The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is ...unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance.
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•BET inhibitors release the Mediator complex from specific enhancers and promoters•Mediator eviction correlates with transcriptional changes caused by JQ1•Genetic knockdown of specific Mediator subunits phenocopies BRD4 inhibition•BRD4 and Mediator maintain expression of a common gene regulatory network
In this study, Bhagwat et al. show that the Mediator complex and BRD4 are linked coactivators that support gene-specific transcriptional activation in leukemia cells. They provide evidence that small-molecule inhibitors of BRD4 exert anti-leukemia effects by interfering with Mediator function to suppress transcription.
Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the ...deaminase superfamily, members of which have found application in gene-editing techniques
. Because previously described cytidine deaminases operate on single-stranded nucleic acids
, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria
. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases
.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.
Biomolecule‐templated or biotemplated metal nanoclusters (NCs) are ultrasmall (<2 nm) metal (Au, Ag) particles stabilized by a certain type of biomolecular template (e.g., peptides, proteins, and ...DNA). Due to their unique physiochemical properties, biotemplated metal NCs have been widely used in sensing, imaging, delivery and therapy. The overwhelming applications in these individual areas imply the great promise of harnessing biotemplated metal NCs in more advanced biomedical aspects such as theranostics. Although applications of biotemplated metal NCs as theranostic agents are trending, the rational design of biomolecular templates suitable for the synthesis of multifunctional metal NCs for theranostics is comparatively underexplored. This progress report first identifies the essential attributes of biotemplated metal NCs for theranostics by reviewing the state‐of‐art applications in each of the four modalities of theranostics, namely sensing, imaging, delivery and therapy. To achieve high efficacy in these modalities, we elucidate the design principles underlying the use of biomolecules (proteins, peptides and nucleic acids) to control the NC size, emission color and surface chemistries for post‐functionalization of therapeutic moieties. We then propose a unified strategy to engineer biomolecular templates that combine all these modalities to produce multifunctional biotemplated metal NCs that can serve as the next‐generation personalized theranostic agents.
This progress report reviews the state‐of‐the‐art applications of biotemplated metal nanoclusters (NCs) in imaging, sensing, delivery and therapeutics, highlights the prerequisites of biotemplates in each modality of theranostics, and proposes a systematic framework to rationally design customizable biotemplates that incorporate multiple functionalities into a single NC for the development of next‐generation, personalized nanomedicine.
Although base editors are widely used to install targeted point mutations, the factors that determine base editing outcomes are not well understood. We characterized sequence-activity relationships ...of 11 cytosine and adenine base editors (CBEs and ABEs) on 38,538 genomically integrated targets in mammalian cells and used the resulting outcomes to train BE-Hive, a machine learning model that accurately predicts base editing genotypic outcomes (R ≈ 0.9) and efficiency (R ≈ 0.7). We corrected 3,388 disease-associated SNVs with ≥90% precision, including 675 alleles with bystander nucleotides that BE-Hive correctly predicted would not be edited. We discovered determinants of previously unpredictable C-to-G, or C-to-A editing and used these discoveries to correct coding sequences of 174 pathogenic transversion SNVs with ≥90% precision. Finally, we used insights from BE-Hive to engineer novel CBE variants that modulate editing outcomes. These discoveries illuminate base editing, enable editing at previously intractable targets, and provide new base editors with improved editing capabilities.
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•Base editing outcome precision and efficiency are frequently unintuitive•Machine learning model (BE-Hive) accurately predicts base editing efficiency and editing patterns•Base editor engineering can increase and reduce aberrant transversion editing•We precisely correct 3,388 pathogenic SNVs, many previously considered intractable
A comprehensive look at CRISPR base editing efficiencies and outcomes across target sequences, cell lines, and base editing effectors yields machine learning models and a web-based tool for users to predict the editing efficiency, bystander edits, and the best base editor to use for a sequence of interest.
Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product ...purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles. We characterized ten promising CGBEs on a library of 10,638 genomically integrated target sites in mammalian cells and trained machine learning models that accurately predict the purity and yield of editing outcomes (R = 0.90) using these data. These CGBEs enable correction to the wild-type coding sequence of 546 disease-related transversion single-nucleotide variants (SNVs) with >90% precision (mean 96%) and up to 70% efficiency (mean 14%). Computational prediction of optimal CGBE-single-guide RNA pairs enables high-purity transversion base editing at over fourfold more target sites than achieved using any single CGBE variant.
The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule ...inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.
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•Developed high-throughput assays for SpCas9 and performed a small-molecule screen•Identified reversible and cell-permeable inhibitors that disrupt SpCas9-DNA binding•Inhibitors allow dose and temporal control of (non)-nuclease-based SpCas9 systems•Identified a pharmacophore for SpCas9 inhibition using structure-activity studies
A suite of high-throughput assays enables discovery of small-molecule inhibitors of CRISPR-Cas9 that are cell permeable, and non-toxic, providing a chemical means to control SpCas9-based tools.
The ability to convert a target nucleotide sequence into any desired nucleotide sequence has been a longstanding goal in genome editing. RNA-guided CRISPR-Cas systems have transformed this field ...because genome editing agents could now be directed to almost any target sequence by simply varying the choice of a guide RNA. CRISPR-Cas systems have since been engineered extensively by our lab and others to perform a myriad of precise DNA modifications, including introduction of a single base pair change using base editing, and performing targeted insertion, deletion and multiple base pair replacement using prime editing. Apart from the nuclear genome, the mitochondrion contains its own genome that encodes for proteins and RNAs critical for energy production. Pathogenic point mutations in the mitochondrial genome (mtDNA) have been identified for mitochondrial disorders including MELAS, LHON, MERRF and Leigh’s disease. Thousands of somatic mtDNA mutations remain uncharacterized for their association with human diseases and ageing. Given the importance of the mtDNA in human health, there is a critical need to develop tools that enable precise mtDNA manipulation. While base editors and prime editors have been shown to edit the nuclear DNA in living cells with high efficiencies, the challenge of RNA delivery to the mitochondrial matrix have precluded the use of CRISPR-based systems for mtDNA engineering. This dissertation seeks to address the challenge of precision mtDNA editing. We developed a CRISPR-free mitochondrial base editor (DdCBE) that enables the first precise C•G to T•A base pair conversion within the mtDNA. DdCBE contains a bacterial deaminase toxin, DddA, that exhibits unprecedented double-stranded DNA cytidine deaminase activity. We engineered non-toxic split halves of DddA, then fused them to programmable DNA-binding TALE array proteins to reassemble active DddA at target DNA site, resulting in efficient and sequence-specific base editing in both human mtDNA and nuclear DNA. Next, we used laboratory evolution to generate DdCBE variants that result in improved activity and expanded targeting scope. The canonical DdCBE showed modest editing efficiencies at selected mtDNA sites and was limited to editing cytosines in a TC context. Using phage-assisted continuous evolution (PACE), we evolved DdCBEs for higher editing activity at TC and non-TC targets. Compared to canonical DdCBEs containing wild-type DddA, those with DddA6 improved mtDNA base editing efficiencies at TC by an average of 3.3-fold across nine tested loci. DdCBEs containing DddA11 offered substantially broadened HC (H = A, C, or T) target sequence context compatibility for both mitochondrial and nuclear base editing. We next used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites, resulting in cells with impaired oxidative phosphorylation and reduced respiration rates. Finally, we observed a modest increase in average mitochondrial genome off-target editing associated with DddA6 and DddA11 for specific TALE designs, but overall ratios of on-target: off-target editing efficiencies of evolved DddA variants were comparable to those of canonical DdCBE. DdCBE enables the installation of disease-associated mtDNA mutations in human cells lines and animal models to accelerate preclinical research. Its potential as a future therapeutic for debilitating mitochondrial disorders may be realized with further developments and innovations.
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