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Base-editing mutagenesis maps alleles to tune human T cell functions.
CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.
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Development of multiplexed orthogonal base editor (MOBE) systems.
Base editors (BEs) enable efficient, programmable installation of point mutations while avoiding the use of double-strand breaks. Simultaneous application of two or more different BEs, such as an adenine BE (which converts A·T base pairs to G·C) and a cytosine BE (which converts C·G base pairs to T·A), is not feasible because guide RNA crosstalk results in non-orthogonal editing, with all BEs modifying all target loci. Here we engineer both adenine BEs and cytosine BEs that can be orthogonally multiplexed by using RNA aptamer-coat protein systems to recruit the DNA-modifying enzymes directly to the guide RNAs. We generate four multiplexed orthogonal BE systems that enable rates of precise co-occurring edits of up to 7.1% in the same DNA strand without enrichment or selection strategies. The addition of a fluorescent enrichment strategy increases co-occurring edit rates up to 24.8% in human cells. These systems are compatible with expanded protospacer adjacent motif and high-fidelity Cas9 variants, function well in multiple cell types, have equivalent or reduced off-target propensities compared with their parental systems and can model disease-relevant point mutation combinations.
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In Vivo Hematopoietic Stem Cell Genome Editing: Perspectives and Limitations.
The tremendous evolution of genome-editing tools in the last two decades has provided innovative and effective approaches for gene therapy of congenital and acquired diseases. Zinc-finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs) and CRISPR-Cas9 have been already applied by ex vivo hematopoietic stem cell (HSC) gene therapy in genetic diseases (i.e., Hemoglobinopathies, Fanconi anemia and hereditary Immunodeficiencies) as well as infectious diseases (i.e., HIV), and the recent development of CRISPR-Cas9-based systems using base and prime editors as well as epigenome editors has provided safer tools for gene therapy. The ex vivo approach for gene addition or editing of HSCs, however, is complex, invasive, technically challenging, costly and not free of toxicity. In vivo gene addition or editing promise to transform gene therapy from a highly sophisticated strategy to a "user-friendly' approach to eventually become a broadly available, highly accessible and potentially affordable treatment modality. In the present review article, based on the lessons gained by more than 3 decades of ex vivo HSC gene therapy, we discuss the concept, the tools, the progress made and the challenges to clinical translation of in vivo HSC gene editing.
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Base editors in the clinic.
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Off-target challenge for base editor-mediated genome editing.
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Bone-marrow-homing lipid nanoparticles for genome editing in diseased and malignant haematopoietic stem cells.
Therapeutic genome editing of haematopoietic stem cells (HSCs) would provide long-lasting treatments for multiple diseases. However, the in vivo delivery of genetic medicines to HSCs remains challenging, especially in diseased and malignant settings. Here we report on a series of bone-marrow-homing lipid nanoparticles that deliver mRNA to a broad group of at least 14 unique cell types in the bone marrow, including healthy and diseased HSCs, leukaemic stem cells, B cells, T cells, macrophages and leukaemia cells. CRISPR/Cas and base editing is achieved in a mouse model expressing human sickle cell disease phenotypes for potential foetal haemoglobin reactivation and conversion from sickle to non-sickle alleles. Bone-marrow-homing lipid nanoparticles were also able to achieve Cre-recombinase-mediated genetic deletion in bone-marrow-engrafted leukaemic stem cells and leukaemia cells. We show evidence that diverse cell types in the bone marrow niche can be edited using bone-marrow-homing lipid nanoparticles.
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Efficient allelic replacement in rice by gene editing: A case study of the NRT1.1B gene.
Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene-editing technology. No additional selective pressure was needed to enrich the precise replacement events. This work demonstrates the feasibility of replacing any genes with elite alleles within one generation, greatly expanding our ability to improve agriculturally important traits.
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Automated design of CRISPR prime editors for 56,000 human pathogenic variants.
Prime editors (PEs) are clustered regularly interspaced short palindromic repeats (CRISPR)-based genome engineering tools that can introduce precise base-pair edits. We developed an automated pipeline to correct (therapeutic editing) or introduce (disease modeling) human pathogenic variants from ClinVar that optimizes the design of several RNA constructs required for prime editing and avoids predicted off-targets in the human genome. However, using optimal PE design criteria, we find that only a small fraction of these pathogenic variants can be targeted. Through the use of alternative Cas9 enzymes and extended templates, we increase the number of targetable pathogenic variants from 32,000 to 56,000 variants and make these pre-designed PE constructs accessible through a web-based portal (http://primeedit.nygenome.org). Given the tremendous potential for therapeutic gene editing, we also assessed the possibility of developing universal PE constructs, finding that common genetic variants impact only a small minority of designed PEs.
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Editing Plant Genomes: a new era of crop improvement.
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Genome editing in the butterfly type-species Papilio machaon.
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