA within cells (Lafontaine et al., 2021;Rippe, 2022).Intracellular condensates, characterized by the dynamic aggregation of biomolecules, play pivotal roles in orchestrating various cellular processes (Lu et al., 2018;Strom et al., 2017).These liquid-liquid phaseseparated compartments enable the spatial organization of cellular components, offering a dynamic platform for biochemical reactions and signaling cascades.

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Current Protocols interactions generate liquid-like compartments, facilitating the concentration and sequestration of molecules, thereby enhancing reaction efficiency and specificity.Examples of phase-separated compartments include stress granules, nucleoli, and P bodies, each fulfilling distinct cellular functions (Riback et al., 2020;Yang et al., 2020).Importantly, the recognition of phase separation's role in cellular processes has expanded our understanding of gene regulation, protein homeostasis, and the stress response.

SIMBA system
The SIMBA (Simultaneous Imaging and Manipulation with Biomolecular Assemblies) system represents a remarkable integration of CRISPR-Cas9 technology, phase separation principles, and BA dynamics (Peng et al., 2022).At its core, the SIMBA system leverages the dCas9-SunTag fusion to achieve precise locus-specific targeting.The FKBP-mCherry-HP1α reporting unit enables the visualization and manipulation of targeted genomic loci, whereas the scFv-FRB serves as a bridging component to allow the assembly of the system in response to rapamycin, triggering dynamic gene manipulation events.
To establish the SIMBA system, several key steps are involved.First, the dCas9-SunTag construct is generated and validated through cloning and functional assays.The FRB-mCherry-HP1α fusion is then introduced, enabling fluorescence-based readouts of gene-targeting events, as well as HP1αmediated transcriptional manipulation.The scFv-FKBP bridge is incorporated, ensuring the inducible assembly of biomolecules upon rapamycin treatment.The culmination of these components creates a synergistic platform that merges live-cell imaging with precise gene manipulation.
One of the remarkable features that distinguish the SIMBA system is its inherent modular design, offering the flexibility to tailor both the targeting and the sensing units to address specific experimental requirements.This modular architecture empowers researchers to customize the system according to their investigational needs, thereby expanding its applicability and versatility.For instance, the targeting unit within the SIMBA system can be seamlessly substituted with different molecular entities to achieve locus-specific gene manipulation.A prime illustration of this flexibility is the integration of transcription factors like NFAT1.Leveraging NFAT1's inherent binding specificity, it becomes pos-sible to precisely target downstream genomic loci and exert a controlled influence on gene expression patterns.This ability to introduce different targeting modules not only demonstrates the versatility of the SIMBA system but also opens avenues for probing intricate gene regulatory networks within living cells.
Moreover, the underlying strength that magnifies the signal of low-repeat or singlecopy loci within the SIMBA system is the orchestrated assembly of HP1α, a component facilitated by the phenomenon of phase separation.This remarkable effect enhances the localization and concentration of HP1α, leading to an amplified readout at specific genomic sites.By harnessing phase separation, the SIMBA system transcends the limitations of low-repeat or single-repeat regions, enabling precise investigations even in challenging genetic contexts.Furthermore, the potential utilization of IDRs, such as the commonly used FUS protein, introduces an additional layer of adaptability to the SIMBA system.As demonstrated in our experiments, the incorporation of FUS N as part of the system showcases its compatibility without inducing disruptive chromatin remodeling.
In essence, the SIMBA system represents a paradigm shift in live-cell imaging and gene manipulation.Its unique fusion of theoretical underpinnings and practical implementation unlocks a new dimension of cellular exploration, as well as promising transformative insights into the complexity of living systems.

Critical Parameters
The SIMBA system approach includes the following basic requirements for DNA cloning, lentivirus packaging, cell transfection, and puncta induction for imaging and manipulation.
For basic cloning of the constructs for SIMBA system delivery, the following need to be considered: 1. DNA quality and storage: High-quality template DNA or fragment of cassettes and vector backbone is crucial for effective construct assembly.Store DNA from clones of varying insert sizes at −20°C to preserve its integrity.
2. Clone verification: Selecting 2 to 3 colonies from each recombineering reaction enhances the identification of correct clones.Validate using restriction enzyme DNA fingerprinting for robust confirmation.
3. Enzyme selection: Opt for efficient and cost-effective restriction enzymes for confirming recombineering outcomes.Compare with Chen et al.

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Current Protocols unmodified clones and perform parallel digestions for accurate verification.
4. Gel electrophoresis and uncut DNA: Employ agarose gel electrophoresis with uncut DNA samples alongside plasmid digestions.
Beyond the realm of DNA recombination, achieving efficient delivery of recombinant DNA into target cells stands as another pivotal facet of the experimental process.This critical step encompasses both transient transfections facilitated by Lipofectamine 3000 and stable transfection via lentiviral vectors, each requiring distinct considerations to optimize successful gene transfer.
In the case of transient transfection utilizing Lipofectamine 3000, several key parameters profoundly impact the process.The choice of transfection reagent concentration, DNAto-reagent ratio, and cell density during transfection significantly influences transfection efficiency.Optimization of these factors ensures optimal DNA uptake and expression.Moreover, the duration of transfection plays a crucial role; a balance must be struck to achieve sufficient gene expression without inducing cellular stress.Co-transfection with reporter genes aids in monitoring transfection efficiency and facilitating downstream analyses.
Alternatively, stable transfection through lentiviral vectors offers a more enduring genetic modification.Critical parameters here include titration of lentiviral particles to determine the optimal multiplicity of infection (MOI) for target cells, ensuring effective gene delivery without inducing cytotoxic effects.The selection of appropriate selection markers, such as antibiotic resistance or fluorescence, is vital for isolating and maintaining cells with stable transgene expression.Proper timing of selection pressure application and proper maintenance duration guarantee robust establishment of stable transfectants.
In both transient and stable transfection approaches, the choice of target cell type is paramount.Considerations encompassing cell proliferation rate, susceptibility to transfection, and compatibility with downstream applications dictate the most suitable cellular context for the experiment.Maintenance of consistent cell culture conditions, including medium composition, temperature, and CO 2 levels, further ensures reproducibility and reliability across experiments.
The key for best imaging and gene manipulation after successful delivery of the SIMBA system is the induction of particle assembly by rapamycin.Thorough gene expression monitoring, transfection validation, and downstream assays validate the relevance of introduced DNA.Effective delivery of recombinant DNA involves optimizing parameters like transfection reagents, ratios, cell density, duration, and selection markers.Precise target cell selection and post-transfection analysis ensure successful SIMBA puncta formation in response to rapamycin, aligning with the experiment's goal.

Troubleshooting
For a list of common problems with different aspects of the protocols, their causes, and potential solutions, please see Table 5.

Understanding Results
Our experimental design hinges on the foundational process of construct cloning (Basic Protocol 1), which serves as the linchpin of the SIMBA system's implementation.Cloning stands as the essential framework upon which the entire endeavor is built.Careful attention is paid to the nuances of each cloning step, employing high-fidelity enzymes to ensure accurate replication of genetic components.Rigorous validation methods, such as PCR and enzymatic fingerprinting, bolster the reliability of each clone, ultimately confirmed by Sanger DNA sequencing to ensure precision.The pivotal factor underpinning the effective utilization of the SIMBA system, as guided by Basic Protocols 2 to 4, revolves around the precise targeting of repetitive or non-repetitive sequences through the sensing unit of the dCas9-sgRNA complex or transcription factors such as NFAT1 aimed at its downstream genes.Additionally, the assembly of fluorescence molecules is triggered by the induction of rapamycin.The initial step involves the validation of dot formation using established methods to ensure robust SNR representation (Fig. 4).
The formation of SIMBA puncta is readily observable following a brief, 30-min rapamycin induction.These puncta become visually evident under the lens of an epifluorescence microscope, providing a direct and tangible visualization of the underlying cellular processes.Once these distinctive dots materialize, their dynamic progression can be conveniently tracked (Fig. 5).This enables the real-time monitoring of their behavior, shedding light on the intricate dynamics of chromatin within the cellular milieu.
Following imaging validation, the potential for gene manipulation within the SIMBA system becomes evident.Utilizing conventional   transformation and induction techniques, we can assess variations in transcription levels induced by different SIMBA strategies.Whether through HP1a-mediated chromatin rearrangement or the NFAT1 transcription factor's downstream gene network regulation, both single-gene and network-level modifications of cellular expression can be achieved.This underscores the versatility of the SIMBA system, enabling not only visualization but also controlled manipulation of cellular processes at both individual gene and network levels.

Construct cloning
Upon receipt of the required primers and plasmids, the preparation of recombineering templates for construct cloning in Basic Protocol 1 can be accomplished within an estimated time frame of ∼5 days.This process unfolds as follows: Day 1: PCR amplification is executed to generate recombineering cassettes for SIMBA puncta formation.Anneal the sgRNA oligos for Golden Gate Assembly.Day 2: Either Gibson Assembly or Golden Gate Assembly is employed for the assembly of PCR-amplified recombineering cassettes.This involves chemical transformation of the assembly reaction and plating onto LB agar plates to generate individual colonies.
Day 3: The colonies obtained from the previous day are established on plates, and cultures are initiated to facilitate the subsequent isolation of plasmids encoding the recombineering templates.
Day 4: Plasmid isolation is carried out, followed by the verification of these plasmids through restriction enzyme DNA fingerprinting.Subsequently, the verified plasmids are submitted for Sanger DNA sequencing to ensure their accuracy and fidelity.
Day 5: Analysis of the Sanger DNA sequencing results is conducted, and recombineering templates are linearized to prepare them for subsequent usage.

Transient transfection
Upon acquisition of the requisite cell lines and transfection reagents, the process of transient cell transfection in Basic Chen et al.

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Current Protocols Protocol 2 unfolds over an estimated 3-day period: Day 1: Cells are seeded in appropriate culture vessels to achieve optimal confluency for transfection.Transfection complexes are prepared by combining plasmids with transfection reagents and are subsequently added to the cells.
Day 2: The transfection process is allowed to proceed, enabling the introduction of various SIMBA components into the cells.This period also facilitates the establishment of gene expression and the necessary cellular responses.
Day 3: Post-transfection analysis is conducted, encompassing the assessment of gene expression, transfection efficiency, and other relevant parameters.This step confirms the successful delivery of recombinant DNA and sets the stage for subsequent experimentation.

Stable cell line generation
The creation of stable cell lines in Basic Protocol 3 spans an approximate duration of 3 to 4 weeks, depending on cell growth and selection dynamics: Week 1: Cells are cultured and maintained, ensuring optimal growth and confluency.Transduction with lentiviral vectors containing the SIMBA system components is performed, initiating the integration of these elements into the host cell genome.
Week 2: Following transduction, cells undergo a selection process using appropriate markers or antibiotics.This step facilitates the enrichment of cells harboring the integrated SIMBA system components.
Weeks 3 to 4: Selected cells are expanded and characterized for stable integration and expression of the SIMBA system.Verification may involve gene expression analysis, fluorescence imaging, or other relevant assays.Successful stable cell line generation establishes a robust foundation for subsequent experimentation.

Evaluating gene manipulation effects with SIMBA system
Gene manipulation with the SIMBA system followed by RNA-seq or qPCR usually takes 1 week's time: Days 1 to 2: Cells are seeded in culture vessels to achieve optimal confluency for subsequent transfection.Then, they are prepared for transfection.
Day 3: Post-transfection analysis is conducted, including the assessment of gene ex-pression, transfection efficiency, and other relevant parameters.
This step confirms the successful delivery of recombinant DNA and sets the stage for subsequent experimentation.
Day 4: Positively expressing cells are sorted based on the established parameters from the post-transfection analysis.The sorting process ensures the isolation of cells expressing SIMBA system components.
Day 5 onward: RNA sample is extracted from cells and qPCR or RNA-seq analysis is performed.qPCR is performed to quantify gene expression levels, providing insights into the success of SIMBA system expression.If utilizing RNA-seq, library preparation is conducted.RNA-seq analysis is performed to comprehensively explore gene expression changes induced by the SIMBA system.

Figure 1
Figure1Illustration of the essential protocols for implementing the SIMBA (Simultaneous Imaging and Manipulation of Genomic Loci by Biomolecular Assemblies) system, enabling the real-time visualization and manipulation of genomic loci in living cells.Basic Protocol 1 involves the design and construction of genetic constructs.The process begins with gene subcloning into a vector, followed by vector linearization.Recombination techniques are then applied to create the desired genetic construct, which undergoes a further verification.In Basic Protocol 2, plasmids containing genetic constructs are transfected into target mammalian cells.This allows for live-cell imaging, enabling real-time visualization of the system's components, including dCas9-SunTag and FRB-mCherry-HP1α.Basic Protocol 3 involves the production of lentiviral vectors carrying SIMBA system components.These lentiviruses are used to infect target cells, and positively expressing cells are subsequently sorted and isolated, establishing a stable cell population with consistent SIMBA component expression.In Basic Protocol 4, either transiently transfected cells or stable cell lines expressing the SIMBA system components can result in the formation of biomolecular assemblies (BAs) at specific genomic loci upon rapamycin induction.This assembly undertakes simultaneous gene labeling and manipulation.Imaging or RNA-seq analysis may be performed to investigate chromatin dynamics and gene expression changes.

Figure 2
Figure 2 SIMBA puncta induction and imaging.(A) Schematic of the formation of SIMBA BAs upon rapamycin induction.The dCas9-sgRNA complex recognizes and specifically targets the gene loci and forms granular fluorescence signal after induction.(B) SIMBA dots formed upon 1 hr of rapamycin induction targeting MUC4-E4 in HEK 293T cells (imaged with Nikon Ti2-E microscopy).Scale bar, 5 μm.

Figure 3
Figure3Flowchart of the design and timeline for the NFAT1-associated transcriptomic RNA-seq experiment.NFAT1 could be recruited to corresponding binding sites on genomic DNA after ATP induction.Further rapamycin treatment initiates the dimerization of FKBP-FRB, which leads to BA assembly for fluorescent granule formation and HP1α-induced chromatin remodeling.That specific manipulation at NFAT1-regulated downstream genes could be examined by the RNA-seq method.

Table 1
Basic Constructs

Table 2
Primers Used for Construct Cloning Chen et al.

Table 3 sgRNAs
Used in the SIMBA System

Table 4
Plasmids Used for Locus-Specific Imaging NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

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Dissolve rapamycin powder in DMSO to a concentration of 100 μM (1000× stock).Dispense in aliquots and store ≤3 months at −20°C.Avoid repeated freeze-thaw cycles.

Table 5
Troubleshooting Guide for SIMBA System Construction, Delivery, and Induction