Early concerns regarding unintended effects of CRISPR/Cas9 indicated that gene editing might not be specific enough. Scientists are working to define, assess, and limit the mechanisms driving the creation of CRISPR “off-target” effects during the gene editing process, which include unintended deletions, unintended rearrangements, and non-homology end joining.[1] These off-target effects may cause considerable harm, for example, by editing functional genes to render them inactive.[2] The resulting mutations may alter or tamper with genes that have protective effects in humans. These include oncogenes (genes that regulate transformation of normal cells into benign or malignant neoplasms or cancer) and tumor suppressor genes (genes that prevent cancer).[3] According to recent studies, CRISPR gene editing can result in decreasing activity and inhibition of p53 – a tumor suppressor gene that helps prevent the transformation of normal and healthy cells into cancer cells.[4] Furthermore, some cells (human pluripotent stem cells (hPSCs)) cease to proliferate subsequent to genetic manipulation via CRISPR.
Image: Misreplication of DNA in human fibroblast nucleus. Wellcome Collection. Credit: Ezequiel Miron, University of Oxford. CC BY [10]
Retracted research studies in which off-target effects harbored questions regarding whether CRISPR effected changes in a single nucleotide sequence in an entire genome at one point seemed to diminish the impact of off-target effects.[5] [6] Sampling of the genetically modified cells with regard to the desired gene edits may have been highly inconstant, resulting in negative implications for biomedical research discoveries. Another study in which “off-target” effects were purportedly cited lacked scientific validity, as they included less than sufficient sample sizes.[7]
Still, the possibility of off-target effects cannot be excluded. Yet, recent research has shown that off-target effects can be minimized with an increase in gene editing precision.[8] [9]
The long-term effects of CRISPR with regard to curing or lessening the effects of disease are still unknown as the embryos must be observed over time as they develop into humans. Even then, one does not know if the genetic repairs will last for the full span of a normal human lifetime. Furthermore, it is difficult to say if CRISPR’s genetic repairs will result in DNA that maintains the edited genetic corrections or if the edits are lost over time with cell division or through genetic evolution. Still, the potential benefits of CRISPR likely outweigh the concerns, as technology develops to mitigate the current complications of CRISPR technology.
Dorkina Myrick, MD, PhD, MPP, is a physician-scientist trained at the National Institutes of Health in Bethesda, Maryland. Dr. Myrick is currently a JD candidate at the Boston University School of Law.
References:
[1] Akcakaya et al. In vivo CRISPR editing with no detectable genome wide off-target mutations. Nature. Volume 561, pp. 416–419 (2018). Online: https://www.nature.com/articles/s41586-018-0500-9.
[2] Liang et al. “CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.” Protein & Cell. May 2015, Volume 6, Issue 5, pp 363-372.
[3] Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol Ther Nucleic Acids 2015; 4: e264. Online: https://www.cell.com/molecular-therapy-family/nucleic-acids/pdf/S2162-2531(16)30049-X.pdf
[4] Haapaniemi et al. Nature Medicine. Online: https://www.nature.com/articles/s41591-018-0049-z.epdf?referrer_access_token=fwW6sFGp0setmHDPu1kJJ9RgN0jAjWel9jnR3ZoTv0MRjuB3dEnTctGtoy16n3DDbmISsvbln9SCISHVDd73tdQRNS7LB8qBlX1vpbLE0nK_CwKThDGcf344KR6RAm9kszOwqjpU9yuzUtck-RhaKAJ4c76SENGJJEzqbsvSUMUsjARFKb-20Yr7Fem3XZ-B . Retrieved June 11, 2018.
[5] Schaefer KA et al. Addendum: Nature Methods Editorial Expression of Concern: Unexpected mutations after CRISPR–Cas9 editing in vivo. Nature Methods 14(6):547-548. May 2017 Online:
[6] Editorial. CRISPR Off Targets: A Reassessment. Nature Methods. volume 15, pages 229–230 (30 March 2018). Online: https://www.nature.com/articles/nmeth.4664
[7] Schaefer KA et al. Nature Methods Retraction: Unexpected mutations after CRISPR–Cas9 editing in vivo. Nature Methods volume 15, page 394 (2018). Online: https://www.nature.com/articles/nmeth0518-394a.
[8] Lawson-Jones et al. (29 September 2017) “Kinetics of dCas9 target search in Escherichia coli.” Science. vol. 357, Issue 6358, pp. 1420-1424. Online: science.sciencemag.org/cgi/doi … 1126/science.aah7084 . Retrieved July 10, 2018.
[9] Carlson-Stervermer. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nature Communications. volume 8, Article number: 1711. Online: http://www.nature.com/articles/s41467-017-01875-9#Sec9. Retrieved July 9, 2018.
[10] Image: Misreplication of DNA in human fibroblast nucleus. Wellcome Collection. Credit: Ezequiel Miron, University of Oxford. CC BY. Description: Super-resolution optical micrograph of DNA stain in a human foetal lung fibroblast nucleus acquired with a 3D structured illumination microscope (3D SIM). A rare phenomenon has occurred in this image; the majority of the genome has been caught in misreplication. If a single chromosome becomes caught and pulled between the two new cells, it can lead to the presence of small chromatin threads/bridges joining adjacent nuclei. This leads to clearly demarcated chromatin fibres visible throughout the interior of the nucleus, and as the new cells move apart the tension distributed by the cable-like chromatin has deformed the entire nuclear envelope. The width of the image is 84 micrometers. Wellcome Image Awards 2017.”
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