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Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a “protecting oligo”), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNAin vivo. Finally, we achievedin vivogenome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. High-throughput mutagenesis is emerging as a powerful tool to probe the varying consequences of different amino acid substitutions across the length of a protein or protein domain, however it is currently limited to specific functional readouts such as target protein abundance or functional assays. Studying the effects of genetic perturbations on cellular programs and fitness has been challenging using traditional pooled screens. Over the last few years, there has been a surge of interest in Perturb-seq style assays to measure the transcriptional consequences of genetic perturbations ranging from whole gene knockout to amino acid substitutions in single cells. While providing greater function insight, these sequencing-based methods are not yet scalable to exhaustive mutagenesis, necessitating selection of target mutations. In this work, we hypothesized that examining the consequences of perturbing distinct protein interactions could provide a useful abstraction of the phenotypic space reachable by individual amino acid substitutions. To explore this hypothesis, we employed a Perturb-seq style approach to generate mutations at physical interfaces of the transcription factor RUNX1, with the potential to perturb different interactions, and therefore produce transcriptional readouts implicating different aspects of the RUNX1 regulon. We analyzed these readouts to identify functionally distinct groups of RUNX1 mutations, characterize their effects on cellular programs and study the implications for cancer mutations. Our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions. Citation Format: Kivilcim Ozturk, Rebecca Panwala, Jeanna Sheen, Prashant Mali, Hannah Carter. Interface-guided phenotyping of coding variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB065.

Summary Gene fragments derived from structural domains mediating physical interactions can modulate biological functions. Utilizing this, we developed lentiviral overexpression libraries of peptides comprehensively tiling high-confidence cancer driver genes. Toward inhibiting cancer growth, we assayed ~66,000 peptides, tiling 65 cancer drivers and 579 mutant alleles. Pooled fitness screens in two breast cancer cell lines revealed peptides, which selectively reduced cellular proliferation, implicating oncogenic protein domains important for cell fitness. Coupling of cell-penetrating motifs to these peptides enabled drug-like function, with peptides derived from EGFR and RAF1 inhibiting cell growth at IC50s of 27–63 μM. We anticipate that this peptide-tiling (PepTile) approach will enable rapid de novo mapping of bioactive protein domains and associated interfering peptides.

CRISPR-Cas clinical trials have begun, offering a first glimpse at how DNA and RNA targeting could enable therapies for many genetic and epigenetic human diseases. The speedy progress of CRISPR-Cas from discovery and adoption to clinical use is built on decades of traditional gene therapy research and belies the multiple challenges that could derail the successful translation of these new modalities. Here, we review how CRISPR-Cas therapeutics are translated from technological systems to therapeutic modalities, paying particular attention to the therapeutic cascade from cargo to delivery vector, manufacturing, administration, pipelines, safety, and therapeutic target profiles. We also explore potential solutions to some of the obstacles facing successful CRISPR-Cas translation. We hope to illuminate how CRISPR-Cas is brought from the academic bench toward use in the clinic.