The spotlight continues to shine on an innovative technology, CRISPR-Cas9, due to its efficient site-directed mutagenesis. An example includes introducing variant Cas9 nucleases which affect the product of the studied protein. Also, it is utilized for RNA editing as opposed to DNA editing. The traditional use for Cas9 nuclease introduces double-strand DNA breaks that are repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanics. These cause spontaneous insertions or deletions (InDels) which may give unwanted phenotypes.
Here, we highlight two new approaches known as CRISPR-STOP and CRISPR-SKIP which alter the genome by introducing single nucleotide edits. These single-based editors are a more controllable gene editing tool because they can result in specific alternative splicing and not off-site target edits as previously discussed. Alternative splicing provides temporal and tissue-specific control which may be vital during biological development. Having the ability to be selective in skipping mutated exons can be significant for biomedical procedures.
CRISPR-STOP truncates proteins by introducing stop codons using a C to T substitution by a cytidine deaminase-dependent CRISPR complex. Billion P., et al., (2017), found that CRISPR-STOP can convert four codons (CAA, CAG, CGA, and TGG) into STOP codons (TAG, TAA, or TGA), which can be easily detected by restriction digest. Additionally, Kuscu C., et al., (2017) found that early stop codons can be introduced in approximately 17,000 human genes with around 216,000 unique inducible stop (iSTOP) sgRNAs. An online database that aids in searching and selecting sgSTOPs can be accessed at http://www.ciccialab-database.com/istop
A few advantages of the iSTOP alternative include: (Billion P., et al., (2017); Kuscu C., et al., (2017))
- iSTOP is not affected by undesired cytosine deamination events
- 97-99% of genes in eight eukaryotes are targetable, including higher copy regions
- Applicable model for over 32,000 human cancer-associated nonsense mutations
- No double-stranded breaks, thus increasing the success rate of knockout models because of the reduction in cell death
- No NHEJ frameshift mutations, thus resulting in no mosaic populations
- No synthetic DNA donor oligos are needed
However, the design of sgSTOPs pose a limitation by requiring the presence of CAA, CAG, CGA, and TGG codons and a PAM located 13–17 bps away from the targeted base(s).
CRISPR-STOP is a powerful alternative for applications that are considered challenging for canonical CRISPR systems. Not only does iSTOP disrupt ORFs without altering gene structure, it may allow the separation of coding from non-coding functions of genes and introduce edited amino acids into proteins. Being able to inactivate thousands of genes on a genome-wide scale while modeling cancer-related mutations is a powerful technology to consider when designing new models.
In contrast, CRISPR-SKIP can modulate different gene products rather than generating a synthetic on-off switch. Gapinske M., et al, (2018) found that CRISPR-SKIP disrupts the splice acceptor consensus sequence. Since almost all introns end in a guanosine, this enables programmable exon skipping. In addition, they found CRISPR-SKIP is not cell line-specific. An online software tool that identifies potential sgRNAs given a desired target gene or exon to be skipped is available at http://song.igb.illinois.edu/crispr-skip/
A few advantages of the exon-skipping alternative are: (Gapinske M., et al, (2018))
- Successful at simultaneous exon skipping
- Used to study function of lncRNAs with precision
- Applicable for studying monogenic diseases, especially Duchenne muscular dystrophy
- Permanent modifications with no genome double-stranded breaks
- A therapeutic model for a wide variety of human diseases
One limitation of CRISPR-SKIP is the dependency on the presence of the PAM site, as previously stated with CRISPR-STOP. To overcome this, different Cas9 scaffolds are reviewed to recognize different PAM motifs. Further understanding is needed of exon–intron architecture and their recognition by the spliceosome machinery in order to enable more efficient targeting. Similarly, advancements in base-editing technologies will likely improve the rate of skipping and the number of targeted exons in the future.
According to Dr. Elizabeth C. Bryda, CRISPR technology facilitates the ability to select species rendering the research being studied and model personalized approaches that address the exact questions asked. Specifically, base-editing technology is extremely useful in somatic cell treatments and genetic diseases that have few, if any, current treatments. Since CRISPR-Cas9 technology is innovative, with each optimization, the manipulation of the genome could be precisely studied finally with base editors, thus accelerating research for rare diseases and biomedical procedures.