Chonghua Ren

• Professor
• Deputy Director of the Institute of Insect Science and Technology at South China Normal University

Inter-organelle communication coordinates cellular homeostasis and function. Juvenile hormone (JH) is produced in the corpora allata (CA) and acts as a gonadotrophic hormone in most insects. Using transcriptomic, biochemical, molecular, and genetic analyses, here we investigated the underlying mechanism how inter-organelle communication dynamically...

Appendage regeneration occurs within the opaque exoskeleton in arthropods, making it challenging to visualize the regenerative processes dynamically. In this protocol, we present a strategy to scan and capture the high-resolution details of microstructural tissues at certain regeneration points through micro-computed tomography (micro-CT). We descr...

Appendage regeneration relies on the formation of blastema, a heterogeneous cellular structure formed at the injury site. However, little is known about the early injury-activated signaling pathways that trigger blastema formation during appendage regeneration. Here, we provide compelling evidence that the extracellular signal–regulated kinase (ERK...

DNA methylation at the fifth position of cytosine (5-methylcytosine, 5mC) is a crucial epigenetic modification for regulating gene expression, but little is known about how it regulates gene expression in insects. Here, we pursue the detailed molecular mechanism by which DNMT1-mediated 5mC maintenance regulates female reproduction in the German coc...

The mystery of appendage regeneration has fascinated humans for centuries, while the regulatory mechanisms remain unclear. In this study, a transcriptional landscape of regenerating leg was established in the American cockroach, Periplaneta americana, an ideal model for appendage regeneration with remarkable regeneration capacity. Through a large-s...

Blattodea, which includes cockroaches and termites, possesses high developmental plasticity that is mainly controlled by nutritional conditions and insect hormones. Insulin/insulin-like growth factor signaling (IIS), target of rapamycin complex 1 (TORC1), and AMP-activated protein complex (AMPK) are three primary nutrition-responsive signals. Juven...

Regeneration, as a fascinating scientific field, refers to the ability of animals replacing lost tissue or body parts. Many metazoan organisms have been reported with the regeneration phenomena, but showing evolutionarily variable abilities. As the most diverse metazoan taxon, hundreds of insects show strong appendage regeneration ability. The rege...

Insects have evolved numerous adaptations and colonized diverse terrestrial environments. Several polyneopterans, including dictyopterans (cockroaches and mantids) and locusts, have developed oothecae, but little is known about the molecular mechanism, physiological function, and evolutionary significance of ootheca formation. Here, we demonstrate...

Tissue regeneration and wound healing are still serious clinical complications globally and lack satisfactory cures. Inspired by the impressive regeneration ability of the post-injury earthworms and their widely accepted medicinal properties, we screened and identified a novel collagen-like peptide from the amputated earthworms using high-throughpu...

Vitellogenesis, including vitellogenin (Vg) production in the fat body and Vg uptake by maturing oocytes, is of great importance for the successful reproduction of adult females. The endocrinal and nutritional regulation of vitellogenesis differs distinctly in insects. Here, the complex crosstalk between juvenile hormone (JH) and the two nutrient s...

Vitellogenesis, including vitellogenin (Vg) production in the fat body and Vg uptake by maturing oocytes, is of great importance for the successful reproduction of adult females. The endocrinal and nutritional regulation of vitellogenesis differs distinctly in insects. Here, the complex crosstalk between juvenile hormone (JH) and the two nutrient s...

The transcription factor grainy head (Grh) functions in the protection of the epithelium against the external environment by generating strongly adhesive layers, and this function is conserved in vertebrates and invertebrates. In Drosophila, the top model for holometabolous insects, Grh is necessary during embryonic development, epidermal different...

In insects, nutrition and hormones coordinately regulate lifespan and reproduction, which might affect each other. We here investigated how nutrition, insulin, and juvenile hormone (JH) signal genes affect lifespan and reproduction in the German cockroach, Blattella germanica, a serious urban pest throughout the world. Starvation as well as alterin...

Using engineered nucleases such as zinc-finger nucleases (ZFNs) and TALE nuclease (TALEN) to accomplish genome editing often causes high cellular toxicity because of the consistent expression of artificial nucleases and off-targeting effect. And lacking selection marker in modified cells makes it hard to enrich these positive cells. Here we introdu...

Artificial transcription factors based on zinc finger, TALE, and CRISPR/Cas9 programmable DNA-binding platforms have been widely used to regulate the expression of specific genes in cultured cells, but their delivery into organs such as the brain represents a critical challenge to apply such tools in live animals. In previous work, we developed a z...

Programmable artificial nucleases have transitioned over the past decade from ZFNs and TALENs to CRISPR/Cas systems, which have been ubiquitously used with great success to modify genomes. The efficiencies of knockout and knockin vary widely among distinct cell types and genomic loci and depend on the nuclease delivery and cleavage efficiencies. Mo...

Prokaryotic Argonaute proteins (pAgos) have been proposed as an alternative to the CRISPR/Cas9 platform for gene editing. Although Argonaute from Natronobacterium gregoryi (NgAgo) was recently shown unable to cleave genomic DNA in mammalian cells, the utility of NgAgo or other pAgos as a targetable DNA-binding platform for epigenetic editing has no...

List of oligonucleotide sequences for activity assay. (PDF)

Indel analysis of PCR amplified genomic target sites using 5'-phosphorylated guide DNAs. (PDF)

Protein sequences of hNgAgo and hTtAgo used in this study. (PDF)

hTtAgo cleaves ssDNA using ssDNA guides in vitro. A. hTtAgo was expressed in HEK293 cells and purified by immunoprecipitation (IP) with Flag antibody. A control IP was performed using rabbit IgG. Protein extract from transfected cells was loaded as a control. B. Copurification of nucleic acids with hTtAgo in HEK293 cells. Nucleic acids were resolve...

Design of hTtAgo guide DNAs. Diagram illustrating the three different gDNA constellations that were tested: forward (FW) guide only, forward (FW) and reverse (RV) guides or forward (FW) and reverse (RV) guides separated by a 15-nt spacer sequence. The genomic RPL13A target site is indicated in blue and complementary ssDNA guides are indicated in re...

List of oligonucleotide sequences of guide DNAs and gRNA target sites. (PDF)

List of oligonucleotide sequences for amplification of genomic target regions. (PDF)

Indel analysis of amplified genomic target sites comparing cleavage ability of RNA-guided SpCas9 and DNA-guided TtAgo and NgAgo in HeK293-c18 cells. (PDF)

Indel analysis of amplified genomic targets using 5'-phosphorylated gDNAs with phosphorothioate modifications. (PDF)

Many cockroach species have adapted to urban environments, and some have been serious pests of public health in the tropics and subtropics. Here, we present the 3.38-Gb genome and a consensus gene set of the American cockroach, Periplaneta americana. We report insights from both genomic and functional investigations into the underlying basis of its...

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and men...

Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-as...

Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanc...

The CRISPR/Cas9 system has recently emerged as a simple, yet powerful genome engineering tool, which has been widely used for genome modification in various organisms and cell types. However, screening biallelic genome modified cells are often time-consuming and technically challenging. In this study, we incorporated two different surrogate reporte...

The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology, as it requires only two key components (Cas9 protein and sgRNA) to function and further enables multiplex genome targeting and homology-directed repair (HDR) based precise genome editing in a wide vari...

CRISPR/Cas9 system has become a new versatile technology for genome engineering in various species. To achieve targeted modifications at the same site in both human and mice genomes by a CRISPR/Cas9 nuclease, we designed two target sites in conserved regions of vitamin D receptor (VDR) gene, which cover more than 17 kb of chromosome region dependin...

Validating activity of CRISPR/Cas9 in HEK293T cells. HEK293T cells were transfected with expression vectors and their corresponding reporter vectors to validate nuclease activities. Red fluorescence indicated vectors delivery into cells successfully. And green fluorescence implied designed CRISPR/Cas9 nuclease cut target sites in reporter vectors....

PCR primers for target and off target sequences amplification. (DOCX)

The sequences of potential off-target sites in mouse genome. *PAM is indicated in underline. Mismatches Nucleotide between the target sequence and the potential off-target sequences are in lower-case. (DOCX)

Schematic diagram of reporter vector characteristics. DsRed gene was a reporter gene to test the transfection efficiency of CRISPR/Cas9 system. During the introduction of CRISPR/Cas9, the disrupted puromycin resistance gene (PuroR) was repaired by single strand annealing (SSA), resulting in restored PuroR and eGFP. (TIF)

Oligonucleotides of sgRNA for CRISPR/Cas9 expression plasmids construction. * Restriction enzyme recognition sequences are in lower-case. (DOCX)

Oligonucleotides of target site for report vector construction. * Restriction enzyme recognition sequences are in lower-case. Underlined nucleotides are nucleotides in the protospacer adjacent motif (PAM) following the 20-ntsgRNA targeting sequence. (DOCX)

CRISPR/Cas9 system has become a new versatile technology for genome engineering. It utilizes a single guide RNA (sgRNA) to recognize target sequences in genome function, and activates Cas9 endonucleases to cut the locus. In this study, we designed two target sites from conserved regions of vitamin D receptor ( VDR ) gene in mammalian cells, which c...

In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other orga...

Isolation of genetically modified cells generated by designed nucleases are challenging, since they are often phenotypically indistinguishable from their parental cells. To efficiently enrich genetically modified cells, we developed two dual-reporter surrogate systems, namely NHEJ-RPG and SSA-RPG based on NHEJ and SSA repair mechanisms, respectivel...

Genome editing with engineered nucleases, such as zinc-finger nucleases (ZFNs) and TALE nucleases, remains confronted with a high risk of cellular toxicity induced by off-targeting. Here we describe the construction of a suicidal nuclease expression vector in which a pair of ZFNs genes were flanked of its target sites. To further enrich the targete...

The Streptococcus thermophilus CRISPR3-Cas (StCas9) system has been shown to mediate DNA cleavage in its original host and in E. coli as well as in vitro. Here, we have reconstituted the StCas9 system in yeast and conducted a systematic optimization of the sgRNA structure, including the minimal length of tracrRNA, loop structure, Match II region, B...

Zinc finger nuclease (ZFN) technology is a powerful molecular tool for targeted genome modifications and genetic engineering. However, screening for specific ZFs and validation of ZFN activity are labor intensive and time consuming. We previously designed a yeast-based ZFN screening and validation system by inserting a ZFN binding site flanked by a...

Prior to the development of zinc-finger nuclease technology, genetic manipulation by gene targeting achieved limited success in mammals, with the exception of mice and rat. Although ZFNs demonstrated highly effective gene targeted disruption in various model organisms, the activity of ZFNs in large domestic animals may be very low, and the probabil...

Zinc finger nucleases (ZFNs) have been successfully used for genome modification in various cell types and species. However, construction of an effective ZFN remained challenging. Previous studies all focused on obtaining specific zinc finger proteins (ZFPs) first via bacterial 2-hybrid approach, and then fusing selected ZFPs to FokI nuclease domai...

Background Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells...

Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated...