Question: I recently read an article about a group of Chinese scientists that used the CRISPR/Cas9 germline modification system to edit genomes of human embryos. I became interested in how exactly this process makes edits and what changes are made to the DNA, if any, and how this system applies to the world of biochemistry and its human system related applications.
Answer: CRISPR/Cas9 is a genome editing system discovered in 2012 capable of modifying genes in human, animal and bacterial applications in an effort to explore and correct mutations caused by disease. Fundamentally, this mechanism is a prokaryotic response in which bacteria gain resistance to specific foreign genetic elements such as plasmids and phages. CRISPRs are sections of short DNA sequences that can be found in many bacteria and archaea. These “guide” RNAs direct the Cas9 protein to the target site – for example viral DNA – and the protein cleaves the invading DNA. As a virus attacks a host, bacteria work to incorporate sequences of viral DNA into their genetic material by placing them between the CRISPR repeats and therefore gain immunity. This tool is especially promising because as bacteria encounter the virus, it uses the DNA from the clusters to produce RNAs capable of distinguishing the matching viral sequences.
The scientists in this specific study used whole-exome sequencing and a T7 endonuclease (T7E1) assay for on-target CRISPR/Cas9 events in cultured cells to analyze the efficiency of homologous recombination directed repair (HDR) and the CRISPR cleaving process of the endogenous B-globin gene (HBB). Although it was observed that the system correctly cleaved the HBB gene, the analyses revealed several instances of off-target cleaving that occurred in the tripronuclear zygotes. Further, the team observed that repairs were made at alternate sites in the embryos, indicating that cleaving of HBB completed by the HDR mechanism repair was minimal. According to the review by Liang, et. al, results suggested that an alternative homologous gene to HBB “competed with exogenous donor oligos by acting as the interim repair template” revealing excess mutations after the CRISPR/Cas9 mechanism. There are extensive ethical concerns regarding the CRISPR/Cas9 use in the human model, however, this editing tool may become a key component to the eradication of disease in multiple organisms. Although it is currently used in several plant models, clinical and laboratory research and development is needed to improve the system’s specificity to yield consistent results that minimize the observed unexpected mutations. This type of protein biochemistry has proven to be revolutionary, however, the current high-profile patent dispute on this technology’s invention by CRISPR Therapeutics may lead to hurdles in the research and development process to come. The abundance of CRISPRs in the genomes of bacteria and archaea continues to allow researchers to gain further knowledge in the processes surrounding gene manipulation and significantly advanced the general understanding of genes.
- Carla T.
Abbott, Alison (27 May 2016). The quiet revolutionary: How the co-discovery of CRISPR explosively changed Emmanuelle Charpentier’s life. Nature, 532 (7600). Web.
Cyranoski, D. and S. Reardon (2015). Chinese scientists genetically modify human embryos. Nature, News. Web.
Liang, P., Y. Xu, X. Zhang, C. Ding, R. Huang, Z. Zhang, J. Lv, X. Xie, Y. Chen, Y. Li, Y.Sun, Y. Bai, Z. Songyang, W. Ma, C.Zhou, and J. Huang (May 2015). CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & Cell, 6 (5): 363-372.