Elevating CRISPR-Cas9

By GMU Biodefense graduate student Janet Marroquin

The contentious debate regarding the precision of the commercially available gene-editing tool, CRISPR-Cas9, continues as Nature recently retracted a study published last year on the unintended effects of off-target mutagenesis.  The 2017 study used CRISPR-Cas9 to edit a genetic mutation causing blindness in mice but the researchers also observed CRISPR-induced mutations in other genes due to off-target editing at higher rates than previously documented.  In their report, the researchers expressed their concern over the alarming target imprecision of the widely-used gene-editing tool and its effect on unwanted mutations in gene therapy.  However, further evaluation of the study by other researchers raised questions about the validity of the study results given the possibility of natural genetic variation introducing mutations independent of CRISPR use.  After eight months of deliberation, Nature determined the cause of the unexpected mutations to be natural genetic variability, as the mice were not genetic clones, and not the pronounced off-target effects of the CRISPR-Cas9 system used, ultimately leading to the retraction of the study from the journal.

What does this mean for the evaluation of off-target effects on gene-editing?  Are off-target effects really a problem or are they negligible?  And what are off-target effects anyway?  The debate remains unresolved as CRISPR continues to be sought as a tool for myriad uses including gene-therapy and cancer treatments.[i]  Experts familiar with the CRISPR-Cas9 system have recognized that off-targeting occurs with varying frequency in different organisms and, as such, validate their results through methods such as whole-genome sequencing in order to discern possible unintended mutations.[ii]  When using gene-editing technology, the experimenter must first design a guide RNA (gRNA) segment that specifically targets the gene of interest for the insertion or deletion of the desired/undesired mutation.  Unfortunately, binding of the gRNA may occur at undesired sites if the base pair mismatch is low enough, resulting in unwanted off-target effects.[iii]  To counter the possibility of off-target editing, researchers have developed computational algorithms to identify potential off-target sites prior to use, thus improving precision in the technique.[iv]

Joining independent research teams in the application of data analytics and machine learning to gene-editing is Microsoft’s artificial intelligence tool, Elevation, designed specifically for the prediction and reduction of off-target effects by CRISPR.  In addition to developing the algorithm for the experimenter, Elevation further commoditizes its service by allowing the experimenter to enter the target RNA sequence online for step-by-step guidance on designing the gRNA.  According to the results of its endorsed study, Microsoft has built an effective and efficient platform available to the general public for precise gene-editing.[v]

The investment of resources in gene-editing technology by commercial industry confirms the enormous potential that CRISPR-Cas9 holds for biology, medicine, and science as a whole. Its functional versatility has brought continuous attention to potential applications but, like all dual-use research, while the benefits from gene-editing are high, so are the associated risks.  The commercialization of CRISPR, coupled with the commercialization of an AI tool that facilitates its use and improves its accuracy, has heavy implications for biosecurity.  Without institutional oversight or regulatory guidance, gene-editing technology is easily accessible to the general public, including malevolent actors with perverse intentions.  As advances in biotechnology continue to democratize and lower the barrier of entry, it is critical for the biosecurity community to find a balance in policy on dual-use technology that will foster scientific discovery while ensuring security against nefarious actions.


[i]Barrangou, Rodolphe., Jennifer Doudna. Applications of CRISPR technologies in research and beyond.  Nature Biotechnology34 (2016): 933-941. doi:10.1038/nbt.3659

[ii]Zhang, Qiang, Hui-Li Xing, Zhi-Ping Wang, Hai-Yan Zhang, Fang Yang, Xue-Chen Wang, Qi-Jun Chen. Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention. Plant Molecular Biology96, (February 2018): 445-456. https://doi.org/10.1007/s11103-018-0709-x

[iii]Zhang, Xiao-Hui., Louis Y. Tee, Xiao-Gang Wang, Qun-Shan Huang, Shi-Hua Yang. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering.  Molecular Therapy-Nucleic Acids4, no.e264 (2015):1-8. https://doi.org/10.1038/mtna.2015.37

[iv]Zhou, Hong., Michael Zhou, Daisy Li, Joseph Manthey, Ekaterina Lioutikova, Hong Wang, Xiao Zeng. Whole genome analysis of CRISPR Cas9 sgRNA off-target homologies via an efficient computational algorithm. BMC Genomics 18, no.9 (November 2017): 31-38. doi: 10.1186/s12864-017-4225-1

[v]Listgarten, Jennifer., Michael Weinstein, Benjamin P. Kleinstiver. Alexander A. Sousa, J. Keith Joung, et al. Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs. Nature Biomedical Engineering2, no.1 (January 2018): 38-47. https://doi.org/10.1038/s41551-017-0178-6

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