Applications of the genome editing system CRISPR are appearing at a furious pace, and gathering momentum toward therapeutic use in human cells. Indeed, Chinese scientists recently began a human clinical trial using CRISPR-edited cells to fight lung cancer, and U.S. clinical trials are slated to begin in 2017. But leading up to this exciting milestone, researchers performed some editing on the CRISPR system itself.
Strategies used to modulate Cas9 activity. (Computational and Structural Biotechnology Journal – Programmable Genome Editing Tools and their Regulation for Efficient Genome Engineering)
(a) Group II intron (GII)-based switch,
(b) separating Cas9 into two peptides, termed split-Cas9,
(c) Tetracycline-inducible and reversible expression system, and
(d) ligand-dependent dimerization of split-Cas9.
(e) Light-dependent dimerization of split-Cas9, termed photoactivatable Cas9 (paCas9),
(f) intein-Cas9, which are activated by splicing of a ligand-dependent intein,
(g) and unstable destabilizing domain-Cas9 (DD-Cas9) fusions, which are degraded unless provided with the ligand, Shield.
CAG = cytomegalovirus early enhancer/chicken β-actin promoter;
Cas9 = clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9;
Cas9′ = partial Cas9;
dCas9 = dead Cas9;
FKBP = FK506 binding protein;
FRB = FKBP-rapamycin binding;
IPTG = isopropyl β-D-1-thiogalactopyranoside;
KRAB = Krüppel-associated box; MN = meganuclease;
mRNA = messenger RNA;
rtTA = reverse tetracycline-controlled transcriptional activator;
sgRNA = single-guide RNA; TRE = tetracycline response element;
T7 = T7 RNA polymerase promoter;
4-HT = 4-hydroxytamoxifen;
DD = destabilizing domain;
nMag = negative Magnet;
pMag = positive Magnet;
sgRNA = single-guide ribonucleic acid.
Scientists quickly began tweaking CRISPR in the right places, and now innovative molecular features are making it work even better and for more cell types. The rapid emergence of CRISPR applications means that clinical trials related to HIV, cancer, sickle cell disease, and other diseases are on the horizon.
Today CRISPR is a cutting-edge tool for many more researchers, and more suitable for future therapeutic use than other gene-modulating methods. “Back when RNA interference [RNAi] hit, it went into hyperdrive,” says Mark Behlke, senior vice president and chief scientific officer at Integrated DNA Technologies (IDT), which supplies RNAi and CRISPR reagents. “But now CRISPR makes that look like a child’s game—it’s just mind-blowing.”
CRISPR reagents get a makeover
Genome editing reagents are being developed and employed at a rapid rate. In order to increase specificity and avoid or reduce toxicity issues due to off-target activities strategies are now being developed to provide temporal control over the DNA-cutting activities of genome editing tools. There are a variety of novel approaches that have been employed so far but there is tremendous potential that is offered by nucleic acid-based regulatory switches that could be incorporated into the expression vectors of genome editing reagents. The development of programmable genome editing tools along with the ability of controlling the temporal and spatial expression of such editing reagents promises to be a very active and challenging research area.
Faster, cheaper, and easier to use than gene editing methods such as TALENs (transcription activator-like effector nucleases) or zinc-finger nucleases, CRISPR was quickly seized upon by researchers in many fields. For example, cancer researchers transformed cell lines with plasmids containing DNA that encoded CRISPR guide RNA (gRNA) and Cas9 (CRISPR-associated protein 9) to create different cancer cell lines for study.
But Matt Porteus, a physician and associate professor of pediatrics at Stanford University School of Medicine, had a different initial experience with CRISPR. “Everyone was saying that CRISPR would solve all the problems of the world, but when we tried to use CRISPR DNA plasmids in cells that we thought were important for therapeutic applications, like hematopoietic cell lines or other primary human cell types, the system didn’t work at all,” he says. So the Porteus lab developed a different delivery method for CRISPR/Cas9 editing in human primary cells, one that doesn’t require DNA plasmids.
Variations of this method exist that introduce CRISPR/Cas9 reagents into cells in the form of ribonucleoproteins (RNPs).
“The researcher combines these reagents [gRNA and Cas9 protein] and allows them to form a complex for five to ten minutes” to create RNPs, says Jon Chesnut, senior director of synthetic biology R and D at Thermo Fisher Scientific. “The CRISPR RNPs can then be delivered to the cell by lipid nanoparticles or electroporation.”
One of the keys to improving CRISPR in primary cells, as well as other cell types, is the recent enhancement of reagents. IDT developed chemically modified gRNAs that are resistant to nuclease degradation inside cells. The company also manufactures gRNAs in the form of two shorter RNAs (as in the original bacterial system) that form a complex, instead of a single, longer gRNA. MilliporeSigma also plans to offer two-part synthetic gRNAs as “SygRNAs.”
Other new tools include transfection chemistries for better delivery of CRISPR reagents into cells. MTI-GlobalStem’s new EditPro Stem Transfection Reagent supports the delivery of CRISPR tools into stem cells, and its EditPro Transfection Reagent enables delivery into human primary cells and cell lines. “The new EditPro chemistry has a wide range of tunable dosage, in terms of the amount of mRNA that will translate into higher protein translation,” says James Kehler, director of scientific alliances at MTI-GlobalStem.
MilliporeSigma also offers lentiviral-based CRISPR tools for whole-genome screening. In collaboration with the Wellcome Trust Sanger Institute, MilliporeSigma also recently constructed arrayed whole-genome CRISPR libraries for human and mouse genomes that offer flexibility in format, delivery, and scope (i.e., single genes, gene families, or whole genomes).
Agilent Technologies recently released pooled CRISPR guide libraries for screening, including the genome-scale CRISPR knockout (GeCKO) SureGuide Catalog human and mouse libraries delivered via lentiviral vector. Agilent also offers preamplified and nonamplified custom libraries for full flexibility. “Our CRISPR pooled libraries are most often used in functional screening, using CRISPR/Cas9 to generate knockouts across the genome,” says Caroline Tsou, Agilent’s global marketing director for molecular and synthetic biology in the Diagnostics and Genomics Group. “Usually these knockouts serve to identify genes involved in cellular responses, such as in signaling pathways, or to discover the function of novel genes.” Agilent also prints custom oligonucleotides of up to 230 base pairs, giving researchers “the freedom to explore other uses for the libraries,” she says.
Aiming CRISPR at human diseases
Meanwhile, all manner of CRISPR reagents are on deck to fight a variety of diseases—especially using the DNA-free approach. The fast-on, fast-off nature of the RNP method, for example, is well suited to therapeutic applications where the CRISPR reagents cut where directed and then degrade quickly.
But correcting genetic defects isn’t as simple as knocking out a gene, because often the correct functional gene must also be introduced at the right location. The Porteus lab at Stanford recently published proof-of-concept work using CRISPR RNPs to target the beta-globin gene, mutations of which cause sickle cell disease. They showed that they could correct the defective beta-globin gene in human hematopoietic stem cells from patients with this disease (4). Independently, a lab at the University of California, Berkeley, accomplished a similar CRISPR editing result with the beta-globin gene, using a slightly different method to deliver the corrected gene.
Taken together, the work of these and other labs is promising for upcoming human trials. In June, the U.S. National Institutes of Health approved the first trial in the United States, slated for 2017, which will use CRISPR-edited human T cells to help augment cancer therapies.
Meanwhile, the Porteus lab is gearing up to manufacture CRISPR-edited cells for use in patients, in clinical trials that they hope to start in 2018. They will likely target sickle cell disease first, followed by severe combined immune deficiency (SCID). Porteus hopes to use CRISPR not just to correct mutations, but also to “give cells new properties that might treat a disease, such as an immune system that’s resistant to HIV, or to create cells that could deliver a protein to the brain,” he says. “In the ecology of science and medicine, we feel like our role is to try to bring this technology to patients.”
With the development of CRISPR research tools in hyperdrive and U.S. clinical trials set to begin next year, these goals are probably closer to being realized than we imagine.
SOURCES – Journal Science, Computational and Structural Biotechnology Journal – Programmable Genome Editing Tools and their Regulation for Efficient Genome Engineering