CRISPR/Cas9, originally discovered in 1987 by a team of Japanese scientists and later refined by Jennifer Doudna in 2012, is a gene-editing tool that can cut and paste any genomic sequence, either in vitro or in vivo. It’s a system that relies on clustered regularly interspaced short palindromic repeats (CRISPR) to recognize foreign DNA and is mainly used in bacteria to fight off viral infection. This tool has garnered a lot of attention recently as researchers have tailored CRISPR/Cas9 to edit animal genomes in ways that were previously impossible or inefficient, revolutionizing genetic and biomedical research. CRISPR/Cas9 has become a crucial resource for labs who require stable cell lines or mice with knockouts, knock-ins, or gene mutations, able to drive constitutive gene activation or to edit micro-RNA and long-noncoding RNA.

An update on CRISPR/Cas9 technology

Within the last few months, several interesting papers characterizing new CRISPR-based applications have been published. In August, Michael Gapinske and his colleagues at the University of Illinois at Urbana-Champaign published a method of skipping the transcription of entire exons by mutating specific bases within splice sites. This new technique, aptly named CRISPR-SKIP, uses a web-based software to help scientists determine where the appropriate mutations should be made, and could be useful for studying the formation and function of alternatively spliced genes or even long-noncoding RNAs, which have multiple exons spliced in a similar fashion to protein-coding mRNAs.¹ This sort of technology has the potential to control which parts of a gene are expressed, so that segments that result in disease when expressed could be skipped over entirely.

Another new use for CRISPR technology comes in the form of CRISPR-modified skin patches engineered to produce an enzyme that degrades cocaine. A team at the University of Chicago inserted a gene for a hyper-efficient version of the cocaine-degrading enzyme, butyrylcholinesterase, into skin epidermal stem cells and produced skin grafts capable of secreting the protein into the bloodstream. The technique could provide a therapeutic avenue to prevent overdoses and to help treat cocaine addiction.²

Finally, scientists from the University of Washington have characterized nearly 4000 different mutations in the BRCA1 gene to help identify which mutations are responsible for increased breast cancer risk. They used CRISPR to make 3,893 distinct variants of BRCA1 and tested them in haploid cells that would die if the corresponding BRCA1 protein didn’t work. The study was validated using well-known variants that caused breast cancer, which correlated with other nonfunctional BRCA1 mutations found in their analysis.³ Ideally, this large set of data could be used to identify patients—some of whom may have uncommon or rare BRCA1 mutations—who are at a high risk for developing the disease.

Will we ever see CRISPR in the clinic?

Clinical trials for CRISPR-based therapies have only just begun, including studies using CRISPR technology to treat malignancies and genetic disease, like β-thalassemia, which is a rare blood disorder caused by mutations in the HBB gene that encodes the beta subunit of hemoglobin. CRISPR hit a rut earlier this year as a study came out highlighting the off-target effects of the technology, suggesting that CRISPR might cut out or modify many non-targeted genes, potentially increasing the risk of cancer.⁴ Companies that specialize in developing CRISPR-based clinical therapies took a steep stock hit, losing more than $300 million of value in July. Fortunately, September brought about a newly published study from a collaboration between scientists at AstraZeneca and Harvard University detailing a novel way of checking for off-target mutations caused by CRISPR gene editing in vivo.⁵ Their process, called VIVO, assesses the number of additional off-target mutations in vitro then confirms the presence of these sites in target tissues, making it possible for scientists to develop CRISPR-based therapeutics using a safe and well-controlled system in the near future.

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References:

1. Gapinske M, Luu A, Winter J, et al. CRISPR-SKIP: Programmable gene splicing with single base editors. Genome Biol. 2018.
2. Li, Y.; Kong, Q.; Yue, J.; Gou, X.; Xu, M.; Wu X. Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose. Nat Biomed Eng. 2018.
3. Findlay GM, Daza RM, Martin B, et al. Accurate functional classification of thousands of BRCA1 variants with saturation genome editing. bioRxiv. 2018.
4. Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018.
5. Akcakaya P, Bobbin ML, Guo JA, et al. In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations. bioRxiv. 2018.