CRISPR-based genome editing technologies are poised to enable countless new therapies to prevent, treat, or cure diseases with a genetic basis. However, the safe and effective delivery of genome ...editing enzymes represents a substantial challenge that must be tackled to enable the next generation of genetic therapies. In this Review, we summarize recent progress in developing enzymatic tools to combat genetic disease and examine current efforts to deliver these enzymes to the cells in need of correction. Viral vectors already in use for traditional gene therapy are being applied to enable in vivo CRISPR-based therapeutics, as are emerging technologies such as nanoparticle-based delivery of CRISPR components and direct delivery of preassembled RNA–protein complexes. Success in these areas will allow CRISPR-based genome editing therapeutics to reach their full potential.
A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff—a programmable epigenetic memory writer ...consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
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•CRISPRoff is a single fusion protein that programs heritable epigenetic memory•CRISPRoff can heritably silence most genes, including genes without CpG islands•CRISPRoff is highly specific and has a broad targeting window across gene promoters•CRISPRoff epigenetic memory persists through differentiation of iPSCs into neurons
CRISPRoff programs heritable epigenetic memory and is able to heritably silence most genes, including genes without CpG islands. This heritable silencing is highly specific and persists through differentiation of iPSCs into neurons.
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying ...their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib’s target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.
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•Combined CRISPRi/a chemical-genetic screening reveals targets of therapeutic agents•Focused chemical-genetic profiling rapidly classifies agents by mechanism of action•Chemical-genetic screens with rigosertib reveal a microtubule-destabilizing signature•Targeted in vivo and in vitro approaches confirm rigosertib’s mechanism of action
Jost et al. present a two-tiered strategy to identify molecular targets of bioactive compounds using CRISPRi/a-mediated chemical-genetic screens. Application to rigosertib, an anti-cancer drug with an unclear mechanism of action, points to rigosertib being a microtubule-destabilizing agent. Targeted cell biological, biochemical, and structural approaches confirm this mechanism of action.
Cancer cells rely on the chaperone heat shock protein 70 (Hsp70) for survival and proliferation. Recently, benzothiazole rhodacyanines have been shown to bind an allosteric site on Hsp70, ...interrupting its binding to nucleotide-exchange factors (NEFs) and promoting cell death in breast cancer cell lines. However, proof-of-concept molecules, such as JG-98, have relatively modest potency (EC50 ≈ 0.7–0.4 μM) and are rapidly metabolized in animals. Here, we explored this chemical series through structure- and property-based design of ∼300 analogs, showing that the most potent had >10-fold improved EC50 values (∼0.05 to 0.03 μM) against two breast cancer cells. Biomarkers and whole genome CRISPRi screens confirmed members of the Hsp70 family as cellular targets. On the basis of these results, JG-231 was found to reduce tumor burden in an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies support the hypothesis that Hsp70 may be a promising target for anticancer therapeutics.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize ...drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR‐associated 9 (dCas9)–positive mouse, cyclization recombination–inducible (Cre) CRISPRa system for cell type–specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high‐throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type–specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto‐oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto‐oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene‐inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. Conclusion: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).
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