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| Open AccessReprogramming mechanism dissection and trophoblast replacement application in monkey somatic cell nuclear transfer
Somatic cloning of rhesus monkey has not been successful until now. Here, authors report epigenetic abnormalities in SCNT embryos and placentas and develop a trophoblast replacement method that enables them to successful clone of a healthy male rhesus monkey.
- Zhaodi Liao
- , Jixiang Zhang
- & Qiang Sun
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Article
| Open AccessInduced formation of primordial germ cells from zebrafish blastomeres by germplasm factors
Primordial germ cell (PGC) transplantation is an important technology for developmental biology and genetic breeding. Here, Wang et al. develop an approach for PGC induction that enhances the efficiency of PGC transplantation in zebrafish.
- Xiaosi Wang
- , Junwen Zhu
- & Yonghua Sun
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Article
| Open AccessCRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus
Culex mosquitoes are carriers of major diseases like West Nile virus and are a public health concern. Here the authors present a CRISPR-Cas9 gene drive as a control technology in the Culex quinquefasciatus mosquito species.
- Tim Harvey-Samuel
- , Xuechun Feng
- & Valentino M. Gantz
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Article
| Open AccessCreating resistance to avian influenza infection through genome editing of the ANP32 gene family
In chickens, influenza A virus relies on host protein ANP32A. Here the authors use CRISPR/Cas9 to generate homozygous gene edited chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction.
- Alewo Idoko-Akoh
- , Daniel H. Goldhill
- & Mike J. McGrew
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Article
| Open AccessDurable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene
This study demonstrates the safety and long-term efficacy of a single-dose, injectable contraceptive in female domestic cats. Treated females remained contracepted for over two years, and did not ovulate or produce kittens when paired with males.
- Lindsey M. Vansandt
- , Marie-Charlotte Meinsohn
- & David Pépin
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Article
| Open AccessA CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias
CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Here the authors use gene linkage to show that in males inheritance bias of wGDe did not occur by homing, rather through increased propagation of the donor drive element.
- Sebald A. N. Verkuijl
- , Estela Gonzalez
- & Luke Alphey
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Article
| Open AccessA doxycycline- and light-inducible Cre recombinase mouse model for optogenetic genome editing
Achieving spatial control of gene expression is important. Here the authors report an optimised photoactivatable Cre recombinase system, doxycycline- and light-inducible Cre recombinase (DiLiCre), and generate a DiLiCre mouse line which they use for mutagenesis in vivo and positional cell-tracing.
- Miguel Vizoso
- , Colin E. J. Pritchard
- & Jacco van Rheenen
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Article
| Open AccessWax worm saliva and the enzymes therein are the key to polyethylene degradation by Galleria mellonella
The crucial first step in the biodegradation of polyethylene plastic is oxidation of the polymer. This has traditionally required abiotic pre-treatment, but now Bertocchini and colleagues report two wax worm enzymes capable of catalyzing this oxidation and subsequent degradation at room temperature.
- A. Sanluis-Verdes
- , P. Colomer-Vidal
- & F. Bertocchini
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Article
| Open AccessHealthy cloned offspring derived from freeze-dried somatic cells
The development of safe preservation methods for genetic resources is important. Here, the authors successfully produce cloned mice from freeze-dried somatic cells, demonstrating the possibility of safe and low-cost preservation of genetic resources.
- Sayaka Wakayama
- , Daiyu Ito
- & Teruhiko Wakayama
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Article
| Open AccessDisease modeling by efficient genome editing using a near PAM-less base editor in vivo
Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. Here the authors describe a near PAM-less base editor and its application in zebrafish to efficiently create disease models harbouring specific point mutations.
- Marion Rosello
- , Malo Serafini
- & Filippo Del Bene
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Article
| Open AccessEfficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci
Trans-chromosomic (Tc) mice have helped the development of therapeutic antibodies, but chromosome instability limits its application. Here the authors develop a new line of Tc mice with full human Ig heavy and kappa loci integrated into the mouse artificial chromosome for stable passage, and confirm efficient generation of B cell responses and specific antibodies.
- Hiroyuki Satofuka
- , Satoshi Abe
- & Yasuhiro Kazuki
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Article
| Open AccessReversing insecticide resistance with allelic-drive in Drosophila melanogaster
Insecticide resistance (IR) poses a major global health challenge. Here, the authors generate common IR mutations in laboratory Drosophila strains and use a CRISPR-based allelic-drive to replace an IR allele with a susceptible wild-type counterpart, providing a potent new tool for vector control.
- Bhagyashree Kaduskar
- , Raja Babu Singh Kushwah
- & Ethan Bier
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Article
| Open AccessExploiting a Y chromosome-linked Cas9 for sex selection and gene drive
CRISPR-based engineering can be used to bias sex ratios. Here the authors develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome and functionally characterize the utility of this strain for both sex selection and gene drive.
- Stephanie Gamez
- , Duverney Chaverra-Rodriguez
- & Omar S. Akbari
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Article
| Open AccessCRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes
In areas such as animal research and agriculture a single sex is often required in abundance, leading to wasted resources and ethical considerations. Here the authors develop a CRISPR/Cas9 mediated synthetic lethal system that enables the production of single sex offspring that can be repurposed for use in multiple organisms.
- Charlotte Douglas
- , Valdone Maciulyte
- & James M. A. Turner
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Article
| Open AccessCryopreservation method for Drosophila melanogaster embryos
The development of a widely adopted cryopreservation method remains a major challenge in Drosophila melanogaster research. Here the authors report a robust cryopreservation protocol of Drosophila embryos and showcase its implementation in 25 distinct strains from different sources.
- Li Zhan
- , Min-gang Li
- & John Bischof
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Article
| Open AccessInherently confinable split-drive systems in Drosophila
NHEJ alleles and Cas9 remnants after a gene drive introduction are scientific and public concerns. Here, the authors use split drives with recoded rescue elements to target essential genes and minimize the appearance of NHEJ alleles while also leaving no trace of Cas9.
- Gerard Terradas
- , Anna B. Buchman
- & Ethan Bier
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Article
| Open AccessBlastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats
The uptake of donor pluripotent stem cells (PSCs) in hosts of different species and subsequent germline transmission is very inefficient. Here, the authors show, using Prdm14 gene depleted rat host blastocysts to remove functional sperm, that germline transmission from donor rat or mouse PSCs is possible.
- Toshihiro Kobayashi
- , Teppei Goto
- & Masumi Hirabayashi
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Article
| Open AccessMitochondrial DNA editing in mice with DddA-TALE fusion deaminases
Split DddA-derived base editors fused to TALEs enable mitochondrial DNA editing. Here the authors demonstrate their use in mouse embryos with germline transmission.
- Hyunji Lee
- , Seonghyun Lee
- & Jin-Soo Kim
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Article
| Open AccessDirect allele introgression into pure chicken breeds using Sire Dam Surrogate (SDS) mating
Chicken are a biological model and an important agricultural animal. Here, the authors demonstrate that pure breed genome edited chicks can be produced for any chicken breed by direct mating of sterile surrogate hosts carrying donor genome edited germ cells.
- Maeve Ballantyne
- , Mark Woodcock
- & Mike J. McGrew
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Article
| Open AccessPolyandry blocks gene drive in a wild house mouse population
This study resolves a long-standing mystery of why t haplotypes, an example of selfish genes, have persisted at unexpectedly low frequencies in wild mouse populations. It shows that multiple mating by females, which is more common at higher mouse population densities, decreases the frequency of driving t haplotypes.
- Andri Manser
- , Barbara König
- & Anna K. Lindholm
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Article
| Open AccessEfficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi
Gene drives may be impeded by the generation of resistant alleles following NHEJ. Here the authors develop a recoded gene-drive rescue system for the malaria mosquito, Anopheles stephensi, that targets the drive to the kynurenine hydroxylase gene for negative selection against mutated alleles.
- Adriana Adolfi
- , Valentino M. Gantz
- & Anthony A. James
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Review Article
| Open AccessACE2 mouse models: a toolbox for cardiovascular and pulmonary research
Angiotensin-converting enzyme 2 (ACE2) is a cell surface enzyme previously shown to mediate SARS-CoV, and now SARS-CoV-2, entry into host cells. Here the authors review existing mouse ACE2 models expressing humanized, transgenic, knockout, knockin, conditional and reporter alleles to provide a toolbox for COVID-19 research.
- Hongpeng Jia
- , Xinping Yue
- & Eric Lazartigues
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Article
| Open AccessPhotoactivatable Cre recombinase 3.0 for in vivo mouse applications
Previous versions of photoactivatable Cre recombinase (PA-Cre) suffered from unintentional recombination in dark conditions. Here, the authors develop an improved version of PA-Cre, called PA-Cre 3.0, which shows reduced leakiness and improved efficiency upon activation, and make mouse lines that express PA-Cre 3.0 conditionally.
- Kumi Morikawa
- , Kazuhiro Furuhashi
- & Masayuki Yazawa
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Article
| Open AccessA drug-inducible sex-separation technique for insects
Sorting insects based on sex is error prone and frequently labour intensive. Here, the authors present a drug-inducible sex separation system based on sex-specific rescue from antibotic toxicity.
- Nikolay P. Kandul
- , Junru Liu
- & Omar S. Akbari
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Article
| Open AccessA transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment
Gene drives raise safety concerns around unintended propagation. Here the authors present a trans-complementing split-gene drive that requires inheritance of separate transgenes to assemble a fully functional drive.
- Víctor López Del Amo
- , Alena L. Bishop
- & Valentino M. Gantz
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Article
| Open AccessMonkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease
Most cases of autosomal dominant polycystic kidney disease (ADPKD) are due to mutations in PKD1. Here, Tsukiyama et al. generate monkeys with mutations in PKD1 and show that animals recapitulate key pathological features of the human disease, suggesting these may provide insights into ADPKD pathogenesis and contribute to the development of future therapeutic strategies.
- Tomoyuki Tsukiyama
- , Kenichi Kobayashi
- & Masatsugu Ema
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Article
| Open AccessAn engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence
Lee et al. report an engineered IgG1 Fc domain that behaves like an hFcRn binding pH toggle switch. The authors show that this new half-life extension Fc domain confers improved pharmacokinetics in new humanized knock-in mouse strains that recapitulate the key processes for antibody persistence in circulation.
- Chang-Han Lee
- , Tae Hyun Kang
- & George Georgiou
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Article
| Open AccessGenerating viable mice with heritable embryonically lethal mutations using the CRISPR-Cas9 system in two-cell embryos
Roughly 25% of mouse genes are embryonically lethal when knocked out, preventing the generation of viable mouse models. Here the authors use CRISPR-Cas9 to edit one blastomere of a two-cell embryo to generate viable chimeric mice.
- Yi Wu
- , Jing Zhang
- & Songlin Wang
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Article
| Open AccessEfficient base editing for multiple genes and loci in pigs using base editors
Base editors can efficiently produce single nucleotide alterations without requiring a double-strand break. Here the authors show base editing at multiple sites simultaneously in pigs.
- Jingke Xie
- , Weikai Ge
- & Liangxue Lai
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Article
| Open AccessComparative oncogenomics identifies combinations of driver genes and drug targets in BRCA1-mutated breast cancer
It is difficult to identify cancer driver genes in cancers, for instance BRCA1 mutated breast cancer, that are characterised by large scale genomic alterations. Here, the authors develop genetically engineered mouse models of BRCA1-deficient breast cancer that allow highthroughput in vivo perturbation of candidate driver genes, validating drivers Myc, Met, Pten and Rb1, and identifying MCL1 as a collaborating driver whose targeting can impact efficacy of PARP inhibition.
- Stefano Annunziato
- , Julian R. de Ruiter
- & Jos Jonkers
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Article
| Open AccessNoninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions
Most approaches to control gene expression in vivo require generation of knock-in mouse lines and often lack spatiotemporal control. Here the authors develop a photo-activatable Flp recombinase system and demonstrate its use by controlling object-exploration behavior in mice through Cav3.1 silencing.
- Hyunjin Jung
- , Seong-Wook Kim
- & Won Do Heo
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Article
| Open AccessStreamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses
CRISPR-Cas9 has been widely adopted for genetically manipulating rodents for scientific research. Here the authors transduce mouse embryos with CRISPR-Cas9 components using rAAVs in explant culture or in vivo to produce gene-edited animals.
- Yeonsoo Yoon
- , Dan Wang
- & Jaime A. Rivera-Pérez
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Article
| Open AccessAn automated platform for high-throughput mouse behavior and physiology with voluntary head-fixation
Transgenic approaches and improvements in functional imaging have necessitated an advance in the behavioral toolkit. Here the authors describe an automated high-throughput voluntary head fixation system for training mice on complex psychophysical decision tasks compatible with concurrent two-photon microscopy.
- Ryo Aoki
- , Tadashi Tsubota
- & Andrea Benucci
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Article
| Open AccessDNA-binding of the Tet-transactivator curtails antigen-induced lymphocyte activation in mice
Tet-transactivators are used for direct regulation of gene expression, RNA interference and for CRISPR/Cas9-based systems. Here the authors show that DNA-bound Tet-transactivators can induce cell death in antigen-activated lymphocytes in vivo, putting into question the use of, and in vivo data generated with, these molecular tools.
- Eleonora Ottina
- , Victor Peperzak
- & Andreas Villunger
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Article
| Open AccessTwo mouse models reveal an actionable PARP1 dependence in aggressive chronic lymphocytic leukemia
ATM and TP53 mutations are associated with poor prognosis in chronic lymphocytic leukaemia (CLL). Here the authors generate mouse models of Tp53- and Atm-defective CLL mimicking the high-risk form of human disease and show that Atm-deficient CLL is sensitive to PARP1 inhibition.
- Gero Knittel
- , Tim Rehkämper
- & H. Christian Reinhardt
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Article
| Open AccessCRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome
CRISPR/Cas9 gene editing has been used to generate mutations in several mouse genes. Here, the authors show that targeting events using single guide RNAs cause large deletions at 17 sites in the mouse genome, suggesting that careful genotyping is needed and sequential targeting may avoid such deletions.
- Ha Youn Shin
- , Chaochen Wang
- & Lothar Hennighausen
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Article
| Open AccessExpanding the genetic code of Mus musculus
Expanding the genetic code with unnatural amino acids in model organisms is a powerful tool for investigatingin vivocellular proteins and processes. Here the authors re-engineer the amber stop codon in laboratory mice to allow site-specific incorporation of unnatural amino acids in target proteins.
- Songmi Han
- , Aerin Yang
- & Hee-Sung Park
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Article
| Open AccessHealthy ageing of cloned sheep
Since the birth of the first cloned animal, Dolly the sheep, concerns have been raised about potential long-term health consequences of cloning. Here the authors report on a cohort of 13 aged cloned sheep, including four created from the same cells as Dolly, and find they are healthy and seem to age normally.
- K. D. Sinclair
- , S. A. Corr
- & D. S. Gardner
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Article
| Open AccessGeneration of mice with longer and better preserved telomeres in the absence of genetic manipulations
Telomere shortening has been linked to some aspects of organismal ageing. Here the authors create chimaeric mice that contain a mix of cells with normal or unnaturally long telomeres, and show chimaeric mice are protected from some forms of ageing-associated cellular damage and have accelerated wound-healing.
- Elisa Varela
- , Miguel A. Muñoz-Lorente
- & Maria A. Blasco
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Article
| Open AccessCas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters
Genomic engineering often requires the cloning of long DNA segments that contain large gene clusters. Here, the authors describe an RNA-guided Cas9 nuclease assistedin vitrotechnique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb in a single step.
- Wenjun Jiang
- , Xuejin Zhao
- & Ting F. Zhu
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Article
| Open AccessZinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows
Zinc-finger nickases are programmable nucleases that can be used to generate site-specific single-strand breaks in DNA. Liu et al. use this technology to insert an antimicrobial gene into the endogenous beta-casein locus in cloned cows, with the aim of providing protection against mastitis.
- Xu Liu
- , Yongsheng Wang
- & Yong Zhang
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Article
| Open AccessA visible dominant marker for insect transgenesis
Genetic modification in insects mostly involves the use of fluorescent markers to identify successful transformation. Here Osanai-Futahashi et al.report a marker system based on changes in melanin pigmentation that allows the identification of genetically modified insects with the naked eye.
- Mizuko Osanai-Futahashi
- , Takahiro Ohde
- & Hideki Sezutsu
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Article |
Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model
Calcium-sensing fluorescent proteins such as TN-XXL are valuable tools for studying cellular function but, when expressed in mice, may affect animal physiology and behaviour. The authors of this paper create transgenic mice expressing TN-XXL and show that long-term expression of TN-XXL is tolerated well.
- Stephan Direnberger
- , Marsilius Mues
- & Oliver Griesbeck