Author: Eleanor Sheekey
CRISPR-Cas9 gene editing (CRISPR) has become a renowned genome editing tool that has greatly enhanced scientific research but has also caused much controversy and concern over its potential applications. With the potential of becoming a standard medical tool to fix incurable genetic diseases, the safety and efficiency of CRISPR needs to be extensively investigated to ensure that only the desired DNA modifications are made. Recent studies published in Nature have discovered how editing efficiency in cells can be influenced by the DNA damage response pathway 1,2 and are raising concerns after finding that CRISPR cuts can result in larger unwanted DNA rearrangements than previously thought 3.
Figure 1 Simplified diagram of the application and consequences of CRISPR-Cas9 gene editing
What is CRISPR?
Taken from a bacterial defense mechanism, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-associated protein 9 (Cas9), is a tool that can be employed to cause precise DNA changes in the genome of a cell. Specificity depends on recognition of DNA by an engineered guide RNA strand, making the CRISPR technology much easier to employ than previous protein-based gene editing techniques 4. The ease of design and specificity of edits launched CRISPR into the limelight as a useful tool to use for scientific research in addition to potential global uses such as medicinal treatments and agriculture.
Efficiency improves without the DNA damage response
Before CRISPR can edit the DNA sequence, it must create breaks in the strand. DNA damage can also be induced by exposure to UV radiation and carcinogens such as those found in tobacco. To repair any damage, cells activate a DNA damage response pathway. The DNA damage response therefore antagonizes the principal application of CRISPR as a gene-editing tool resulting in a reduced editing efficiency. A low efficiency could have implications both in the lab and in clinical studies. This antagonism was explored further by two recent studies published in Nature by the teams of Taipale1 and Kaykas2. Both teams associated this antagonism with a protein vital to the DNA damage response, p53. Kaykas’s group showed that DNA damage induced by CRISPR resulted in upregulation of programmed cell death, a downstream event of p53 activation. Reducing the activity of p53 increased editing efficiency and decreased cell toxicity to CRISPR, matching the findings of Taipale’s study. Transiently reducing p53 activity is a potential solution to improve the efficacy of CRISPR; however, controlling the reduction would be critical to prevent unwanted side effects, such as increased mutation frequency due to a disrupted DNA damage response.
More damage than expected
CRISPR technology is designed to induce strand breaks in DNA at specific areas of interest in the genome. Whil3 strand breaks are made at the correct sites there is also the potential for CRISPR to induce breaks at off-target sites. Additionally, Kosicki et al.3 have reported that unwanted on target DNA changes can also occur, such as large deletions and sequence rearrangements. The team performed an extensive analysis to identify these changes that were previously overlooked. By analyzing different clones of cells, it was observed that the extent of the unwanted changes varied greatly, and this diversity was observed in both mouse and human cell lines, validating the group’s findings. The consequences of these changes will require further studying to improve the safety of CRISPR.
What’s in store?
While the results – particularly those reported by Kosicki’s team – have raised concerns over the safety of CRISPR as a tool for treating human cells, it is important that these findings have been recognized to allow for the development of safer versions of the technology. With the Nuffield Council on Bioethics recent announcement that genetically modified babies could be ‘morally permissible’ 5, more comprehensive genomic analyses of cells following CRISPR treatment in combination with improved modifications of the CRISPR-Cas9 techniques is evidently needed before CRISPR is used in further clinical contexts. However, while safety is important, so is efficiency, and improving our understanding of the cellular response to CRISPR is essential before these potential medical advances can be considered.
References:
1. Haapaniemi, Emma; Botla, Sandeep; Persson, Jenna; Schmierer, Bernhard; Taipale, J. CRISPR–Cas9 genome editing induces a p53 mediated DNA damage response. Nature medicine 24, 927–930 (2018).
2. Ihry, R. J. et al. p53 inhibits CRISPR – Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 1–8 (2018).
3. Kosicki, M. & Bradley, A. Repair of CRISPR–Cas9-induced double-stranded breaks leads to large deletions and complex rearrangements. Nat. Publ. Gr. (2018). doi:10.1038/nbt.4192
4. https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr
5. https://www.theguardian.com/science/2018/jul/17/genetically-modified-babies-given-go-ahead-by-uk-ethics-body?CMP=share_btn_tw