Bringing the bench one step closer to the bedside with modified CRISPR/Cas9 machinery
posted May 11, 2017
Clustered regularly interspaced short palindromic repeats (CRISPR) gene-editing technology has created tremendous new possibilities for scientific research and medical advancement. CRISPR is a bacterial adaptive immune mechanism that protects the host from phage infections. During infections, bacteria incorporate pieces of the phage DNA into their own genome and when a new infection occurs, these phage sequences are processed into single guide RNAs (sgRNAs) that form a complex with the endonuclease protein CRISPR-associated protein 9 (Cas9). Guided by sgRNA, Cas9 scans the phage genome and cuts the DNA where complementarity is established. Scientists at MIT and Stanford have adapted this brilliant system to create gene knockouts in mammalian cells by tailoring the sgRNAs to gene targets of interest. Through the error-prone repair mechanism of non-homologous end joining, the resulting DNA is left with a frame-shift mutation that ablates the protein product. At the time of DNA cleavage, if given a gene sequence flanked by homology arms, the cell can incorporate the sequence via homologous recombination and thereby knock-in a foreign gene of interest1.
The high precision and efficiency of this system have once more revived hopes for gene therapy. However, as promising as the results have been in cell lines, it is still challenging to edit primary human cells. Moreover, safety remains a concern due to the commonly used delivery tool of DNA plasmids, which stably insert into the recipient genome. The constitutive expression of Cas9 and sgRNAs may result in potential off-target effects, although the chances of which are extremely low using appropriately designed sequences. Additionally, the microbial origin of Cas9 could cause adverse immune responses in the mammalian system. To address these concerns, a group of researchers at Stanford University showed that chemically modified sgRNAs (cm-sgRNAs) co-delivered with Cas9 mRNA or protein induced high levels of genome editing in primary human T cells, and CD34+ hematopoietic stem and progenitor cells (HSPCs)2. The researchers attached various stabilizing chemical groups onto the three terminal nucleotides at both 5’ and 3’ ends of the sgRNA. The cm-sgRNAs resulted in substantially higher editing efficiency than the unmodified sgRNA using the DNA plasmid delivery route. Similar trends were observed when the sgRNAs were co-delivered with Cas9 mRNA or complexed with Cas9 protein. Unlike stably integrated DNA, mRNA and protein are rapidly degraded in the cytosol, leaving no CRISPR machinery floating around in the cell. Intriguingly, as the active window for the editing machinery is now smaller, increased stability of the cm-sgRNAs showed considerable advantage of 66-87% editing rate over a 7% of the unmodified. Furthermore, while un-stimulated primary human T cells have been reported to be more resistant to editing, co-delivery with Cas9 mRNA showed a higher editing rate than by plasmid delivery, with an apparent advantage of cm-sgRNA over the unmodified sgRNA. Similar results were found in CD34+ HSPCs using Cas9 mRNA and cm-sgRNA, with surprisingly higher overall editing efficiency than that in T cells.
However, in terms of the important question regarding specificity, results from this study were inconclusive. Although certain chemical modifications showed increased but low-level off-target effects, the on-to-off-target ratios were often improved over the unmodified sgRNA. This non-specific editing seemed to be gene sequence-dependent. Perhaps the key here is to find the appropriate loci position to target, as well as to use the optimal type of chemical modification.
In addition to the synthesis convenience of using cm-sgRNA to co-deliver with Cas9 mRNA or protein, the non-DNA platform offers flexibility in exploring sgRNA designs for improved editing efficacy, and most important of all, low cytotoxicity. As the biology of CRISPR/Cas9 rapidly unfolds, scientists are moving towards the “bench to bedside” transition by exploring means to modify, improve, and deliver the system. Hopefully by the time the next significant breakthrough comes around, the ethics discussion will have caught up to the technology, so that it can truly benefit the ones who need it the most.
1. Doudna, J.A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science 346(2014).
2. Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotech 33, 985-989 (2015).