Search

Although liver-humanized animals are desirable tools for drug development and expansion of human hepatocytes in large quantities, their development is restricted to mice. In animals larger than mice, a precondition for efficient liver humanization remains preliminary because of different xeno-repopulation kinetics in livers of larger sizes. Since rats are ten times larger than mice and widely used in pharmacological studies, liver-humanized rats are more preferable. Here, Fah -/- Rag2 -/- IL2rg -/- (FRG) rats are generated by CRISPR/Cas9, showing accelerated liver failure and lagged liver xeno-repopulation compared to FRG mice. A survival-assured liver injury preconditioning (SALIC) protocol, which consists of retrorsine pretreatment and cycling 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) administration by defined concentrations and time intervals, is developed to reduce the mortality of FRG rats and induce a regenerative microenvironment for xeno-repopulation. Human hepatocyte repopulation is boosted to 31 ± 4% in rat livers at 7 months after transplantation, equivalent to approximately a 1200-fold expansion. Human liver features of transcriptome and zonation are reproduced in humanized rats. Remarkably, they provide sufficient samples for the pharmacokinetic profiling of human-specific metabolites. This model is thus preferred for pharmacological studies and human hepatocyte production. SALIC may also be informative to hepatocyte transplantation in other large-sized species., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Reactivation of fetal haemoglobin (HbF) expression is an effective way to treat β-thalassaemia and sickle cell anaemia. In the present study, we identified a novel GATA zinc finger domain-containing protein 2A (GATAD2A) mutation, which contributed to the elevation of HbF and ameliorated clinical severity in a patient with β-thalassaemia, by targeted next-generation sequencing. Knockout of GATAD2A led to a significant induction of HbF in both human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) and human cluster of differentiation (CD)34 + cells with a detectable impact on erythroid differentiation. Furthermore, heterozygous knockout of GATAD2A impaired recruitment of chromodomain helicase DNA-binding protein 4 (CHD4) to the methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex. Our present data suggest that mutations causing the haploinsufficiency of GATAD2A might contribute to amelioration of clinical severity in patients with β-thalassaemia., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of β-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.

SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.

The zinc-finger motif found in many transcription factors is thought to be important for human heart development and diseases. In this study, we have identified and characterized a novel zinc-finger gene named ZNF480 using degenerate primers from an early human embryo heart cDNA library. ZNF480 contains a KRAB-A box and 12 C2H2 zinc fingers. The cDNA sequence contains an open reading frame of 1551 bp, encoding a putative protein of 516 amino acid residues with a predicted molecular mass of 57 kDa. Northern blot analysis indicates that a 4.7kb transcript specific for ZNF480 is expressed only in embryonic heart. In the adult tissues, the expression of ZNF480 is restricted largely to heart, skeletal muscle, pancreas, and placenta. Overexpression of ZNF480 in cells activates the transcriptional activities of AP-1 and SRE. Therefore, our data suggest that ZNF480 may act as a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE.

Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner. The zinc-finger-containing transcription factor has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Mitogen-activated protein kinase (MAPK) signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation. The MAPKs function inside the nucleus and target transcription factors that are prebound to DNA. Many transcription factors are probably important MAPK targets. Here, we have cloned a new zinc-finger gene named ZNF411 using degenerate primers from an early embryo heart cDNA library, which mapped to 19p13.11. The ZNF411 gene consists of 2360 nucleotides and encodes a protein of 499 amino acids with an amino-terminal KRAB domain and eleven carboxy-terminal C2H2 zinc-finger units. Northern blot analysis indicates that a 2.4 kb transcript specific for ZNF411 is expressed in heart, skeletal muscle, and placenta at adult stage and is expressed in most of the examined embryonic tissues, especially at a higher level in skeletal muscle, heart, and pancreas. ZNF411 protein distributes evenly in nuclei when overexpressed in the cells. Reporter gene assays show that ZNF411 is a transcriptional repressor and overexpression of ZNF411 in the COS-7 cells inhibits the transcriptional activities of AP-1 and SRE. These results indicate that ZNF411 is a member of the zinc-finger transcription factor family and may be involved in the heart development, and it probably works as a negative regulator in MAPK signaling pathway.

The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications. [Display omitted] • Engineered IscB and ωRNA significantly increase programmable DNA cleavage activity • Fusion of DNA deaminases with eIscB-D efficiently induces base conversions • eIscB-D/eωRNA enables robust DNA editing in mouse embryos to generate disease models Xue et al. develop a highly efficient, miniature genome-editing system through extensive engineering of ωRNA and IscB nuclease via amino acid substitutions and HMG-D domain fusion. eIscB-D can be adapted to base editors to achieve robust A-to-G or C-to-T conversion, and it is highly efficient in mouse embryos to generate disease models. [ABSTRACT FROM AUTHOR]