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Perspectives on the Application of Genome-Editing Technologies in Crop Breeding.
Most conventional and modern crop-improvement methods exploit natural or artificially induced genetic variations and require laborious characterization of the progenies of multiple generations derived from time-consuming genetic crosses. Genome-editing systems, in contrast, provide the means to rapidly modify genomes in a precise and predictable way, making it possible to introduce improvements directly into elite varieties. Here, we describe the range of applications available to agricultural researchers using existing genome-editing tools. In addition to providing examples of genome-editing applications in crop breeding, we discuss the technical and social challenges faced by breeders using genome-editing tools for crop improvement.
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Advancing gene targeting for primary immune deficiencies: Adenine base editing of the human IL2RG locus for correction of SCID-X1.
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Programmable gene modulation networks for Parkinson's disease using nanotechnology enabled CRISPR/Cas brain delivery.
Parkinson's disease is a progressive neurodegenerative disorder driven by interconnected molecular pathways, including α-synuclein aggregation, mitochondrial dysfunction, impaired proteostasis, and neuroinflammation. Current therapies are primarily symptomatic and have not consistently demonstrated prevention of disease progression. This review introduces Programmable gene modulation networks, a systems-level framework that integrates CRISPR/Cas technologies with nanotechnology-enabled brain delivery for precision intervention in Parkinson's disease. Advanced CRISPR modalities, including CRISPR interference, activation, base editing, prime editing, and epigenetic editing, are evaluated for reversible and targeted modulation of disease-relevant gene networks. Non-viral nanocarrier platforms, such as lipid nanoparticles, polymeric systems, and exosome-mimetic vesicles, are discussed for overcoming blood-brain barrier limitations and improving brain-specific delivery. The review further emphasizes translational challenges, including delivery efficiency, off-target effects, long-term safety, manufacturing scalability, and regulatory considerations. By integrating molecular network biology, programmable gene regulation, and translational decision-making, this review provides a roadmap for developing next-generation disease-modifying therapies for Parkinson's disease.
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Functional interrogation of DNA damage response variants with base editing screens.
Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.
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Precision genome editing using cytosine and adenine base editors in mammalian cells.
Genome editing has transformed the life sciences and has exciting prospects for use in treating genetic diseases. Our laboratory developed base editing to enable precise and efficient genome editing while minimizing undesired byproducts and toxicity associated with double-stranded DNA breaks. Adenine and cytosine base editors mediate targeted A•T-to-G•C or C•G-to-T•A base pair changes, respectively, which can theoretically address most human disease-associated single-nucleotide polymorphisms. Current base editors can achieve high editing efficiencies-for example, approaching 100% in cultured mammalian cells or 70% in adult mouse neurons in vivo. Since their initial description, a large set of base editor variants have been developed with different on-target and off-target editing characteristics. Here, we describe a protocol for using base editing in cultured mammalian cells. We provide guidelines for choosing target sites, appropriate base editor variants and delivery strategies to best suit a desired application. We further describe standard base-editing experiments in HEK293T cells, along with computational analysis of base-editing outcomes using CRISPResso2. Beginning with target DNA site selection, base-editing experiments in mammalian cells can typically be completed within 1-3 weeks and require only standard molecular biology techniques and readily available plasmid constructs.
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AlleleProfileR: A versatile tool to identify and profile sequence variants in edited genomes.
Gene editing strategies, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9), are revolutionizing biology. However, quantitative and sensitive detection of targeted mutations are required to evaluate and quantify the genome editing outcomes. Here we present AlleleProfileR, a new analysis tool, written in a combination of R and C++, with the ability to batch process the sequence analysis of large and complex genome editing experiments, including the recently developed base editing technologies.
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A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants.
CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.
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Genome editor-directed in vivo library diversification.
The generation of a library of variant genes is a prerequisite of directed evolution, a powerful tool for biomolecular engineering. As the number of all possible sequences often far exceeds the diversity of a practical library, methods that allow efficient library diversification in living cells are essential for in vivo directed evolution technologies to effectively sample the sequence space and allow hits to emerge. While traditional whole-genome mutagenesis often results in toxicity and the emergence of "cheater" mutations, recent developments that exploit the targeting and editing abilities of genome editors to facilitate in vivo library diversification have allowed for precise mutagenesis focused on specific genes of interest, higher mutational density, and reduced the occurrence of cheater mutations. This minireview summarizes recent advances in genome editor-directed in vivo library diversification and provides an outlook on their future applications in chemical biology.
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Efficient C-to-G Base Editing with Improved Target Compatibility Using Engineered Deaminase-nCas9 Fusions.
CRISPR-guided DNA base editors (BEs) are potent genome editing tools in biotechnology and medicine. However, conventional cytosine and adenine BEs can only induce base transitions (C-to-T and A-to-G) and cannot induce base transversions. Recently, several C-to-G base editors (CGBEs) were generated and applied in human cells. By comparing them, we found that engineered deaminases rather than additional base excision repair proteins significantly improved the C-to-G efficiency. In addition, significant increase in C-to-G transversions in the GC context were determined by using rationally engineered eAID deaminase. The genome-targeting scope of CGBEs were further expanded by using SpRY Cas9 variant, which then successfully induced stop codon (TAC to TAG) to disrupt Tyr gene in mouse embryos. Taken together, these new CGBEs with engineered deaminase-nCas9 fusions broaden the BE toolsets for efficient gene modification and therapeutic applications.
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Photoactivatable base editors for spatiotemporally controlled genome editing in vivo.
CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.
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