CAST

Engineering CRISPR associated transposases for use in Eukaryotes.

After reading some thingys I realized there is a lot of optimism regarding using CRISPR/Cas9 for genetic engineering.  According to Dockrill (2018), a study done by the Sanger institute shows that CRISPR/Cas9 is causing many mutations and that these mutations wouldn’t be detected in a standard DNA tests.  The optimism surrounding CRISPR/Cas9 should be tempered with the stark reality that CRISPR/Cas9 gene-editing technology causes many off-target and on-target mutations including splicing disruptions and mutations several kilobases away from target sites (Kosicki et al., 2018).  Since homologous recombination repair is directly tied to mitosis it does not address every cell.   Many times, CRISPR Cas9 double-stranded cut results in the target cell using non-homologous end-joining.   Resultant from this is the formation of indels which if in a protein-coding sequence can cause frameshift mutations.  These frameshifts or other mutations could result in the induction of oncogenesis or neoplasms.  Thus, non-templetive repair events should be mitigated if CRISPR systems are to be used in-vivo.  Another confounding factor is that genetic variation between individuals and species can change off-target/on-target mutation frequency.

One bright spot is a new form of CRISPR editing that uses inactive Cas for point mutations targeted by cytidine or adenine deaminases, this is of course restricted to nucleotide substitutions (Streckler et al., 2019).  Although mutations from genetic drift greatly exceeded off target and on target mutations, in-vivo use of this technology specifically in humans at this point would be unethical due to violation of the non-maleficence tenet of ethics.  Therefore, a more targeted and humane approach should be considered.

Now that we know some of the problems associated with CRISPR/Cas9, we should also investigate solutions.  Recent research has indicated that CRISPR associated transposases represent a more accurate and less mutagenic form of genetic engineering in prokaryotes (Strecker et al, 2019).  Their work shows that there is a way to use CRISPR without the CAS nuclease activity thereby avoiding homologous recombination.  Streckler et al., (2019) were able to bind nickase Cas9(D10A) to ssDNA transposase TnpA of H. pylori and created TnpA, Cas9, sgRNA, targeted DNA insertions in vitro and in E. coli.  Further research into CRISPR associated transposase systems of microbial genomes could show many previously unknown targeting profiles.  This investigation could unleash a revolution similar to CRISPR itself.  Transposase systems allow for the insertion of full-length genes which could be targeted to just before damaged genes, which would enable their replacement.  If we find that the transposase systems can be used in eukaryotes just as the CRISPR/Cas complex was what will the applications be?   There are many steps ahead in researching this potentially lucrative new form of genetic editing.  I am suggesting that we research into this topic with urgency so that opportunities are not missed.

References

Dockrill, P. (2018, July 16). BREAKING: CRISPR Could Be Causing Extensive Mutations And Genetic Damage After All. Retrieved May 1, 2020, from https://www.sciencealert.com/crispr-editing-causes-frequent-extensive-mutations-genetic-damage-target-deletion-site

Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nature biotechnology. 2018;36(8):765-771. doi:10.1038/nbt.4192.

Strecker, J., Ladha, A., Gardner, Z., Schmid-Burgk, J. L., Makarova, K. S., Koonin, E. V., & Zhang, F. (2019). RNA-guided DNA insertion with CRISPR-associated transposases. Science, 365(6448), 48–53. https://doi-org.proxy.library.kent.edu/10.1126/science.aax9181