How can we make CRISPR therapies safer?
Image credit: Arek Socha/Pixabay
- There are off-target effects, is there anything else that could make CRISPR therapies potentially unsafe?
- How could we make CRISPR therapies safer (especially the in vivo ones)?
- Should we stick to ex vivo CRISPR therapies instead of the in vivo CRISPR therapies?
Terns, Michael P., and Rebecca M. Terns. "CRISPR-based adaptive immune systems." Current opinion in microbiology 14.3 (2011): 321-327.
Anderson, Keith R., et al. "CRISPR off-target analysis in genetically engineered rats and mice." Nature methods 15.7 (2018): 512-514.
Why gene editing precision and safety is not only determined by off-target effects.
- NHEJ is the most common DSB repair pathway in higher eukaryotes, being active in all phases of the cell cycle, but it is most active during the G1 phase . In short, this system involves several DNA repair factors that, after recognition of the DSB, lead to filling of the gap using random nucleotides. This system is very efficient at quickly repairing the DSB but lacks precision, usually resulting in insertion or deletion (INDELs) of random bases around the cleavage site. This feature of NHEJ is exploited in some gene editing approaches in order to alter the reading frame and thus knock out expression of targeted genes.
- HDR is mostly active in dividing cells, particularly in the G2 and S phases of the cell cycle. It is associated to DNA damage during chromosomal replication. After recognition of the DSB, HDR factors employ the sister chromatid as a template for strand invasion and homologous recombination followed by polymerase-mediated extension of the single-stranded DNA. This leads to precise repair of the DSB. This mechanism can be exploited in dividing cells by delivering a homologous recombination template flanked by homology arms. This allows correction of mutations as well as integration of exogenous sequences in targeted loci. This approach has been used extensively in vivo but can only be used in dividing cells and thus is very inefficient in most differentiated tissues of the organism.
- MMEJ is considered to be a mix between NHEJ and HDR. It involves proteins from both pathways but is active through all cell cycle phases, which implies it can be independent of both NHEJ and HDR. In short, the DNA repair machinery can detect microhomologous sequences flanking the site of the DSB and facilitate recombination between those sequences, generating a small and precise deletion between the two microhomologous regions. If possible, this mechanism is quite efficient and leads to predictable INDELs.
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.
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.
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.
Anguela, X.M., et al., Robust ZFN-mediated genome editing in adult hemophilic mice. Blood, 2013. 122(19): p. 3283-7.
Li, H., et al., In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature, 2011. 475(7355): p. 217-21.
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.
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.
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
- They can be tailored for delivering different forms of the CRISPR–Cas9 system.
- Viral vectors are smaller and the gene-editing system needs to be divided and separately packed in two vectors.
- Cells may have a preexisting immunity against viral vectors. The immunocompatibility of synthetic materials can be improved by optimizing the size, shape, coating, and surface chemistry and charge.
- Retroviral and lentiviral vectors have a large genome of size. However, these vectors may integrate the transgene into the host genome, disrupting functional genes, and increasing off-target gene editing. Synthetic-material-mediated delivery can avoid such problems although random integration has been observed.
- Synthesis/ production of the synthetic material and its conversion to nanoparticles is more cost-effective and suitable for large-scale production than viral vectors.
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
- it allows controlling edited cells without the need to integrate bulky construct in the cell. Like a safety-switch as you would do for CART cells for example. So you can envision to perform along with the editing at the desired target also the KO of the UMPS gene. This means that if after re-infusing the edited cells in the patient if some side effects are observed ( maybe as a consequence of undetected off-targets ), withdrawing the organic compound will be enough to get rid of the cells
- if you infuse such cells in the patient and the patient benefits from them, you will need to keep these cells alive. How you do it? By supplying for example Uridine. Unfortunately, even if Uridine has been used in humans for the treatment of hereditary orotic aciduria and fluoropyrimidine toxicity, it is poorly absorbed and broken down in the liver. This can be circumvented by the administration as the prodrug uridine triacetate (UTA; also known as PN401), which has approval from the US Food and Drug Administration (FDA). So, you are maybe solving one problem but you are creating a dependency on the patients on constant Uridine supplement. At least until the edited cells have completed their job
- to create cells holding both the UMPS gene KO and the modification in the gene of interest you will need to introduce two DNA double-stranded break (DSB) at the same time. This increases the probability to introduce gross DNA rearrangements like translocations or head-to-head chromosomic fusions .
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
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.
- They inhibit the guide RNA and Cas complex.
- They prevent Cas binding to the DNA.
- They prevent DNA cleavage by Cas.
Marino, Nicole D., et al. "Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies." Nature Methods (2020): 1-9.
Shin, Jiyung, et al. "Disabling Cas9 by an anti-CRISPR DNA mimic." Science advances 3.7 (2017): e1701620.
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.
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
- why CRISPR is already safe enough
- strategies used to refine CRISPR precision
- why although precise, CRISPR has an inner “ defect “
- alternative to CRISPR to address the previous point.
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
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
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
The point of this
Perhaps drug-induced CRISPR systems could make CRISPR safer.
Zhang, Jingfang, et al. "Drug inducible CRISPR/Cas systems." Computational and Structural Biotechnology Journal 17 (2019): 1171-1177.
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.
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.
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.
Merienne, N., et al., The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes. Cell Rep, 2017. 20(12): p. 2980-2991.
Suzuki, K., et al., In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature, 2016. 540(7631): p. 144-149.