How can we make CRISPR therapies safer?
Image credit: Arek Socha/Pixabay

[1]Terns, Michael P., and Rebecca M. Terns. "CRISPR-based adaptive immune systems." Current opinion in microbiology 14.3 (2011): 321-327.
[2]Anderson, Keith R., et al. "CRISPR off-target analysis in genetically engineered rats and mice." Nature methods 15.7 (2018): 512-514.
Creative contributions
Why gene editing precision and safety is not only determined by off-target effects.

[1]Iyama, T. and D.M. Wilson, 3rd, DNA repair mechanisms in dividing and non-dividing cells. DNA Repair (Amst), 2013. 12(8): p. 620-36.
[2]Takata, M., et al., Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J, 1998. 17(18): p. 5497-508.
[3]Delacote, F. and B.S. Lopez, Importance of the cell cycle phase for the choice of the appropriate DSB repair pathway, for genome stability maintenance: the trans-S double-strand break repair model. Cell Cycle, 2008. 7(1): p. 33-8.
[4]Anguela, X.M., et al., Robust ZFN-mediated genome editing in adult hemophilic mice. Blood, 2013. 122(19): p. 3283-7.
[5]Li, H., et al., In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature, 2011. 475(7355): p. 217-21.
[6]Sharma, R., et al., In vivo genome editing of the albumin locus as a platform for protein replacement therapy. Blood, 2015. 126(15): p. 1777-84.
[7]McVey, M. and S.E. Lee, MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. Trends Genet, 2008. 24(11): p. 529-38.
[8]Shen, M.W., et al., Predictable and precise template-free CRISPR editing of pathogenic variants. Nature, 2018. 563(7733): p. 646-651.


Using a synthetic material-based delivery system
[1]Tong, S., Moyo, B., Lee, C.M. et al. Engineered materials for in vivo delivery of genome-editing machinery. Nat Rev Mater 4, 726–737 (2019). https://doi.org/10.1038/s41578-019-0145-9


Controlling CRISPR edited cells via Auxotrophy

[1]Wiebking, V., Patterson, J.O., Martin, R. et al. Metabolic engineering generates a transgene-free safety switch for cell therapy. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0580-6
[2]CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods Emily Rayner, Mary-Anne Durin, Rachael Thomas, Daniela Moralli, Sean M. O'Cathail, Ian Tomlinson, Catherine M. Green, and Annabelle Lewis The CRISPR Journal 2019 2:6, 406-416

Use Anti-CRISPRs as an antidote to CRISPR.

[1]Marino, Nicole D., et al. "Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies." Nature Methods (2020): 1-9.
[2]Shin, Jiyung, et al. "Disabling Cas9 by an anti-CRISPR DNA mimic." Science advances 3.7 (2017): e1701620.
[3]Li, Chang, et al. "HDAd5/35++ adenovirus vector expressing anti-CRISPR peptides decreases CRISPR/Cas9 toxicity in human hematopoietic stem cells." Molecular Therapy-Methods & Clinical Development 9 (2018): 390-401.
[4]Lee, Jooyoung, et al. "Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins." RNA 25.11 (2019): 1421-1431.


Why CRISPR is already safe but it may still need improvements

[1]Hirakawa MP, Krishnakumar R, Timlin JA, Carney JP, Butler KS. Gene editing and CRISPR in the clinic: current and future perspectives. Biosci Rep. 2020;40(4):BSR20200127. doi:10.1042/BSR20200127
[2]Zhang, J., Li, X., Neises, A. et al. Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency. Sci Rep 6, 28566 (2016). https://doi.org/10.1038/srep28566
[3]Havlicek S, Shen Y, Alpagu Y, et al. Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells. Mol Ther. 2017;25(2):342-355. doi:10.1016/j.ymthe.2016.11.007


Bioinformatics and sequencing to increase the safety of CRISPR
[1]https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi
[2]https://genome.ucsc.edu/cgi-bin/hgPcr


Perhaps drug-induced CRISPR systems could make CRISPR safer.

[1]Zhang, Jingfang, et al. "Drug inducible CRISPR/Cas systems." Computational and Structural Biotechnology Journal 17 (2019): 1171-1177.
[2]Davis, Kevin M., et al. "Small molecule–triggered Cas9 protein with improved genome-editing specificity." Nature chemical biology 11.5 (2015): 316-318.

Temporary and tissue-specific expression of Cas9 could be crucial to improve its safety.

[1]Zhang, H.X., Y. Zhang, and H. Yin, Genome Editing with mRNA Encoding ZFN, TALEN, and Cas9. Mol Ther, 2019. 27(4): p. 735-746.
[2]Zuris, J.A., et al., Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol, 2015. 33(1): p. 73-80.
[3]Merienne, N., et al., The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes. Cell Rep, 2017. 20(12): p. 2980-2991.
[4]Suzuki, K., et al., In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature, 2016. 540(7631): p. 144-149.

