CRISPR genome editing is transforming biological research, and experimental controls are the backbone of reliable, reproducible results. In this article, we explore the essential role of CRISPR controls and supporting reagents in optimizing editing efficiency, troubleshooting workflows, and ensuring consistency across experiments. Whether you're editing immortalized cells, iPSCs, or primary cells, CRISPR positive and negative controls are key to unlocking the full potential of your gene editing experiments.
Why CRISPR Controls Matter
CRISPR controls are not just “nice to have”—they are fundamental to every step of a genome editing experiment:
- Optimization: Use controls to fine-tune transfection parameters and editing protocols across different cell types and workflows. Controls help establish optimal conditions before investing in gene-specific reagents.
- Assay Development: Optimize transfection conditions and editing performance before targeting your gene of interest.
- Screening and Validation: Confirm editing efficiency and distinguish true biological effects from technical artifacts.
- Troubleshooting: Identify potential problems with delivery, reagent performance, or cell health.
Without appropriate controls, even the most promising CRISPR screen can yield ambiguous or misleading results.
Figure 1. Having gRNA controls that allow for comparison between treatment cells and the expected phenotypes that controls provide is vital, especially with high-throughput screening where reproducibility is key to applications like target identification and validation.
CRISPR Controls: Driving Biological Reproducibility
Recent research underscores the importance of CRISPR controls in driving reproducibility, assay optimization, and biological discovery.
Positive Controls: Benchmarking Success
Positive controls are pre-validated sgRNAs with high editing efficiency across multiple cell types. They help establish editing baselines, assess efficiency across workflows, and validate experimental conditions.
One of the largest CRISPR screening efforts to date, the Cancer Dependency Map (DepMap) project uses standardized positive and non-targeting control sgRNAs across hundreds of cancer cell lines. These controls were crucial for calibrating knockout efficiency and minimizing false positives, enabling robust identification of cancer vulnerabilities. Many of the 2,000 cell lines in the DepMap collection have been genetically and pharmacologically characterized by systematically knocking out individual genes using CRISPR-Cas9 (Project Achilles) — a project that could only be completed with rigorous quality control, including the use of positive sgRNA controls.
Figure 2. A wide variety of knockout cancer cell lines within the DepMap collection's thousands have been characterized via Project Achilles. (Credit: Broad Institute DepMap)
EditCo's positive controls are ideal for building and optimizing your assay, optimizing your experimental conditions, monitoring the gene editing efficiency across different cell lines or workflows, and troubleshooting. They can be used in any cell type, including immortalized, iPSC, and primary cells.
Negative Controls: Identifying Background Noise
CRISPR Negative or Non-targeting controls are designed not to induce any edits in the genome. These are critical for distinguishing the effects of specific gene edits from nonspecific changes introduced during the editing process.
Non-targeting controls can be extremely important to the success and understanding of results coming from high-throughput screens. In 2021, one such study, published in Nature Communications Biology was the first genome-wide screen to identify modulators of endogenous Tau protein levels. Tau has been the target of extensive research due to its role and aggregation as a pathological hallmark of over 20 neurodegenerative diseases— including most prominently Alzheimer's. These screens rely on negative controls to confirm that phenotypes are due to CRISPR knockouts, not unrelated experimental effects.
Figure 3. To compare genome-wide CRISPR-screened phenotypes modulating the Tau protein in neurodegenerative disease, a non-targeting gRNA control ("control gRNA") and the gRNA targeting the gene encoding Tau, MAPT, were used as negative and positive controls, respectively. (Credit: Sanchez et al. 2021)
EditCo's negative controls are essential in gene-editing experiments to compare the phenotype of cells treated with a gene-targeting guide RNA to those treated with a non-targeting guide RNA. They are commonly included in CRISPR screens to help identify potential false positives.
AAVS1 Controls: The Safe Harbor Standard
AAVS1 controls target a known “safe harbor” site in the human genome. These dual-purpose CRISPR controls act as both positive (for editing validation) and negative (for phenotypic neutrality) references, offering high efficiency without disrupting cellular function.
AAVS1-targeting CRISPR reagents have been widely used in stem cell and T cell engineering, particularly in CAR-T therapy development. These safe harbor controls help demonstrate editing without phenotypic disruption, serving as a baseline in clinical research settings. In a 2022 Nature paper, a team from UC San Francisco did a genome-wide CRISPR screen to identify genes that could be targeted to prevent T cell dysfunction for cancer therapies. From that screen, they created and introduced two knockout CAR T cell lines into a leukemia mouse model: one disrupting a gene discovered in the screen, RASA2, and the other a control with a knockout of the safe harbor locus AAVS1. This gold standard for CRISPR controls enabled their Nature publication and, most importantly, revealed a promising new target that can resist various inhibitory checkpoints that can diminish the efficacy of adoptive T cell therapies.
Figure 4. Compared to the AAVS1 knockout control cell line, RASA2 Knockout T cells were compared for the ability to resist T cell regulatory suppression, making it a potential target for T cell-related therapies. (Credit: Carnevale et al. 2022)
EditCo's AAVS controls are essential for evaluating the baseline cellular response to SpCas9 cutting in the absence of a phenotypic readout. They serve as negative controls, helping to distinguish intended gene-editing effects from unintended functional changes in cells. AAVS1 controls, in particular, are widely used in CRISPR screening experiments, where phenotypic readouts are typically assessed. These can be used in any cell type including immortalized, iPSC, and primary cells.
Lethal Controls: Phenotypes You Can See
Targeting essential genes like PLK1 produces a clear and rapid cell-death phenotype, making these controls ideal for transfection optimization or visual confirmation of editing success.
PLK1 (Polo-like kinase 1) lethal CRISPR controls are important in research because they serve as functional indicators of CRISPR editing efficiency and delivery success. PLK1 and other essential gene-targeting guides are standard controls in genome-wide CRISPR screens. When PLK1 is successfully knocked out via CRISPR, cells typically undergo apoptosis within 48–72 hours. The effect is easy to detect—cell viability drops sharply, and this loss can be tracked visually or with viability assays.
Figure 5. Lethal control PLK1 provides high editing efficiency of the cell cycle regulator gene PLK1 and induces cell death within 3 days. A) Knockout score and B) Cell viability for lethal control, PLK1, versus negative control, NTC3.
EditCo's lethal control is ideal for for optimizing gene editing conditions and validating the transfection efficiency since it provides a very clear phenotype easily assessed via microscopy or a simple cell viability assay.
* Each cell line was genetically modified using EditCo’s proprietary process under optimized editing conditions. Editing efficiency was evaluated 72 hours post-nucleofection. The targeted genomic region was amplified via PCR, sequenced using Sanger sequencing, and analyzed with the ICE analysis tool to determine knockout (KO) levels. To assess the impact of CRISPR editing on cell growth, total cell numbers were measured using a fluorescence-based cell counting assay 72 hours post-nucleofection. Hoechst staining was used to label all cells, while dead cells were identified via propidium iodide staining. The number of live cells was determined by subtracting dead cells from the total count.
** Representative data, found on the CRISPR controls product page, show high knockout efficiency and maintained viability across controls.
Controls Enable Confidence — and EditCo Makes It Easy
In genome editing, precision without validation is just guesswork. That’s why CRISPR controls—positive, negative, lethal, and safe harbor—are not optional extras, but essential tools for building reproducible, interpretable experiments. Whether you’re troubleshooting delivery, benchmarking editing performance, or validating phenotypes in complex cell models, controls turn uncertainty into confidence.
At EditCo, we’ve designed our suite of CRISPR controls and supporting reagents to meet researchers where they are—from early assay development to high-throughput screening. In addition to traditional single-guide formats, our unique XDel knockout guide design maximizes protein depletion. With validated sgRNAs, optimized transfection kits, and high-performance SpCas9 protein, we provide a reliable foundation for every experiment. Our controls aren’t just compatible with our Gene Knockout Kits—they complete them, enabling seamless workflows that reduce variability and accelerate discovery.
Confidence in gene editing starts with the right controls. EditCo delivers them—so you can focus on the science, not the troubleshooting.
