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DNA base editing corrects common hemophilia A mutations and restores factor VIII expression in in vitro and ex vivo models.
BACKGROUND: Replacement and nonreplacement therapies effectively control bleeding in hemophilia A (HA) but imply lifelong interventions. Authorized gene addition therapy could provide a cure but still poses questions on durability. FVIIIgene correction would definitively restore factor (F)VIII production, as shown in animal models through nuclease-mediated homologous recombination (HR). However, low efficiency and potential off-target double-strand break still limit HR translatability. OBJECTIVES: To correct common model single point mutations leading to severe HA through the recently developed double-strand break/HR-independent base editing (BE) and prime editing (PE) approaches. METHODS: Screening for efficacy of BE/PE systems in HEK293T cells transiently expressing FVIII variants and validation at DNA (sequencing) and protein (enzyme-linked immunosorbent assay; activated partial thromboplastin time) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells by lentiviral-mediated delivery of BE. RESULTS: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of wild-type recombinant FVIII) for the p.R2166∗ and p.R2228Q mutations. In stable clones, we demonstrated that the mutation reversion on DNA (∼24%) was consistent with the rescue of FVIII secretion and activity of 20% to 30%. The lentiviral-mediated delivery of the selected BE systems was attempted in engineered blood outgrowth endothelial cells harboring the p.R2166∗ and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII. CONCLUSION: Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients through a single intervention.
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Multiplex precise base editing in cynomolgus monkeys.
Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.
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Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST.
Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide-resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.
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Harnessing artificial intelligence to advance CRISPR-based genome editing technologies.
CRISPR-based genome editing technologies, including nuclease-based editing, base editing and prime editing, have revolutionized biological research and modern medicine by enabling precise, programmable modification of the genome and offering new therapeutic strategies for a wide range of genetic diseases. Artificial intelligence (AI), including machine learning and deep learning models, is now further advancing the field by accelerating the optimization of gene editors for diverse targets, guiding the engineering of existing tools and supporting the discovery of novel genome-editing enzymes. In this Review, we summarize key AI methodologies underlying these advances and discuss their recent noteworthy applications to genome editing technologies. We also discuss emerging opportunities, such as AI-powered virtual cell models, which can guide genome editing through target selection or prediction of functional outcomes. Finally, we identify key directions where the integration of AI methods is poised to have a substantial impact going forward.
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Improved RNA base editing with guide RNAs mimicking highly edited endogenous ADAR substrates.
Adenosine deaminase acting on RNA (ADAR)-mediated RNA base editing offers a safer alternative to genome editing for specific clinical applications because of nonpermanent editing of targets. Current guide RNA (gRNA) designs feature a fully complementary specificity domain with an A-C mismatch at the targeted adenosine. However, perfectly matched dsRNA is not the most effective ADAR substrate. Here we introduce MIRROR (mimicking inverted repeats to recruit ADARs using engineered oligoribonucleotides), an approach that implements structural motifs derived from highly edited inverted Alu repeats in human tissues to enable rational gRNA design for ADAR recruitment. We demonstrated that MIRROR is applicable to both short chemically synthesized gRNAs with modifications and long biologically generated gRNAs and surpasses current state-of-the-art approaches in both gRNA forms. It enhances editing efficiency by up to 5.7-fold in multiple human cell types and primary hepatocytes from an alpha-1 antitrypsin deficiency mouse model. Our findings improve programmable RNA editing in vitro and in vivo by rational design through the screening of highly edited natural substrate mimics.
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Expanded targeting scope and enhanced base editing efficiency in rabbit using optimized xCas9(3.7).
Evolved xCas9(3.7) variant with broad PAM compatibility has been reported in cell lines, while its editing efficiency was site-specific. Here, we show that xCas9(3.7) can recognize a broad PAMs including NGG, NGA, and NGT, in both embryos and Founder (F0) rabbits. Furthermore, the codon-optimized xCas9-derived base editors, exBE4 and exABE, can dramatically improve the base editing efficiencies in rabbit embryos. Our results demonstrated that the optimized xCas9 with expanded PAM compatibility and enhanced base editing efficiency could be used for precise gene modifications in organisms.
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In vivo application of base and prime editing to treat inherited retinal diseases.
Inherited retinal diseases (IRDs) are vision-threatening retinal disorders caused by pathogenic variants of genes related to visual functions. Genomic analyses in patients with IRDs have revealed pathogenic variants which affect vision. However, treatment options for IRDs are limited to nutritional supplements regardless of genetic variants or gene-targeting approaches based on antisense oligonucleotides and adeno-associated virus vectors limited to targeting few genes. Genome editing, particularly that involving clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies, can correct pathogenic variants and provide additional treatment opportunities. Recently developed base and prime editing platforms based on CRISPR-Cas9 technologies are promising for therapeutic genome editing because they do not employ double-stranded breaks (DSBs), which are associated with P53 activation, large deletions, and chromosomal translocations. Instead, using attached deaminases and reverse transcriptases, base and prime editing efficiently induces specific base substitutions and intended genetic changes (substitutions, deletions, or insertions), respectively, without DSBs. In this review, we will discuss the recent in vivo application of CRISPR-Cas9 technologies, focusing on base and prime editing, in animal models of IRDs.
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Precision Medicine Trials in Retinal Degenerations.
The beginning of the twenty-first century was marked by the innovative use of pharmacochemical interventions, which have since expanded to include gene-based molecular therapies. For years, treatment has focused on tackling the pathophysiology of monogenic orphan diseases, and one of the first applications of these novel genome editing technologies was the treatment of rare inherited retinal dystrophies. In this review, we present recent, ongoing, and future gene therapy-based treatment trials for choroideremia, X-linked retinitis pigmentosa, Stargardt disease, and age-related macular degeneration. As these trials pave the way toward halting the progression of such devastating diseases, we will begin to see the exciting development of newer, cutting-edge strategies including base editing and prime editing, ushering in a new era of precision medicine.
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Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor.
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Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis.
Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently available genome-editing methods for P. chlororaphis are based on homologous recombination (HR)-dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ-Red-like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time-consuming, laborious and inefficient. Here, we established a CRISPR-assisted base-editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9ppD10A ) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO-inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR-based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non-traditional bacterial hosts.
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