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A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells.
BACKGROUND: Adenine base editors (ABE) enable single nucleotide modifications without the need for double-stranded DNA breaks (DSBs) induced by conventional CRIPSR/Cas9-based approaches. However, most approaches that employ ABEs require inefficient downstream technologies to identify desired targeted mutations within large populations of manipulated cells. In this study, we developed a fluorescence-based method, named "Cas9-mediated adenosine transient reporter for editing enrichment" (CasMAs-TREE; herein abbreviated XMAS-TREE), to facilitate the real-time identification of base-edited cell populations. RESULTS: To establish a fluorescent-based assay able to detect ABE activity within a cell in real time, we designed a construct encoding a mCherry fluorescent protein followed by a stop codon (TGA) preceding the coding sequence for a green fluorescent protein (GFP), allowing translational readthrough and expression of GFP after A-to-G conversion of the codon to "TGG." At several independent loci, we demonstrate that XMAS-TREE can be used for the highly efficient purification of targeted cells. Moreover, we demonstrate that XMAS-TREE can be employed in the context of multiplexed editing strategies to simultaneous modify several genomic loci. In addition, we employ XMAS-TREE to efficiently edit human pluripotent stem cells (hPSCs), a cell type traditionally resistant to genetic modification. Furthermore, we utilize XMAS-TREE to generate clonal isogenic hPSCs at target sites not editable using well-established reporter of transfection (RoT)-based strategies. CONCLUSION: We established a method to detect adenosine base-editing activity within a cell, which increases the efficiency of editing at multiple genomic locations through an enrichment of edited cells. In the future, XMAS-TREE will greatly accelerate the application of ABEs in biomedical research.
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Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.
CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.
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Cas12a Base Editors Induce Efficient and Specific Editing with Low DNA Damage Response.
The advent of base editors (BEs) holds great potential for correcting pathogenic-related point mutations to treat relevant diseases. However, Cas9 nickase (nCas9)-derived BEs lead to DNA double-strand breaks, which can trigger unwanted DNA damage response (DDR). Here, we show that the original version of catalytically dead Cas12a (dCas12a)-conjugated BEs induce a basal level of DNA breaks and minimally activate DDR proteins, including H2AX, ATM, ATR, and p53. By fusing dCas12a with engineered human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A), we further develop the BEACON (base editing induced by human APOBEC3A and Cas12a without DNA break) system to achieve enhanced deamination efficiency and editing specificity. Efficient C-to-T editing is achieved by BEACON in mammalian cells at levels comparable to AncBE4max, with only low levels of DDR and minimal RNA off-target mutations. Importantly, BEACON induces in vivo base editing in mouse embryos, and targeted C-to-T conversions are detected in F0 mice.
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In vivo base editing by a single i.v. vector injection for treatment of hemoglobinopathies.
Individuals with β-thalassemia or sickle cell disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% fetal hemoglobin (HbF) appear to be symptom free. Here, we used a nonintegrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113 A>G HPFH mutation in the γ-globin promoters in healthy CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only s.c. and i.v. injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in > 60% -113 A>G conversion, with 30% γ-globin of β-globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-Seq. In vitro, in HSCs from β-thalassemia and patients with sickle cell disease, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.
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Sequence Context-Agnostic TadA-Derived Cytosine Base Editors for Genome-Wide Editing in Zebrafish.
Single-nucleotide variants (SNVs) represent a significant form of genetic variation linked to various diseases. CRISPR-mediated base editing has emerged as a powerful method for modeling diseases caused by SNVs, particularly in zebrafish, which serve as an excellent platform for investigating disease mechanisms and conducting drug screenings. However, existing cytosine base editors (CBEs) for zebrafish often have broad editing windows and strong sequence preferences, limiting their effectiveness. In this study, zebrafish (z) TadA-derived cytosine base editors, termed zTadA-CBEs, are developed by introducing key mutations into the TadA8e enzyme. These novel editors demonstrate improved efficiency and precision in cytosine base editing. Specifically, zTadA-BE4max and zTadA-BEmv offer complementary editing windows, while zTadA-SpRY-BE4max allows for PAM-flexible editing. Using zTadA-CBEs, a precise disease model for Axenfeld-Rieger syndrome is established, and created two new models for Hermansky-Pudlak syndrome. Additionally, a novel albinism model carrying two pathogenic SNVs in the F0 generation is developed. By employing specifically designed sgRNA, the fmsts± missense mutation is corrected back to the wild-type nucleotide (C > T), successfully restoring macrophage levels to normal. These findings underscore the potential of zTadA-CBEs to enhance genome editing techniques and their applications in developing therapies for SNV-related disorders.
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Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces.
Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces. Of note, eSCBE3-NG-Hypa recognizes NG PAM and exhibits high activity at challenging sites with high GC content or GC motifs, while displaying minimal off-target effects. To illustrate its practicability, we employ eSCBE3-NG-Hypa to achieve precise key amino acid conversion of the dehydratase (DH) domains within the modular polyketide synthase (PKS) responsible for the insecticide avermectins biosynthesis, achieving domains inactivation. The resulting DH-inactivated mutants, while ceasing avermectins production, produce a high yield of oligomycin, indicating competitive relationships among multiple biosynthetic gene clusters (BGCs) in Streptomyces avermitilis. Leveraging this insight, we use eSCBE3-NG-Hypa to introduce premature stop codons into competitor gene cluster of ave in an industrial S. avermitilis, with the mutant Δolm exhibiting the highest 4.45-fold increase in avermectin B1a compared to the control. This work provides a potent tool for modifying biosynthetic pathways and advancing metabolic engineering in Streptomyces.
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A simplified RUBY reporter-enabled hairy-root system for rapid evaluation of genome editing and gene function in grapevine.
The simplified RUBY represents an efficient reporter for monitoring plant transformation and, when combined with hairy-root system in grapevine, can facilitate the application of novel CRISPR technologies and gene functional study. Monitoring successful transformation events is essential for plant transformation and genome editing. The development and application of the RUBY reporter enable effective selection of transformation events based solely on distinct red pigmentation. Here, we report that a simplified version of RUBY (siRUBY), lacking the glucosyltransferase gene, also functions effectively as a visual selection marker for plant transformation. Furthermore, the siRUBY-assisted hairy-root system was established as a rapid and efficient platform for evaluating activity of adenine base editor (ABE) in grapevine. Targeted A-to-G base editing was achieved using ABE8e, with an average efficiency of approximately 14%. Moreover, this platform is also suitable for functional investigation of genes of interest. Knockout of the MYB4a gene promoted lignin deposition, likely through upregulating key lignin biosynthesis genes while repressing transcription of downstream flavonoid biosynthesis genes. Collectively, these results demonstrate that siRUBY coupled with grapevine hairy roots provides a robust platform for rapid and efficient genome editing in grapevine.
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Genome-edited allogeneic CAR-T cells: the next generation of cancer immunotherapies.
Chimeric Antigen Receptor T (CAR-T) cell therapy has revolutionized cancer immunotherapy, particularly in hematological malignancies. However, the clinical application of autologous CAR-T cells faces significant high cost and manufacturing challenges. Universal allogeneic CAR-T cells, derived from healthy donors, represent a promising solution to these obstacles. These "off-the-shelf" therapies aim to reduce the complexity and cost of CAR-T production. Despite exciting advancements in genome-editing technologies and promising clinical trial data, significant challenges remain, including graft-versus-host disease (GVHD), Host-versus-graft reaction (HVGR), off-target effects, genotoxicity, and manufacturing scalability. To address these concerns, genome-editing technologies such as ZFNs, TALENs, Meganucleases, CRISPR systems, base editing, and prime editing are being employed. This review summarizes the progress of universal allogeneic CAR-T cell therapies, addresses the critical challenges, and discusses the future directions for their clinical implementation.
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A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC-Cas9 or cleavage by Cas9 in living cells.
Base editing is an exciting new genome engineering technology. C-to-T mutations in genomic DNA have been achieved using ribonucleoprotein complexes comprised of rat APOBEC1 single-stranded DNA deaminase, Cas9 nickase (Cas9n), uracil DNA glycosylase inhibitor (UGI), and guide (g)RNA. Here, we report the first real-time reporter system for quantification of APOBEC-mediated base editing activity in living mammalian cells. The reporter expresses eGFP constitutively as a marker for transfection or transduction, and editing restores functionality of an upstream mCherry cassette through the simultaneous processing of two gRNA binding regions that each contain an APOBEC-preferred 5'TCA target site. Using this system as both an episomal and a chromosomal editing reporter, we show that human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more efficient than the original rat APOBEC1-Cas9n-UGI construct. We also demonstrate coincident enrichment of editing events at a heterologous chromosomal locus in reporter-edited, mCherry-positive cells. The mCherry reporter also quantifies the double-stranded DNA cleavage activity of Cas9, and may therefore be adaptable for use with many different CRISPR systems. The combination of a rapid, fluorescence-based editing reporter system and more efficient, structurally defined DNA editing enzymes broadens the versatility of the rapidly expanding toolbox of genome editing and engineering technologies.
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Structure and engineering of Brevibacillus laterosporus Cas9.
The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.
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