Automated (iPSC) gene editing using CRISPR-Cas9, Advanced Cell and Immune Therapies

A fully automated induced pluripotent stem cell (iPSC) gene editing experiment using CRISPR-Cas9 involves the use of robotic systems and computerized protocols to carry out all steps of the gene editing process without manual intervention.

• iPSC Culture and Maintenance
• Design of CRISPR Components
• Synthesis and Preparation of CRISPR Components
• Transfection
• Incubation and Gene Editing
• Selection and Cloning
• Verification and Analysis
• Reporting and Data Analysis

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iPSC Culture and Maintenance

Automated systems handle the cultivation of iPSCs, maintaining the necessary conditions for cell growth, such as temperature, CO2 levels, and humidity, as well as media changes.

Induced pluripotent stem cells (iPSCs) are a type of stem cell that can be generated directly from adult cells. The iPSC technology has transformed stem cell research with potential applications in drug development, regenerative medicine, and disease modeling. Here's a detailed protocol for the setup and maintenance of iPSC cultures, including the preparation, seeding, and ongoing care of the cells.

Reagent and Material Preparation

Essential Materials

iPSCs (already reprogrammed and confirmed)

Tissue culture plates (6-well, 12-well, or as required)

Incubator set at 37°C with 5% CO2

Biological safety cabinet (Class II)

Microscope (inverted with phase contrast)

Growth Media

Essential 8 Medium or another iPSC-specific medium

Penicillin-Streptomycin (100 units/mL)

Substrate for Plating

Matrigel or a similar extracellular matrix gel, diluted as per manufacturer's instructions in cold DMEM/F-12

Preparing the Culture Surface

Coating

Thaw the Matrigel on ice.

Dilute the Matrigel in cold DMEM/F-12.

Add enough Matrigel mixture to cover the surface of each well (typically 1 mL per well of a 6-well plate).

Incubate the plates at room temperature for 1 hour or at 4°C overnight.

Washing

After incubation, aspirate the Matrigel.

Wash once with DMEM/F-12.

Thawing and Plating iPSCs

Thawing Cells

Quickly thaw the iPSC vial in a 37°C water bath.

Transfer the cells to a tube containing pre-warmed Essential 8 Medium.

Centrifuge at 200 x g for 5 minutes.

Aspirate the supernatant and resuspend the cell pellet gently in Essential 8 Medium.

Plating Cells

Count the cells using a hemocytometer.

Seed the cells at a density appropriate for the desired confluency (e.g., 10,000 cells/cm²).

Add iPSC medium gently to the wells to avoid detaching the cells.

Change the medium daily.

Daily Maintenance

Medium Change

Aspirate the old medium from each well.

Add fresh, pre-warmed iPSC medium.

Avoid direct streaming of the medium onto the cells.

Passaging Cells

Monitoring Confluency

Monitor the cells daily using an inverted microscope.

Passage the cells before they reach 80% confluency to prevent differentiation.

Passaging Method

Aspirate the medium and wash the cells with DPBS.

Add an enzyme (e.g., EDTA) to detach the cells.

Incubate at 37°C for 5-7 minutes.

Neutralize the enzyme with iPSC medium.

Collect and centrifuge the cells.

Resuspend the cell pellet and replate at a suitable dilution (typically 1:6 to 1:10).

Cryopreservation

Freezing Cells

Prepare a cryopreservation medium (90% FBS + 10% DMSO).

Resuspend the cell pellet in cryopreservation medium.

Dispense into cryovials.

Freeze the vials using a controlled rate freezer or a Mr. Frosty Freezing Container at -80°C overnight before transferring to liquid nitrogen for long-term storage.

Quality Control

Routine Testing

Test for mycoplasma contamination regularly.

Perform karyotyping and pluripotency marker analysis periodically to ensure the cells maintain their undifferentiated state.

This protocol provides a foundation for establishing and maintaining iPSC cultures. Depending on specific research needs, modifications might be necessary. For example, some labs might use feeder layers or different media formulations.

Design of CRISPR Components

Software is used to design guide RNAs (gRNAs) specific to the gene target in the iPSC genome. These gRNAs direct the CRISPR-Cas9 system to the exact location in the DNA where edits are intended.

Designing CRISPR components for genome editing involves several critical steps, from selecting the target site to constructing and delivering the CRISPR/Cas9 components. The CRISPR/Cas9 system is one of the most widely used tools in genetic engineering due to its simplicity and high efficiency. Below is a detailed protocol for designing CRISPR components.

Target Selection

Choose Target Gene

Identify the gene you want to edit. Consider the purpose of the modification (knockout, knock-in, or regulation).

Identify Target Sequence

Select a 20 bp sequence within the gene that precedes a PAM sequence (NGG for Cas9 from Streptococcus pyogenes).

The target sequence should be unique to the gene of interest to avoid off-target effects.

Guide RNA (gRNA) Design

gRNA Synthesis

Use an online tool (e.g., CRISPR Design Tool, Benchling, or CRISPOR) to design gRNA. These tools help identify target sequences with high specificity and minimal off-target sites.

Synthesize the gRNA or order it from a commercial provider.

gRNA Testing

Test the gRNA in a cell line to evaluate its efficacy and specificity.

Cas9 Expression Vector

Vector Selection

Choose a vector for expressing the Cas9 protein. Options include plasmids, adenoviruses, or lentiviruses, depending on the delivery method and cell type.

Ensure the vector has a promoter that is active in your target cell type (e.g., CMV for many mammalian cells).

Construct Preparation

Clone the Cas9 gene into the expression vector.

Include a selection marker if stable expression is required.

Delivery of CRISPR Components

Transfection (for in vitro studies)

Select an appropriate transfection method based on cell type (lipofection, electroporation, etc.).

Co-transfect cells with the Cas9 vector and gRNA.

Viral Transduction (for hard-to-transfect cells)

Produce viral particles containing the CRISPR components.

Infect the target cells with the virus.

Microinjection (for in vivo studies or embryos)

Inject the CRISPR components directly into the target tissue or embryo.

Selection and Screening

Selection

If a selection marker is included, use antibiotics or other selection agents to enrich for cells that have taken up the CRISPR components.

Screening

Use PCR, sequencing, or a reporter assay to confirm the introduction of the desired genetic changes.

Assess off-target effects using genome-wide assays or targeted deep sequencing.

Confirmation of Gene Editing

Validate Editing

Perform Sanger sequencing or next-generation sequencing to confirm the edit at the DNA level.

Use protein assays (Western blot, ELISA) to confirm changes at the protein level if the gene edit affects protein expression or function.

Off-Target Analysis

Off-Target Assessment

Use computational tools to predict potential off-target sites.

Experimentally validate these sites using targeted sequencing or genome-wide off-target detection methods.

This comprehensive approach ensures that the CRISPR components are designed and delivered effectively, with a high chance of success in editing the target gene. To help further, could you tell me about your experience with molecular biology techniques? This will allow me to tailor the information to your specific needs, ensuring clarity and understanding.

Synthesis and Preparation of CRISPR Components

Automated systems synthesize and prepare the CRISPR-Cas9 components, including the gRNAs and Cas9 protein or mRNA, ensuring they are delivered in the correct format for cellular uptake.

Synthesizing and preparing CRISPR components is a critical step in CRISPR-Cas9 mediated genome editing. This involves preparing the guide RNA (gRNA) and Cas9 protein, which form the core components of the CRISPR system. Here is a detailed protocol for synthesizing and preparing these components for a genome editing experiment.

Guide RNA (gRNA) Synthesis

Designing the gRNA

Target Sequence Identification: Choose a 20 bp DNA sequence adjacent to a PAM (Protospacer Adjacent Motif) sequence ('NGG' for Streptococcus pyogenes Cas9). This should be specific to the target gene to avoid off-target effects.

Online Tools: Use online design tools such as CRISPOR, Benchling, or the Broad Institute’s CRISPR design tool to identify the best target sequence and predict off-target risks.

Synthesis of gRNA

In Vitro Transcription (IVT): Order a DNA template containing the T7 promoter sequence followed by your target-specific gRNA sequence. Use this template for in vitro transcription:

Prepare a transcription reaction with T7 RNA polymerase, NTPs, and the DNA template.

Incubate the reaction at 37°C for 2-4 hours.

Treat the reaction with DNase I to remove the DNA template.

Purify the RNA using an RNA purification kit or phenol-chloroform extraction and ethanol precipitation.

Assess the quality and quantity of the RNA using gel electrophoresis and a spectrophotometer.

Cas9 Protein Preparation

Expression of Cas9 Protein

Expression Vector: Use or construct an expression vector containing the Cas9 gene under a strong promoter. Include an epitope tag or fusion (e.g., His-tag) for purification.

Protein Expression:

Transform the expression vector into a suitable bacterial strain (e.g., E. coli BL21).

Induce expression with IPTG when the culture reaches mid-log phase.

Harvest the cells after 3-5 hours of induction.

Purification of Cas9 Protein

Cell Lysis: Resuspend the pellet in lysis buffer containing protease inhibitors and lyse the cells via sonication or chemical lysis.

Purification:

Clear the lysate by centrifugation.

Purify the Cas9 protein using affinity chromatography (e.g., nickel NTA agarose for His-tagged proteins).

Further purify using size-exclusion chromatography if high purity is needed.

Protein Quantification and Storage:

Quantify the protein using a Bradford assay or similar method.

Aliquot and store the protein at -80°C to avoid freeze-thaw cycles that could denature the protein.

Forming the RNP Complex

Assembly of Ribonucleoprotein (RNP) Complex

Complex Formation: Mix purified Cas9 protein with synthesized gRNA at a molar ratio of approximately 1:2.5 (Cas9:gRNA).

Incubation: Incubate the mixture at room temperature for 10-20 minutes to allow the formation of the Cas9-gRNA complex.

Delivery into Target Cells

Choosing a Delivery Method

Transfection: Use lipofection, electroporation, or another method suitable for your cell type.

Microinjection: Directly inject the RNP into cells or embryos, commonly used in animal model studies.

Verification of Editing

Detection of Genome Editing

Surveyor Assay or T7E1 Assay: Use mismatch-sensitive enzymes to detect indels introduced by NHEJ (non-homologous end joining) at the target site.

Sequencing: Use Sanger sequencing or next-generation sequencing to confirm specific edits.

This protocol provides a comprehensive guide to synthesizing and preparing CRISPR components for effective genome editing. Understanding each step's details is crucial to ensuring high efficiency and specificity in your CRISPR experiments. Let me know if you need clarification on any step or have specific conditions or applications in mind!

Transfection

iPSCs are transfected with the CRISPR components using methods such as electroporation, which can also be automated. This step introduces the CRISPR-Cas9 system into the cells to perform gene editing.

Transfecting induced pluripotent stem cells (iPSCs) can be challenging due to their sensitivity to handling and susceptibility to differentiation. However, efficient transfection is crucial for various applications like gene editing, overexpression studies, or RNA interference. Here’s a detailed protocol for the transfection of iPSCs, focusing on a non-viral method, which minimizes the risk of genomic integration and is commonly used due to its relative safety and efficiency.

Preparing iPSCs

Culture Conditions

Maintain iPSCs on a Matrigel-coated plate with iPSC-specific medium (e.g., mTeSR1 or Essential 8).

Passage iPSCs regularly to avoid over-confluence and differentiation. Ideally, iPSCs should be 70-80% confluent at the time of transfection.

Transfection Reagent Preparation

Selection of Transfection Reagent

Choose a reagent: Opt for transfection reagents known for gentleness and effectiveness in iPSCs, like Lipofectamine Stem, Lipofectamine 3000, or equivalents.

Preparation of Reagent

Prepare the reagent according to manufacturer's instructions. Typically, this involves diluting the lipid-based reagent in a serum-free medium such as Opti-MEM.

Plasmid DNA Preparation

Plasmid Quality

Use high-quality, endotoxin-free plasmid DNA. Prepare or purchase plasmid DNA with a high-purity plasmid prep kit.

Quantify the DNA using a spectrophotometer to ensure accurate dosing.

Transfection Procedure

Formation of DNA-Reagent Complex

Dilute the DNA in serum-free medium. For a typical 6-well plate format, use 1-3 µg of DNA per well depending on the transfection reagent and the sensitivity of iPSCs.

Mix the diluted DNA with the prepared transfection reagent. Vortex lightly or pipette to mix.

Incubate the mixture for 15-20 minutes at room temperature to allow complex formation.

Adding Complex to iPSCs

Aspirate the old medium from the iPSC cultures and replace with fresh iPSC medium without antibiotics.

Add the DNA-reagent complex dropwise to the iPSCs, gently swirling the plate to distribute the mixture evenly.

Return the cells to the incubator.

Post-Transfection Care

Medium Change

Change the medium 6-24 hours post-transfection to a fresh iPSC-specific medium to remove any residual transfection reagent and mitigate cytotoxicity.

Monitoring

Monitor cell morphology daily under a microscope. Look for signs of differentiation or cell death, which might indicate excessive toxicity from the transfection.

Analysis of Transfection Efficiency

Evaluation of Transfection

48-72 hours post-transfection, evaluate the efficiency using a reporter gene (e.g., GFP) or perform qPCR or Western blot for the target gene.

Troubleshooting

Adjust DNA and reagent amounts if efficiency is low or cytotoxicity is observed. Transfection conditions may require optimization depending on the specific iPSC line and the construct used.

Follow-Up Experiments

Further Analysis

Conduct downstream applications such as gene expression analysis, immunostaining, or functional assays to assess the effects of gene modulation.

This protocol provides a framework for the transfection of iPSCs using lipid-based reagents. It’s crucial to tailor the protocol based on the specific iPSC line and the purpose of transfection.

Incubation and Gene Editing

After transfection, cells are incubated under controlled conditions. The CRISPR-Cas9 system makes the intended cuts or edits to the genome.

Working with induced pluripotent stem cells (iPSCs) for gene editing involves a meticulous protocol that spans from the culturing of these cells to the actual editing process using tools like CRISPR-Cas9. Below, I'll provide a detailed overview of the protocol, using some clear and straightforward analogies to help grasp each step.

Culturing iPSCs

Equipment and Materials

Laminar flow hood

CO2 incubator

Essential 8 Medium (or similar)

6-well plates

Matrigel or other suitable extracellular matrix

Protocol

Thawing iPSCs:

iPSCs are usually stored in liquid nitrogen. Think of them like seeds frozen in time, waiting to be thawed to spring to life.

Quickly thaw the vial in a 37°C water bath to minimize ice crystal formation (which can be thought of as tiny knives potentially damaging the cells).

Plating:

Prepare plates coated with Matrigel, which acts like a comfortable mattress for iPSCs to attach and spread out.

Seed the cells in Essential 8 Medium, ensuring the cells have all the nutrients they need.

Maintenance:

Change the medium daily. Consider it like changing the water in a fish tank to keep the environment fresh and conducive for growth.

Passage the cells when they reach around 70-80% confluence. This involves detaching and re-plating them at a lower density to prevent over-crowding.

Gene Editing using CRISPR-Cas9

Equipment and Materials

CRISPR-Cas9 components (guide RNA, Cas9 protein)

Transfection reagent

Selection antibiotics (if a selection marker is used)

PCR reagents for genotyping

Protocol

Designing Guide RNA (gRNA):

The gRNA is like a GPS navigator that guides the Cas9 enzyme to the exact location in the genome where edits are needed.

Transfection:

Introduce the CRISPR components into iPSCs. This can be likened to uploading new software into a computer. Various methods can be used, like electroporation (using an electrical field to make the cell membrane permeable) or lipofection (using lipid nanoparticles to encase and deliver the CRISPR components).

Selection and Cloning:

After editing, not all cells will have the desired modification. Antibiotics or fluorescence-activated cell sorting (FACS) can be used to select the successfully edited cells.

Clone the cells to isolate single colonies, ensuring each colony comes from one cell, which is crucial for uniformity in experiments.

Verification:

Perform PCR and sequencing to confirm the edit. Think of this as doing quality control to ensure the product meets the specifications.

Characterization and Differentiation:

Once edited, characterize the iPSCs to ensure they maintain stem cell properties and can differentiate into desired cell types.

Post-Editing Culture and Analysis

Further Analysis: Depending on the gene edited and the purpose, further analysis might be necessary. For instance, functional assays to assess how the edit affects the cell's behavior or properties.

Scale-Up: If the edited cells meet all criteria, they can be scaled up for further studies or potential therapeutic use.

This protocol outlines the high-level steps involved in culturing and gene editing of iPSCs. Each step is critical and requires precise control to ensure success.

Selection and Cloning

Post-editing, cells are screened and selected to identify those that have successfully incorporated the edits. Cloning of edited cells can also be automated to isolate pure populations of edited iPSCs.

Selection and cloning of induced pluripotent stem cells (iPSCs) post-gene editing is a crucial step to ensure you have successfully edited cells with the desired genetic alteration. Here's a detailed protocol that covers this process. To make the process easier to understand, imagine you are sifting through a collection of mystery novels, selecting only those that feature your favorite detective, and then ensuring every copy you have is exactly the same.

Selection and Cloning Protocol for iPSCs

Equipment and Materials

Cultured iPSCs post-CRISPR editing

Selection antibiotics (if using a resistance gene)

Fluorescence-activated cell sorting (FACS) machine (if using a reporter gene)

96-well plates

Essential 8 Medium or equivalent

Microscope

Colony-picking equipment

Steps

Application of Selection Pressure:

With Antibiotics:

If your CRISPR system includes a resistance gene, you'll add a specific antibiotic to the culture medium. This is like setting a trap that only allows iPSCs with the resistance gene (successful edits) to survive.

With Fluorescence-Activated Cell Sorting (FACS):

If you've used a fluorescent reporter gene, you'll employ FACS to sort the cells. Cells that fluoresce have successfully integrated the reporter gene, similar to using a blacklight to find hidden marks on a treasure map.

Isolation and Cloning:

After selection, isolate individual cells to form colonies in a 96-well plate. This is akin to picking out seeds to plant individually in pots, ensuring each pot gets only one seed.

Use a microscope to ensure that each well receives only one cell. Precision here is key to ensure clonal purity.

Expansion of Clones:

Monitor the wells regularly under a microscope for colony growth. Once colonies are visible, they need careful maintenance with daily medium changes.

When colonies reach a substantial size but before overgrowing, they should be passaged. This involves transferring them to a larger well or plate.

Screening of Clones:

Once colonies are established, you'll pick several for genetic screening. This involves using PCR and sequencing to confirm the presence and correctness of the gene edit.

Think of this like verifying that each book in your collection features the correct detective. It's ensuring that the changes you made to the genome are precisely what you intended.

Secondary Confirmation:

For clones that pass the genetic screening, perform a secondary confirmation, often involving further PCR, sequencing, or functional assays.

This ensures the gene edit not only is present but also functions as expected in the cellular environment.

Banking and Further Expansion:

Successful clones can be expanded further and stored in liquid nitrogen for future use. This is akin to making multiple copies of your favorite detective novel to ensure you always have it on hand.

Characterization:

Finally, characterize the selected clones for typical iPSC properties, such as pluripotency markers and differentiation potential. This confirms that despite the genetic modification, the cells still retain their essential stem cell characteristics.

Verification and Analysis

Automated systems then perform assays such as sequencing or PCR to verify that the correct gene edits have been made, assessing the efficiency and specificity of the CRISPR editing.

Induced pluripotent stem cells (iPSCs) are derived from non-pluripotent cells, such as adult somatic cells, by introducing genes that reprogram the cells back into a pluripotent state. This process is highly valuable for research, therapeutic, and drug development purposes. Once iPSCs are generated and isolated, it’s crucial to verify their identity and quality before they can be used in experiments or therapies. Here’s a detailed protocol for the verification and analysis after selection and cloning of iPSCs:

Initial Screening and Isolation

Selection and Cloning: After reprogramming, select individual colonies that appear morphologically similar to embryonic stem cells (round, tight colonies with high nucleus-to-cytoplasm ratio).

Expansion: Expand these clones to establish individual cell lines.

Morphological Assessment

Visual Inspection: Regularly inspect iPSC colonies under a microscope. Ideal iPSC colonies should be compact, with clearly defined edges and a high nucleus-to-cytoplasm ratio.

Live Imaging: Utilize live-cell imaging to monitor colony morphology over time without disrupting the culture.

Genetic Analysis

Karyotyping: Perform karyotyping to check for chromosomal stability and integrity. This can be done using G-banding.

PCR/RT-PCR: Screen for the presence and expression of reprogramming factors and pluripotency markers such as OCT4, SOX2, KLF4, c-MYC (also known as OSKM factors).

Pluripotency Tests

Immunofluorescence/Immunocytochemistry: Stain for pluripotency markers (e.g., SSEA4, TRA-1-60, TRA-1-81, NANOG, SOX2).

Flow Cytometry: Quantitatively assess the expression of surface markers on a larger number of cells to confirm uniform expression of pluripotency markers.

In Vitro Differentiation

Embryoid Body Formation: Induce differentiation in vitro by forming embryoid bodies (EBs) in suspension culture. This assesses the ability of iPSCs to differentiate into the three germ layers: ectoderm, mesoderm, and endoderm.

Directed Differentiation: Guide iPSCs to differentiate into specific cell types to verify functional pluripotency.

In Vivo Differentiation

Teratoma Formation Assay: Inject iPSCs into immunodeficient mice to observe the formation of teratomas that contain tissues from all three germ layers, confirming pluripotency.

Genomic Integrity

Whole Genome Sequencing: Optionally perform whole-genome sequencing to detect any genetic abnormalities or mutations acquired during the reprogramming process.

Copy Number Variation (CNV) Analysis: Check for genomic integrity and the presence of duplications or deletions that may have occurred during cell culture.

Epigenetic Status

DNA Methylation Analysis: Analyze the methylation status of pluripotency genes to ensure that iPSCs have an epigenetic state similar to embryonic stem cells.

Mycoplasma Testing

Regular Screening: Regularly test cultures for mycoplasma contamination, which can affect cell growth and differentiation.

Record Keeping and Documentation

Documentation: Maintain detailed records of cell line derivation, characterization, passage number, and all test results for reference and reproducibility.

This protocol outlines a comprehensive approach to ensure that iPSCs are pluripotent, genetically stable, and free from contamination, thereby suitable for further experimental or clinical use.

Reporting and Data Analysis

Data from the entire process is compiled and analyzed using bioinformatics tools. The results are formatted into reports, providing insights into the experiment's success and any potential off-target effects or anomalies.

Once you have completed the selection, cloning, and verification of induced pluripotent stem cells (iPSCs), the next crucial steps involve thorough reporting and data analysis. This phase is essential to ensure that the data collected from various tests are interpreted correctly and documented comprehensively. Here’s a detailed protocol for reporting and data analysis for iPSCs:

Data Compilation

Collect Raw Data: Gather all raw data from genetic, phenotypic, and functional tests, including images, flow cytometry data files, PCR amplifications, karyotype analyses, etc.

Standardize Formats: Convert all data into standard formats that can be easily accessed and analyzed. This may involve digitizing analog records, standardizing file formats, or entering data into a database.

Data Cleaning

Validation Checks: Run checks for data completeness, accuracy, and consistency. Remove or correct any outliers or errors after verifying with original records.

Normalization: Standardize data to remove biases due to differing scales or measurements for comparability.

Statistical Analysis

Descriptive Statistics: Calculate means, medians, standard deviations, and ranges for quantitative data to get a sense of data distribution and central tendencies.

Inferential Statistics: Perform statistical tests such as t-tests, ANOVA, or regression analysis to determine if differences between groups are statistically significant.

Multivariate Analysis: Apply techniques like principal component analysis (PCA) or cluster analysis to understand relationships within data and identify patterns.

Data Visualization

Graphical Representation: Use charts, graphs, and plots to visually summarize and present data. Common types include bar charts, histograms, scatter plots, and heat maps.

Interactive Dashboards: Develop interactive dashboards for dynamic data exploration, which can be particularly useful in presentations or for collaborative projects.

Quality Control

Replication Analysis: Analyze repeatability and reproducibility of experiments to validate findings.

Sensitivity Analysis: Determine how sensitive results are to changes in the method of data collection or analysis parameters.

Interpretation

Contextual Analysis: Interpret data in the context of existing literature and theoretical frameworks. Discuss any deviations from expected results and potential reasons.

Biological Relevance: Focus on the biological implications of the findings, particularly how they affect the understanding or use of iPSCs.

Reporting

Methodology Description: Document all methods and protocols used in detail to allow for reproducibility. Include specifics of data collection, analysis techniques, and software used.

Results Presentation: Present results clearly and logically, including both significant and non-significant findings. Discuss the implications of these results for the field.

Discussion: Compare results with previous studies, discuss possible implications, and suggest future research directions.

Conclusion: Summarize the main findings and their importance to the broader field.

Peer Review and Publication

Internal Review: Have the report reviewed internally by peers to catch any errors or oversights.

External Publication: Prepare and submit a manuscript to a peer-reviewed journal to share findings with the wider scientific community. Ensure compliance with journal guidelines for format and style.

Data Sharing

Data Repositories: Deposit data in public repositories if appropriate, ensuring that data is anonymized and complies with all ethical guidelines.

Supplementary Materials: Include raw data, code, and other materials as supplementary files with publications to enhance transparency and reproducibility.

Regulatory Compliance

Ethical Approval: Ensure all experiments and data collection are in line with ethical standards and have necessary approvals.

Data Protection: Adhere to data protection laws and guidelines, especially when handling sensitive or personal data.

This comprehensive approach ensures that your data is not only rigorously analyzed and correctly interpreted but also well-documented and accessible for future reference, reproduction, and regulatory review.

Fully automated systems for gene editing

Fully automated systems for gene editing in iPSCs are particularly valuable in high-throughput experiments where large numbers of edits are tested, or when precision and reproducibility are critical. These systems reduce human error, increase throughput, and can significantly accelerate research and development in genetic research and therapeutic development.

If you are interested in having tailored protocols designed for your specific needs, please feel free to get in touch with me anytime. Once I know your project specifics, I would be able to consult you in a more targeted way as well as recommend and suggest manufacturers for media, equipment and reagents which I know personally.