For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which ...require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.
Animal interphase chromosomes are organized into topologically associating domains (TADs). How TADs are formed is not fully understood. Here, we combined high-throughput chromosome conformation ...capture and gene silencing to obtain insights into TAD dynamics in Xenopus tropicalis embryos. First, TAD establishment in X. tropicalis is similar to that in mice and flies and does not depend on zygotic genome transcriptional activation. This process is followed by further refinements in active and repressive chromatin compartments and the appearance of loops and stripes. Second, within TADs, higher self-interaction frequencies at one end of the boundary are associated with higher DNA occupancy of the architectural proteins CTCF and Rad21. Third, the chromatin remodeling factor ISWI is required for de novo TAD formation. Finally, TAD structures are variable in different tissues. Our work shows that X. tropicalis is a powerful model for chromosome architecture analysis and suggests that chromatin remodeling plays an essential role in de novo TAD establishment.
Abstract Human multidrug resistance protein 5 (hMRP5) effluxes anticancer and antivirus drugs, driving multidrug resistance. To uncover the mechanism of hMRP5, we determine six distinct cryo-EM ...structures, revealing an autoinhibitory N-terminal peptide that must dissociate to permit subsequent substrate recruitment. Guided by these molecular insights, we design an inhibitory peptide that could block substrate entry into the transport pathway. We also identify a regulatory motif, comprising a positively charged cluster and hydrophobic patches, within the first nucleotide-binding domain that modulates hMRP5 localization by engaging with membranes. By integrating our structural, biochemical, computational, and cell biological findings, we propose a model for hMRP5 conformational cycling and localization. Overall, this work provides mechanistic understanding of hMRP5 function, while informing future selective hMRP5 inhibitor development. More broadly, this study advances our understanding of the structural dynamics and inhibition of ABC transporters.
Precise genome editing is essential for both basic and translational research. The recently developed CRISPR/Cas9 system can specifically cleave a designated site of target gene to create a DNA ...double-strand break, which triggers cellular DNA repair mechanism of either inaccurate non-homologous end joining, or site-specific homologous recombination. Unfortunately, homology-directed repair (HDR) is challenging due to its very low efficiency. Herein, we focused on improving the efficiency of HDR using a combination of CRISPR/Cas9, eGFP, DNA ligase IV inhibitor SCR7, and single-stranded oligodeoxynucleotides (ssODN) in human cancer cells.
When Cas9, gRNA and eGFP were assembled into a co-expression vector, the disruption rate more than doubled following GFP-positive cell sorting in transfected cells compared to those unsorted cells. Using ssODNs as templates, SCR7 treatment increased targeted insertion efficiency threefold in transfected cells compared to those without SCR7 treatment. Moreover, this combinatorial approach greatly improved the efficiency of HDR and targeted gene mutation correction at both the GFP-silent mutation and the β-catenin Ser45 deletion mutation cells.
The data of this study suggests that a combination of co-expression vector, ssODN, and ligase IV inhibitor can markedly improve the CRISPR/Cas9-directed gene editing, which should have significant application in targeted gene editing and genetic disease therapy.
In humans, germline TP53 mutations predispose carriers to a wide spectrum of cancers, which is known as Li–Fraumeni syndrome (LFS). To date, the association of melanomas with LFS remains ...unestablished. No melanomas have been reported in any P53‐modified mouse models either. In this study, we show that targeted disruption of P53 at the DNA‐binding domain in Xenopus tropicalis recapitulates LFS, with the formation of soft‐tissue sarcomas and pancreatic ductal adenocarcinoma. Interestingly, 19% of the 14‐month‐old tp53Δ7/Δ7 homozygotes and 18% of tp53+/Δ7 heterozygotes spontaneously developed small nevi and non‐invasive melanomas. Large invasive melanomas were also observed in other older homozygous mutants, with about 7.9% penetrance. Our data suggest that more dermatologic investigation of LFS patients should be able to settle the association of melanoma with LFS in epidemiology. Our model is also valuable for further investigation of the molecular mechanism underlying melanoma progression upon germline alteration of the tp53 locus.
In humans, the association of melanomas and germline TP53 mutations remains unsettled. Melanomas have not been observed in any Trp53‐modified mouse models either. Here, we show that targeted disruption of P53 at the DNA binding domain in Xenopus tropicalis led to nevus and melanoma formation, which recapitulates the process of human cutaneous melanoma development.
Transposable elements (TEs) are abundant in metazoan genomes and have multifaceted effects on host fitness. However, the mechanisms underlying the functions of TEs are still not fully understood. ...Here, we combine Hi-C, ATAC-seq, and ChIP-seq assays to report the existence of multimegabase supersized loop (SSL) clusters in the Xenopus tropicalis sperm. We show that SSL anchors are inaccessible and devoid of the architectural protein CTCF, RNA polymerase II, and modified histones. Nearly all SSL anchors are marked by Helitrons, a class II DNA transposon. Molecular dynamics simulations indicate that SSL clusters are likely formed via a molecular agent-mediated chromatin condensation process. However, only slightly more SSL anchor-associated genes are expressed at late embryo development stages, suggesting that SSL anchors might only function in sperm. Our work shows an evolutionarily distinct and sperm-specific genome structure marked by a subset of Helitrons, whose establishment and function remain to be explored.
Display omitted
•Multimegabase supersized loops (SSLs) form clusters in mature X. tropicalis sperm•SSL anchors are inaccessible and devoid of CTCF, RNAPII, and modified histones•SSL anchors are explicitly enriched with class II DNA transposon of Helitrons•Molecular dynamics simulation indicates SSL clusters may form via phase separation
Through systematic comparison and comprehensive analysis of chromosome conformation, Shi et al. show an evolutionarily distinct and sperm-specific genome structure of supersized loop clusters in Xenopus tropicalis that are demarcated by transposon Helitrons, suggesting the existence of a link between the control of Helitrons and genome architecture during spermatogenesis.
The true diploid frog, Xenopus tropicalis (X. tropicalis) is an excellent genetic model organism. To date, the CRISPR/Cas-mediated genome editing methods established in this species are mostly based ...on SpCas9 that requires the stringent NGG protospacer-adjacent motif (PAM) for target recognition, which limits its genome editing scope. Thus, it is highly desirable to circumvent this limitation. Through one-cell stage injection of Cas/gRNAs into X. tropicalis embryos, we evaluated the mutagenic efficiency of 8 different Cas variants using T7EI assay, Sanger DNA sequencing, or deep sequencing. Our data indicate that SaCas9 and KKH SaCas9 are highly effective in frogs, which could be used for direct phenotyping in G0 embryos. In contrast, VQR Cas9, xCas9 3.7, SpG Cas9, and SpRY Cas9 were ineffective in X. tropicalis embryos and no activity was detected for iSpyMac Cas9. We also found that LbCas12a/crRNA RNP complexes with paired crRNAs efficiently induced small fragment deletions in X. tropicalis embryos. SaCas9 and KKH SaCas9 are robust genome editing tools in X. tropicalis embryos. LbCas12a/crRNA RNP complexes are useful for inducing DNA fragment deletions in frog embryos. These tools expand the CRISPR/Cas genome editing scope in X. tropicalis and increase the flexibility for various genome editing applications in frogs.
Targeted integration of genes in Xenopus tropicalis Shi, Zhaoying; Tian, Dandan; Xin, Huhu ...
Genesis (New York, N.Y. : 2000),
January 2017, 2017-01-00, 20170101, Letnik:
55, Številka:
1-2
Journal Article
Recenzirano
With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique ...contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous recombination‐mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology‐independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis.
The cornea and sclera are distinct adjacent tissues, yet their stromal cells originate from common neural crest cells (NCCs). Sclerocornea is a disease characterized by an indistinguishable boundary ...between the cornea and sclera. Previously, we identified a RAD21 mutation in a sclerocornea pedigree. Here, we investigated the impacts of RAD21 on NCC activities during eye development. RAD21 deficiency caused upregulation of PCDHGC3. Both RAD21 knockdown and PCDHGC3 upregulation disrupted the migration of NCCs. Transcriptome analysis indicated that WNT9B had 190.9-fold higher expression in scleral stroma than in corneal stroma. WNT9B was also significantly upregulated by both RAD21 knockdown and PCDHGC3 overexpression, and knock down of WNT9B rescued the differentiation and migration of NCCs with RAD21 deficiency. Consistently, overexpressing wnt9b in Xenopus tropicalis led to ocular developmental abnormalities. In summary, WNT9B is a determinant factor during NCC differentiation into corneal keratocytes or scleral stromal cells and is affected by RAD21 expression.
Display omitted
•Established a stable differentiation protocol from hESCs to corneal keratocytes•RAD21 deficiency affected the proliferation and migration ability of NCCs•Increased scleral markers after RAD21 knockdown during NCC differentiation to cornea•WNT9B is a crucial mediator during ocular NCC differentiation
Cell biology; Developmental biology
Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and ...further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.
PDF
