scott.pritchett

Gene modulation tools are invaluable to life science researchers, allowing them to modify gene function by altering gene expression and studying the effects to gain biological insights. Small interfering RNA (siRNA) halts the expression of specific genes by degrading messenger RNA (mRNA) after transcription, producing a transient effect called gene silencing. In contrast, CRISPR permanently modifies DNA by deleting or inserting segments, rewriting the genetic code.

Sanger sequencing and next-generation sequencing (NGS) are two common methods used to assess gene modifications after gene silencing or gene editing is performed. For siRNA, mRNA experiments are often used to verify gene silencing, but mRNA and protein levels may not always correlate. To ensure accurate biological conclusions, it is important to confirm gene modulation success at the protein level. Immunoblotting is commonly used, but this method can be limited by the availability and quality of antibodies. Mass-based proteomics offers a solution by confirming protein-level changes induced by genome engineering and measuring the proteome-wide alterations in a single experiment.

What is LC-MS/MS proteomics?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a bioanalytical tool that can be used to identify and quantify an organism’s entire proteome [2]. A typical mass-based proteomics workflow involves several steps, including sample preparation (protein extraction, protein digestion, and purification of peptides), sample analysis via LC-MS/MS, data-based identification and quantification, and bioinformatics (data interpretation). This workflow provides a detailed understanding of the molecular consequences of gene modulation, crucial for advancing genetic research and developing new therapeutic strategies. LC-MS/MS proteomics is not limited to quantifying target proteins- it also monitors the changes across the entire proteome. Additionally, it does not require antibodies, making it broadly applicable to any target protein in many organisms.

Emerging technologies, such as label-based and label-free quantitative proteomics, enable the identification of changes in protein expression levels under different conditions. By examining the entire proteome, researchers can identify changes in protein levels, modifications, and interactions, providing a comprehensive view of gene modulation’s biological impact. Combining quantitative proteomics with other ‘omics’ data (e.g., transcriptomics) and mapping these to ontological terms offers a holistic understanding of gene expression correlations and interactions. Some common use cases include:

• Identifying and quantifying biomarkers for various diseases, aiding early diagnosis, prognosis, and treatment monitoring [3]. Recently, researchers at Helmholtz Munich in Germany combined in-depth proteomic profiling and CRISPR/Cas9 screens, identifying ADAM10 as a potential novel therapeutic target to treat acute leukemias [4].
• Detecting and quantifying post-translational modifications (PTMs) such as phosphorylation, glycosylation, and ubiquitination, crucial for understanding protein function and regulation [5].
• Increasing understanding of drug mechanisms of action, identifying potential drug targets, and studying drug metabolism and pharmacokinetics [6].

GenScript solutions for gene modulation

GenScript is a leader in nucleic acid production. We offer a range of services for genome engineering and gene regulation, including CRISPR guide RNA (gRNA), ssDNA, closed-end dsDNA, and miniaturized circular dsDNA HDR templates, and siRNA. All of these services are available from RUO and GMP levels to satisfy the needs of customers at different stages of therapy development. Our comprehensive QC testing services, including HPLC, MS, and advanced technologies like LC-MS/MS sequencing, ensure our products meet the quality requirements for a range of downstream applications.

Our in-house bioinformatics experts have developed and optimized gRNA and siRNA design algorithms, enabling customers to design their own constructs online with ease. To confirm and validate gene modifications at the protein level, we can offer a comprehensive LC-MS/MS proteomics workflow as an optional customized service, covering everything from experimental design and sample preparation to data analysis and interpretation. When customers design their constructs with us, we can provide siRNA or gRNA screening, along with protein expression data, which includes a detailed list of identified and quantified proteins, proteome profiles under different conditions (wild type and siRNA or gRNA treated), and ontology information of significantly expressed proteins. For customers involved in drug development or biomarker discovery, we also offer this service independently to support their research goals. With advanced instrumentation and an expert data analysis pipeline, our team is ready to assist in planning and executing these experiments.

Figure 3 shows the proteome profiling changes in cells treated with in-house designed siRNA targeting the epidermal growth factor receptor (EGFR). Compared to non-treated samples, 73 proteins were significantly differentially expressed, with 37 proteins upregulated and 36 proteins downregulated, including the EGFR related protein. We also studied the gene silencing efficiency of siRNA when delivered by various systems (electroporation, lipid nanoparticles (LNPs), antibody-siRNA conjugates (ARC), and peptide nanoparticles (PNP)) and found that LC-MS/MS proteomics revealed similar protein profiling changes with both electroporation and LNP delivery systems.

Future Outlook

Gene modulation tools like CRISPR/Cas9 and siRNA continue to revolutionize biomedical research with highly selective gene modulation capabilities. For downstream product validation, confirmation at the protein level is critical. LC-MS/MS proteomics not only accomplishes protein-level verification with ease, but is also high throughput, eliminates the need for antibodies, and provides a wealth of data. This makes it ideally suited for in-depth studies of any organism following gene editing or gene silencing experiments.

Currently, most verification and validation experiments are conducted in cell lines to assess both product metabolism and gene modulation efficiency. At GenScript, we have an internationally-accredited animal facility and have established an experiment workflow to confirm and validate gene editing efficiency in vivo. We use label-free quantitative proteomics to identify and quantify proteins in biological samples. By comparing protein levels in different states (e.g., wild type vs. treated), we can identify changes linked to specific biological processes. We are also developing a label-based quantitative proteomics workflow to enhance the accuracy and reproducibility of quantification results.

For more information on our LC-MS/MS proteomics services, please reach out to crispr@genscript.com.
Learn more about our gene editing and siRNA solutions here:
Gene Editing
siRNA

References

• 1. Ciencias Españolas KoS, CC BY-SA 3.0via Wikimedia Commons.
• 2. Steven R. Shuken S.R.; An Introduction to Mass Spectrometry-Based Proteomics. J. Proteome Res. 2023, 22, 7, 2151–2171.
• 3. Pejchinovski, M., Magalhães, P.; Metzger, J.; Editorial: Mass spectrometry-based proteomics in drug discovery and development. Front. Med. 11:1448152. doi: 10.3389/fmed.2024.1448152.
• 4. Bahrami, E., Schmid, J.P., Jurinovic, V. et al. Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo. Mol Cancer 22, 107 (2023).
• 5. Neagu, A.-N.; Jayathirtha, M.; Baxter, E.; Donnelly, M.; Petre, B.A.; Darie, C.C.; Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research. Molecules 2022, 27, 2411.
• 6. Cutillas, P.R.; Timms, J.F.; LC-MS/MS in Proteomics: Methods and Applications.