Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has gained momentum due to its potential use in gene editing and genetic engineering. A growing body of research points to its utility in disease research, target discovery and gene therapies.

The successful application of CRISPR in clinical settings depends on its safety profile and consistent effectiveness. Therefore, high-throughput CRISPR-Cas9 arrays must undergo a thorough evaluation with experimental CRISPR controls implemented at transfection and editing. Here, we explore the different types of CRISPR controls and their contribution to validating cleavage efficiency and target specificity.

Understanding CRISPR Technology

What is CRISPR?

CRISPR-based gene editing entails selectively cutting a DNA sequence to disrupt its function or introduce an intentional repair. This mechanism was originally discovered in bacteria that cut viral genome sequences to disrupt invasive viruses' function.¹ In 2012, a research group led by Jennifer Doudna and Emmanuelle Charpentier succeeded in replicating this mechanism, giving rise to the genetic scissor, which can cut specific sequences from a gene.²

Components of the CRISPR/Cas system

The experiments conducted by Doudna and Charpentier shed light on the essential components of CRISPR. Firstly, a group of endonucleases called CRISPR-associated proteins (Cas) are responsible for the cleave process. However, the Cas enzyme is guided by a short sequence of RNA called CRISPR RNA (crRNA) complementary to the target DNA sequence. Furthermore, a longer sequence called transactivating crRNA (tracrRNA) stabilizes the complex.

In designing the CRISPR-Cas9 complex, Doudna and Charpentier fused crRNA and tracrRNA into a single guide RNA (sgRNA) that can facilitate targeted cleaving. A sgRNA recognizes its target based on the complementary sequence and the presence of a protospacer-adjacent motif (PAM) downstream of that sequence.

The outcome of gene editing also relies on the transfection or transduction of the CRISPR system into the host. Direct methods involve the delivery of CRISPR-Cas9 as a ribonucleoprotein complex (RNP) complex via electroporation, microinjection or nanoparticles. Viral vectors can transduce the plasmid vectors to express sgRNA and Cas9 within the cell.

Experimental Controls in CRISPR Research

Importance of experimental controls

CRISPR-based gene editing is a multilayered process that needs to be monitored in several steps. Furthermore, CRISPR knockout experiments must be well-documented to make the technology compliant with regulatory requirements. Researchers must confirm the system’s functionality and reproducibility while ruling out false positives to ensure that the sgRNA induces phenotypic effects. Therefore, CRISPR controls are essential to ensure data validity, minimize variability across experiments, and improve confidence in the reported results.

Positive CRISPR Control

A positive control is essential to ensure that the CRISPR machinery works seamlessly. To that end, researchers can use a control gRNA sequence targeting a well-characterized gene sequence and induce a strong knockout phenotype across different cell types.

Several genes can serve as target regions for positive control, although the ideal positive control gRNA depends on the experiment. In a 2021 study published in Cell, researchers used the well-known tumor suppressor gene PTPN12 as a positive control to test the functionality of their CRISPR-based in vitro transformation assays for assessing the safety of gene therapies.³ In another study, which focused on validating a CRISPR assay for COVID diagnostics, the orf1ab viral gene was identified as a positive control and used to optimize the efficiency of the assay.⁴

Negative CRISPR Control

A negative control is a CRISPR-Cas variant that can identify off-target editing and eliminate false positives by ensuring that the observed phenotype stems from the gRNA-mediated genetic modification, not the CRISPR system itself. The types of negative CRISPR controls are as follows:

1. Scramble Control involves using a gRNA that does not correspond to a complementary sequence in the host genome, with the idea that the Cas nuclease must not make double-stranded breaks (DSB) without a guide.
2. Cas-only Control refers to the delivery of the Cas nuclease without any gRNA. Similarly to scramble control, Cas-only Control aims to confirm that Cas does not carry out non-specific cleavage independently.
3. In gRNA-only Control, the Cas nuclease is removed from the complex to observe whether gene editing would still transpire.
4. Mismatch control is essential for evaluating target specificity. Sequence mismatches are introduced into the gRNA to assess whether the CRISPR-Cas system would tolerate it. Any tolerated mismatch sequence would indicate off-target editing.
5. A special type of negative CRISPR control is mock control, where the cells are transfected with a delivery method, such as lipofection or electroporation, without the actual CRISPR-Cas system. Then, the cell phenotype is compared to the wild-type cells to understand whether any observed phenotype may be because of the transfection conditions instead of the system itself.

Validation techniques

The CRISPR control techniques listed here rely on validation experiments, cellular assays and protein tracking.

On the DNA level, PCR, Sanger Sequencing, next-generation sequencing (NGS) and T7 Endonuclease I assays can incorporate CRISPR controls to assess editing efficiency and detect undesired mutations. Primers are often used to characterize gene knockouts and knock-ins by amplifying target regions and detecting indel mutations.

Reporter systems are powerful tools for quantifying on- and/or off-target editing through fluorescent or bioluminescent assays. For instance, a sgRNA can target a fluorescent reporter gene encoding the green fluorescent protein (GFP), and the decrease in the fluorescence signal can quantify the loss of function.⁵ Furthermore, a bioluminescence resonance energy transfer (BRET) based reporter platform can detect off-target editing caused by mismatch repair activities.⁶

Challenges and Best Practices for CRISPR Controls

Limitations of CRISPR technologies

The threat of unwanted mutations remains a bottleneck in the widespread application of CRISPR. Firstly, off-target edits may occur due to the Cas9 tolerance towards gRNA mismatches and sequences resembling the target site, posing significant risks of genomic instability in gene therapy applications. In addition, successful editing is highly dependent on a PAM sequence, which limits the scope of genomic regions that can be edited. Efficiency can be hampered further by poor sgRNA design, suboptimal delivery and erroneous DNA repair. Although several improvements are proposed to overcome these challenges, robust CRISPR controls remain central to validating CRISPR efficiency.

Best practices for designing CRISPR controls

Positive CRISPR experiment controls can help researchers confirm CRISPR system functionality and fine-tune design protocols to maximize efficiency. Negative controls are equally important, as they help monitor undesired edits and non-specific Cas9 activity. Additional controls can be employed to increase confidence in the CRISPR system. For instance, a rescue experiment can be performed by introducing a copy of the knocked-out gene and observing the phenotype to prove the causality between knockout and phenotype. Finally, transfection controls can be used to visualize and quantify cellular uptake of the CRISPR components.

Conclusion

Experiment controls pave the way for robust, reproducible, impactful gene editing protocols. By implementing rigorous quality control, CRISPR-based gene therapies, diagnostic and research toolkits can earn prominence in research, medicine and genetic engineering.

References

1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007;315(5819):1709-1712.
2. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816-821.
3. Lemmens M, Fischer B, Zogg M, Rodrigues L, Kerr G, del Rio-Espinola A, et al. Evaluation of two in vitro assays for tumorigenicity assessment of CRISPR-Cas9 genome-edited cells. Mol Ther Methods Clin Dev 2021;23:241-253.
4. Hou T, Zeng W, Yang M, Chen W, Ren L, Ai J, et al. Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19. PLoS pathog 2020;16(8):e1008705.
5. Lyu P, Yoo KW, Yadav MK, Atala A, Aartsma-Rus A, Putten Mv, et al. Sensitive and reliable evaluation of single-cut sgRNAs to restore dystrophin by a GFP-reporter assay. PLoS One 2020;15(9):e0239468.
6. Wimmer T, Lorenz A, Hossfeld LT, Ponnam SPG, Lytvynchuk L, Stieger K. Evaluation of CRISPR-Cas9 mismatch activity using a BRET-based reporter system. Microchem J 2025;208:112256.

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