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Reporter-based screening identifies small-molecule CBL0137 as an enhancer of CRISPR cytosine base editor and prime editor via p53 activation and NF-κB inhibition.
Recently developed CRISPR base editors (BEs) and prime editors (PEs) enable precise genome editing without inducing double-strand breaks, making them highly promising tools for therapeutic applications. However, their efficiency remains a major barrier to clinical translation, particularly at difficult-to-target sites. To address this limitation, we used a high-throughput GFP reporter system responsive to cytosine base editor (CBE) activity to screen small molecules involved in DNA damage response, cell cycle, and apoptosis pathways. This screen identified CBL0137 as a candidate that significantly enhanced editing efficiency at both the reporter and endogenous target sites, with up to an 80 % improvement. Mechanistic studies revealed that CBL0137 acts through activation of the p53 pathway and inhibition of NF-κB. Interestingly, the enhancement was largely specific to CBEs, while in PEs, CBL0137 selectively improved multi-site mutations and fragment insertions without affecting single-point edits or deletions. Collectively, these results identify CBL0137 as a selective enhancer of specific genome editing activities, providing a strategy to increase editing efficiency and advancing the translational potential of next-generation genome editing tools for therapeutic applications.
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In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer.
Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.
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A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.
Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.
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Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing.
Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.
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Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces.
CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination (HR) ability. Here, a deaminase-assisted base editor dCas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9 (dCas9), the cytidine deaminase from Petromyzon marinus (PmCDA1), the uracil DNA glycosylase inhibitor (UGI) and the protein degradation tag (LVA tag). Using dCas9-CDA-ULstr, we achieved single-, double- and triple-point mutations (cytosine-to-thymine substitutions) at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-ULstr could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes (with high GC content). Collectively, the base editor dCas9-CDA-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the dCas9-CDA-ULstr-based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.
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Base editor screens for in situ mutational scanning at scale.
A fundamental challenge in biology is understanding the molecular details of protein function. How mutations alter protein activity, regulation, and response to drugs is of critical importance to human health. Recent years have seen the emergence of pooled base editor screens for in situ mutational scanning: the interrogation of protein sequence-function relationships by directly perturbing endogenous proteins in live cells. These studies have revealed the effects of disease-associated mutations, discovered novel drug resistance mechanisms, and generated biochemical insights into protein function. Here, we discuss how this "base editor scanning" approach has been applied to diverse biological questions, compare it with alternative techniques, and describe the emerging challenges that must be addressed to maximize its utility. Given its broad applicability toward profiling mutations across the proteome, base editor scanning promises to revolutionize the investigation of proteins in their native contexts.
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Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.
BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.
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Unlocking the secrets of ABEs: the molecular mechanism behind their specificity.
CRISPR-Cas, the bacterial immune systems, have transformed the field of genome editing by providing efficient, easily programmable, and accessible tools for targeted genome editing. DNA base editors (BE) are state-of-the-art CRISPR-based technology, allowing for targeted modifications of individual nucleobases within the genome. Among the BEs, adenine base editors (ABEs) have shown great potential due to their ability to convert A-to-G with high efficiency. However, current ABEs have limitations in terms of their specificity and targeting range. In this review, we provide an overview of the molecular mechanism of ABEs, with a focus on the mechanism of deoxyadenosine deamination by evolved tRNA-specific adenosine deaminase (TadA). We discuss how mutations and adjustments introduced via both directed evolution as well as rational design have improved ABE efficiency and specificity. This review offers insights into the molecular mechanism of ABEs, providing a roadmap for future developments in the precision genome editing field.
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Intravitreal adenine base editing of RS1 improves vision in a preclinical mouse model of retinoschisis.
Base editing offers high potential for treating genetic diseases, particularly those with limited treatment options. Retinoschisis, an X-linked retinal disease causing progressive vision loss, currently lacks effective therapies. We identified the c.422G>A (p.Arg141His) variant of the RS1 gene in six male patients with retinoschisis and generated a humanized mouse model harboring this variant, which mimicked the disease phenotype. By testing adenine base editors and single-guide RNAs, we identified an optimal combination of high editing efficiency and low bystander editing. Intravitreal injection of adeno-associated viral vectors encoding this adenine base editor achieved ∼40% editing efficiency in all retinal cells, restored retinal layer integrity, and preserved visual functions in 2-week-old male hemizygous mice. These mice exhibited retinal layer splitting at baseline, further validating the model. This study demonstrates a strategy for identifying effective base editing tools for clinical use through the preclinical evaluation of humanized mouse lines with patient-derived mutations and highlights their applicability in treating genetic diseases.
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Engineered hypercompact Fanzor-ωRNA system with enhanced genome editing activity.
As programmable RNA-guided DNA nucleases found in eukaryotes, Fanzors have shown promise in genome editing with their compact size. A wide variety of RuvC-containing Fanzor nucleases have been identified in various eukaryotes and their viruses. However, low editing efficiency limits the application of Fanzor in mammalian genome editing. In this study, we introduce SpuFz1 V4, an engineered RNA-guided DNA endonuclease with vigorous editing activity in the human genome. Furthermore, we demonstrate the substantial potential of the Fanzor system as a base editor. SpuFz1 V4 is currently the most active RNA-guided DNA nuclease of eukaryotic origin, belonging to the Fanzor1 family. Due to its compact size, SpuFz1 V4 can be efficiently delivered via a single adeno-associated virus (AAV) into the retina, achieving robust in vivo genome editing, which has the potential to be applied in both basic research and disease treatment applications.
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