Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR.

Programmable approaches to sense and respond to the presence of specific RNAs in biological systems have broad applications in research, diagnostics, and therapeutics. Here we engineer a programmable RNA-sensing technology, reprogrammable ADAR sensors (RADARS), which harnesses RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a cargo protein by the presence of endogenous RNA transcripts. Introduction of a stop codon in a guide upstream of the cargo makes translation contingent on binding of an endogenous transcript to the guide, leading to ADAR editing of the stop codon and allowing translational readthrough. Through systematic sensor engineering, we achieve 277 fold improvement in sensor activation and engineer RADARS with diverse cargo proteins, including luciferases, fluorescent proteins, recombinases, and caspases, enabling detection sensitivity on endogenous transcripts expressed at levels as low as 13 transcripts per million. We show that RADARS are functional as either expressed DNA or synthetic mRNA and with either exogenous or endogenous ADAR. We apply RADARS in multiple contexts, including tracking transcriptional states, RNA-sensing-induced cell death, cell-type identification, and control of synthetic mRNA translation.

In vivo prime editing of a metabolic liver disease in mice.

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 1014 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label treatment.

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope.

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA.

Base editors are RNA-programmable deaminases that enable precise single-base conversions in genomic DNA. However, off-target activity is a concern in the potential use of base editors to treat genetic diseases. Here, we report unbiased analyses of transcriptome-wide and genome-wide off-target modifications effected by cytidine base editors in the liver of mice with phenylketonuria. The intravenous delivery of intein-split cytidine base editors by dual adeno-associated viruses led to the repair of the disease-causing mutation without generating off-target mutations in the RNA and DNA of the hepatocytes. Moreover, the transient expression of a cytidine base editor mRNA and a relevant single-guide RNA intravenously delivered by lipid nanoparticles led to ~21% on-target editing and to the reversal of the disease phenotype; there were also no detectable transcriptome-wide and genome-wide off-target edits. Our findings support the feasibility of therapeutic cytidine base editing to treat genetic liver diseases.

Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-ß Resistance.

Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-ß-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-ß resistance and attenuate TGF-ß target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.

Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

Growth of Epithelial Organoids in a Defined Hydrogel.

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME.