Base editors, including dual base editors, are innovative techniques for efficient base conversions in genomic DNA. However, the low efficiency of A-to-G base conversion at positions proximal to the protospacer adjacent motif (PAM) and... more
Base editors, including dual base editors, are innovative techniques for efficient base conversions in genomic DNA. However, the low efficiency of A-to-G base conversion at positions proximal to the protospacer adjacent motif (PAM) and the A/C simultaneous conversion of the dual base editor hinder their broad applications. In this study, through fusion of ABE8e with Rad51 DNA-binding domain, we generate a hyperactive ABE (hyABE) which offers improved A-to-G editing efficiency at the region (A10-A15) proximal to the PAM, with 1.2- to 7-fold improvement compared to ABE8e. Similarly, we develop optimized dual base editors (eA&C-BEmax and hyA&C-BEmax) with markedly improved simultaneous A/C conversion efficiency (1.2-fold and 1.5-fold improvement, respectively) compared to A&C-BEmax in human cells. Moreover, these optimized base editors catalyze efficiently nucleotide conversions in zebrafish embryos to mirror human syndrome or in human cells to potentially treat genetic diseases, indic...
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Research Interests:
Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved.... more
Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antise...
Research Interests: Genetics, Biology, Protein, Drug Discovery, Medicine, and 15 moreBiological Sciences, Model, CRISPR, Mutations, Proliferation, Cell, CAS, Genome, Gene, Applied Microbiology and Biotechnology, Zebrafish, Biochemistry and molecular biology, Genome Editing, insertional mutagenesis, and Medical and Health Sciences
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Research Interests:
Teleost zebrafish and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). Although many mitogenic signals that stimulate zebrafish heart... more
Teleost zebrafish and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). Although many mitogenic signals that stimulate zebrafish heart regeneration have been identified, transcriptional programs that restrain injury-induced CM renewal are incompletely understood. Here, we report that mutations in gridlock (grl; also known as hey2), encoding a Hairy-related basic helix-loop-helix transcriptional repressor, enhance CM proliferation and reduce fibrosis following damage. In contrast, myocardial grl induction blunts CM dedifferentiation and regenerative responses to heart injury. RNA sequencing analyses uncover Smyd2 lysine methyltransferase (KMT) as a key transcriptional target repressed by Grl. Reduction in Grl protein levels triggered by injury induces smyd2 expression at the wound myocardium, enhancing CM proliferation. We show that Smyd2 functions as a methyltransferase and modula...
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Heart regeneration occurs by dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). However, the signaling mechanisms by which injury induces CM renewal remain incompletely understood. Here, we find that cardiac injury... more
Heart regeneration occurs by dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). However, the signaling mechanisms by which injury induces CM renewal remain incompletely understood. Here, we find that cardiac injury in zebrafish induces expression of the secreted Wnt inhibitors, including Dickkopf 1 (Dkk1), Dkk3, secreted Frizzled-related protein 1 (sFrp1), and sFrp2, in cardiac tissue adjacent to injury sites. Experimental blocking of Wnt activity via Dkk1 overexpression enhances CM proliferation and heart regeneration, whereas ectopic activation of Wnt8 signaling blunts injury-induced CM dedifferentiation and proliferation. Although Wnt signaling is dampened upon injury, the cytoplasmic β-catenin is unexpectedly increased at disarrayed CM sarcomeres in myocardial wound edges. Our analyses indicated that p21-activated kinase 2 (Pak2) is induced at regenerating CMs, where it phosphorylates cytoplasmic β-catenin at Ser 675 and increases its stability at disassem...
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Pseudoxanthoma elasticum (PXE), caused by ABCC6/MRP6 mutation, is a heritable multisystem disorder in humans. The progressive clinical manifestations of PXE are accompanied by ectopic mineralization in various connective tissues. However,... more
Pseudoxanthoma elasticum (PXE), caused by ABCC6/MRP6 mutation, is a heritable multisystem disorder in humans. The progressive clinical manifestations of PXE are accompanied by ectopic mineralization in various connective tissues. However, the pathomechanisms underlying the PXE multisystem disorder remains obscure, and effective treatment is currently available. In this study, we generated zebrafish abcc6a mutants using the transcription activator-like effector nuclease (TALEN) technique. In young adult zebrafish, abcc6a is expressed in the eyes, heart, intestine, and other tissues. abcc6a mutants exhibit extensive calcification in the ocular sclera and Bruch’s membrane, recapitulating part of the PXE manifestations. Mutations in abcc6a upregulate extracellular matrix (ECM) genes, leading to fibrotic heart with reduced cardiomyocyte number. We found that abcc6a mutation reduced levels of both vitamin K and pyrophosphate (PPi) in the serum and diverse tissues. Vitamin K administration...