RESUMO
The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9's activity, the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by â¼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor's activity.
Assuntos
Proteínas de Bactérias , Sistemas CRISPR-Cas , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas/genética , DNA/metabolismo , Humanos , Aprendizado de Máquina , MutagêneseRESUMO
The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity.
Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes , Staphylococcus aureus , Sistemas CRISPR-Cas , Humanos , Nuclease do Micrococo/genética , RNA Guia de Cinetoplastídeos , Staphylococcus aureus/genéticaRESUMO
Systematic testing of existing drugs and their combinations is an attractive strategy to exploit approved drugs for repurposing and identifying the best actionable treatment options. To expedite the search among many possible drug combinations, we designed a combinatorial CRISPR-Cas9 screen to inhibit druggable targets. Coblockade of the N-methyl-d-aspartate receptor (NMDAR) with targets of first-line kinase inhibitors reduced hepatocellular carcinoma (HCC) cell growth. Clinically, HCC patients with low NMDAR1 expression showed better survival. The clinically approved NMDAR antagonist ifenprodil synergized with sorafenib to induce the unfolded protein response, trigger cell-cycle arrest, downregulate genes associated with WNT signaling and stemness, and reduce self-renewal ability of HCC cells. In multiple HCC patient-derived organoids and human tumor xenograft models, the drug combination, but neither single drug alone, markedly reduced tumor-initiating cancer cell frequency. Because ifenprodil has an established safety history for its use as a vasodilator in humans, our findings support the repurposing of this drug as an adjunct for HCC treatment to improve clinical outcome and reduce tumor recurrence. These results also validate an approach for readily discovering actionable combinations for cancer therapy. SIGNIFICANCE: Combinatorial CRISPR-Cas9 screening identifies actionable targets for HCC therapy, uncovering the potential of combining the clinically approved drugs ifenprodil and sorafenib as a new effective treatment regimen.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Piperidinas/administração & dosagem , Sorafenibe/administração & dosagem , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The CRISPR-Cas system coupled with Combinatorial Genetics En Masse (CombiGEM) enables systematic analysis of high-order genetic perturbations that are important for understanding biological processes and discovering therapeutic target combinations. Here, we present detailed steps and technical considerations for building multiplexed guide RNA libraries and carrying out a combinatorial CRISPR screen in mammalian cells. We also present an analytical pipeline, CombiPIPE, for mapping two- and three-way genetic interactions. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2020).
Assuntos
Sistemas CRISPR-Cas , Testes Genéticos , Animais , Linhagem Celular , Humanos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.
Assuntos
Sistemas CRISPR-Cas , Combinação de Medicamentos , Sistemas de Liberação de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Animais , Antineoplásicos/farmacologia , Drosophila melanogaster , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Doença de Parkinson/tratamento farmacológico , RNA Guia de CinetoplastídeosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.