Featured
-
-
Article
| Open AccessEnhanced regulation of prokaryotic gene expression by a eukaryotic transcriptional activator
Expanded toolkits for prokaryotic synthetic biology can enhance the dynamic range of gene expression. Here the authors move the eukaryotic transcription factor QF into E. coli and integrate it into genetic devices.
- I. Cody MacDonald
- , Travis R. Seamons
- & Tara L. Deans
-
Article
| Open AccessDisplay of the human mucinome with defined O-glycans by gene engineered cells
Mucins play critical roles in maintaining the human microbiome, with their O-glycosylated tandem repeats (TRs) providing important cues for microbiota. Here, the authors develop a cellular platform for producing TRs with defined O-glycan structures to dissect the functions of TR O-glycosylation.
- Rebecca Nason
- , Christian Büll
- & Yoshiki Narimatsu
-
Article
| Open AccessA genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression
Technologies that can halt the spread of gene drives would be highly useful in controlling or reverting their effect. Here the authors use the anti-CRISPR protein AcrIIA4 to inactivate drives in A. gambiae.
- Chrysanthi Taxiarchi
- , Andrea Beaghton
- & Andrea Crisanti
-
Article
| Open AccessAsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines
The utility of AsCas12a can be limited to poor editing efficiency. Here the authors identify a variant, “AsCas12a Ultra”, that has high on-target specificity demonstrated through editing of clinically relevant T cell genes.
- Liyang Zhang
- , John A. Zuris
- & Christopher A. Vakulskas
-
Article
| Open AccessRETRACTED ARTICLE: Selective inheritance of target genes from only one parent of sexually reproduced F1 progeny in Arabidopsis
Unlike insects and mice, CRISPR/Cas9-based gene drives have not been achieved in plants. Here, the authors demonstrate homozygous F1 Arabidopsis plants can be obtained through zygotic conversion using CRISPR/Cas9-mediated homology-directed repair.
- Tao Zhang
- , Michael Mudgett
- & Yunde Zhao
-
Article
| Open AccessNon-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells
Bone marrow chimaeric mice are a valuable tool in research, but require myeloablative conditioning. Here the authors demonstrate efficient FACS-free enrichment of haematopoietic stem and progenitor cells for transplantation into unconditioned recipient mice, as well as for genetic engineering using polyvinyl alcohol based media.
- Kiyosumi Ochi
- , Maiko Morita
- & Satoshi Yamazaki
-
Article
| Open AccessScalable recombinase-based gene expression cascades
There are few robust circuit architectures for sequential gene perturbations. Here, the authors use a modular recombinase-based design that sequentially edits loci, synchronizes cells, and deletes itself.
- Tackhoon Kim
- , Benjamin Weinberg
- & Timothy K. Lu
-
Article
| Open AccessExpanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems
CRISPR-Cas12a is a promising system for targeting AT-rich regions of the genome. Here the authors identify Cas12a orthologs with expanded targeting scope and develop a highly multiplexable editing system in rice.
- Yingxiao Zhang
- , Qiurong Ren
- & Yiping Qi
-
Article
| Open AccessRobust and flexible platform for directed evolution of yeast genetic switches
Eukaryotic genetic switches are more complex than prokaryotic ones, complicating their design. Here the authors present a workflow for parallel screening, selection and evolution of yeast genetic switches.
- Masahiro Tominaga
- , Kenta Nozaki
- & Akihiko Kondo
-
Article
| Open AccessPolymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering
Existing in vivo mutagenesis tools are limited by low mutation diversity and mutation rates. Here the authors present TRIDENT for targeted, continual and inducible diversification of genes of interest using deaminases fused to T7 RNA polymerase.
- Aaron Cravens
- , Osman K. Jamil
- & Christina D. Smolke
-
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
-
Article
| Open AccessProgrammable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins
Many diseases are caused by single-nucleotide polymorphisms. Here, the authors present CRISPR base editors that use the base excision machinery for single-base transversions.
- Liwei Chen
- , Jung Eun Park
- & Wei Leong Chew
-
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
-
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
-
Article
| Open AccessTowards plant resistance to viruses using protein-only RNase P
New approaches to plant disease control are important for pathogens that are difficult to control by existing methods. Here, the authors report a potential strategy to combat plant viruses by cytosolic expressed protein-only RNase P and show its ability for in vitro cleavage of tRNA-like structures existing in many plant viruses.
- Anthony Gobert
- , Yifat Quan
- & Philippe Giegé
-
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
-
Article
| Open AccessTALEN outperforms Cas9 in editing heterochromatin target sites
While Cas9 outperforms TALENs in euchromatin, it is less efficient in heterochromatic regions. Here the authors, using single-molecule imaging, show that Cas9 uses a less efficient search strategy compared to TALENs in these regions.
- Surbhi Jain
- , Saurabh Shukla
- & Huimin Zhao
-
Article
| Open AccessUniversal toxin-based selection for precise genome engineering in human cells
Genome engineering in cell lines or human stem cells often has poor efficiency, limiting the development of research and therapeutic applications. Here, the authors use a toxin-based selection system for precise bi-allelic engineering in cells and in vivo.
- Songyuan Li
- , Nina Akrap
- & Marcello Maresca
-
Article
| Open AccessDissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy
Developmental genes are often regulated by multiple elements, yet their relative contribution to gene expression remains poorly understood. Here the authors apply in vivo CRISPR/Cas9 genome engineering to find two distinct regulatory logics directing Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary.
- A. Hörnblad
- , S. Bastide
- & F. Spitz
-
Article
| Open AccessIn vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9
In vivo mutagenesis systems can often show restricted capabilities and deleterious off-site mutations. Here the authors fuse base deaminases to T7 RNA polymerase to mutate a target sequence and use dCas9 to define the boundaries of the diversified DNA.
- Beatriz Álvarez
- , Mario Mencía
- & Luis Ángel Fernández
-
Article
| Open AccessModelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter
Genetic mosaicism is frequently present in monogenic diseases of the central nervous system. Here the authors design a dual-colour reporter system that can be used to tune the degree of mosaicism in mouse models.
- Francesco Trovato
- , Riccardo Parra
- & Gian Michele Ratto
-
Article
| Open AccessIn vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo
Ex vivo engineering of antigen-specific T cells has shown therapeutic efficacy but can be costly and scarce. Here the authors show that in vitro-transcribed antigen receptor mRNA packaged in nanocarriers can directly induce, in vivo, transient their expression in circulating T cells to provide therapeutic effects in mouse models of cancer or viral infection.
- N. N. Parayath
- , S. B. Stephan
- & M. T. Stephan
-
Article
| Open AccessDocking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination
Current SpCas9 adenine base editors that minimise RNA off-target activities have constrained editing windows. Here the authors use domain insertion of TadA into Cas9 to narrow, expand or shift the editing window with RNA off-target minimization simultaneously
- Shuo Li
- , Bo Yuan
- & Tian-Lin Cheng
-
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
-
Article
| Open AccessPrime editing for functional repair in patient-derived disease models
Prime editing uses Cas9 nickase fused to a reverse transcriptase to edit genetic information. Here, the authors prime edit primary adult stem cells in 3D organoid cultures to show functional correction of pathogenic mutations without genome-wide off-target effects.
- Imre F. Schene
- , Indi P. Joore
- & Sabine A. Fuchs
-
Comment
| Open AccessThe second decade of synthetic biology: 2010–2020
Synthetic biology is among the most hyped research topics this century, and in 2010 it entered its teenage years. But rather than these being a problematic time, we’ve seen synthetic biology blossom and deliver many new technologies and landmark achievements.
- Fankang Meng
- & Tom Ellis
-
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
-
Article
| Open AccessCRISPRoff enables spatio-temporal control of CRISPR editing
Uncontrolled gene editing can lead to off-target effects and chromosomal translocations. Here the authors develop CRISPRoff, light degradable sgRNAs for titratable and spatially defined gene editing.
- Jared Carlson-Stevermer
- , Reed Kelso
- & Travis Maures
-
Article
| Open AccessChanging local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering
The heterochromatic knob (hk4S) on Arabidopsis chromosome 4 prevents the recombination between accessions with and without hk4S. Here, via egg-cell specific expression of the Cas9 nuclease, the authors demonstrate targeted reversal of the 1.1 Mb long hk4S-inversion in Col-0 and restore the crossovers with Ler-1.
- Carla Schmidt
- , Paul Fransz
- & Holger Puchta
-
Article
| Open AccessGene editing and elimination of latent herpes simplex virus in vivo
Herpes simplex virus establishes lifelong latency in ganglionic neurons, which are the source for recurrent infection. Here Aubert et al. report a promising antiviral therapy based on gene editing with adeno-associated virus-delivered meganucleases, which leads to a significant reduction in ganglionic HSV loads and HSV reactivation.
- Martine Aubert
- , Daniel E. Strongin
- & Keith R. Jerome
-
Article
| Open AccessTargeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome
In recent years, hematopoietic stem cells gene editing has emerged as a promising tool to treat blood disorders. Here the authors develop a CRISPR/Cas9-based genome editing strategy that allows the precise correction of Wiskott-Aldrich Syndrome in vitro and in vivo with high efficiency.
- Rajeev Rai
- , Marianna Romito
- & Alessia Cavazza
-
Article
| Open AccessIn vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural product derivatives
Several different genetic strategies have been reported for the modification of polyketide synthases but the highly repetitive modular structure makes this difficult. Here the authors report on an adapted Cas9 reaction and Gibson assembly to edit a target region of the polyketide synthases gene in vitro.
- Kei Kudo
- , Takuya Hashimoto
- & Kazuo Shin-ya
-
Article
| Open AccessA protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering
Fluorescent RNA aptamers could potentially be used as protein-independent reporters of transgene expression in plants. Here, the authors report that an optimized RNA aptamer, developed from Broccoli, can be used to detect transgene expression in stable and transiently transformed plant tissue.
- Jiuyuan Bai
- , Yao Luo
- & Yun Zhao
-
Article
| Open AccessA second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing
Here, the authors engineer an attenuated knock-out Leishmania (LmCen−/−) vaccine that is safe in immunocompromised mice and induces an immune response and protection similar to leishmanization with wild-type Leishmania. Since LmCen−/− is antibiotic resistant marker free, it is a candidate for clinical development.
- Wen-Wei Zhang
- , Subir Karmakar
- & Hira L. Nakhasi
-
Article
| Open AccessSynergistic gene editing in human iPS cells via cell cycle and DNA repair modulation
Precision editing with CRISPR-Cas9 often suffers from poor efficiency. Here the authors identify culture conditions and small molecules that synergize to promote homology-directed repair (HDR) in induced pluripotent stem (iPS) cells.
- Thomas L. Maurissen
- & Knut Woltjen
-
Article
| Open AccessCephalopod-inspired optical engineering of human cells
While organisms like squid can adaptively modulate the optical properties of their tissues, human cells lack analogous abilities. Here the authors engineer human cells to produce protein architectures with tunable light scattering functionalities.
- Atrouli Chatterjee
- , Juana Alejandra Cerna Sanchez
- & Alon A. Gorodetsky
-
Article
| Open AccessSuppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs
Numerous strategies exist to limit the off-target activity of CRISPR-Cas9 nucleases. Here the authors co-administer truncated gRNAs that block both Cas9 and high-fidelity Cas9 variants from cleaving at off-target sites.
- John C. Rose
- , Nicholas A. Popp
- & Douglas M. Fowler
-
Article
| Open AccessAssessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids
The relative contribution of fallopian tube (FT) or ovarian surface epithelium (OSE) to high-grade serous ovarian cancer (HG-SOC) development is unclear. Here, the authors establish organoid models from murine oviductal and OSE tissues that allow cancer modeling via CRISPR-Cas9 genome editing, and report a dual origin of murine HG-SOC.
- Kadi Lõhmussaar
- , Oded Kopper
- & Hans Clevers
-
Article
| Open AccessMultiplex precise base editing in cynomolgus monkeys
Due to the polygenic nature of most diseases, simultaneous correction or introduction of single nucleotide variants is needed. Here, the authors demonstrated the feasibility of multiplex base editing for polygenes disease modeling in cynomolgus monkey embryos with high specificity.
- Wenhui Zhang
- , Tomomi Aida
- & Shihua Yang
-
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
-
Article
| Open AccessTimed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair
Altering cellular responses to double-strand breaks in DNA could rebalance CRISPRediting outcomes. Here, the authors use a pooled CRISPR screen to identify inhibition of CDC7 as a strategy to improve HDR outcomes.
- Beeke Wienert
- , David N. Nguyen
- & Jacob E. Corn
-
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
-
Article
| Open AccessSynthetic biology based construction of biological activity-related library of fungal decalin-containing diterpenoid pyrones
Combining genome mining and heterologous expression in a genetically tractable host can lead to bioactive natural products discovery and production. Here, the authors employ this strategy for new decalin-containing diterpenoid pyrenes production by expressing native, extended, and shunt pathways in Aspergillus oryzae.
- Kento Tsukada
- , Shono Shinki
- & Teigo Asai
-
Article
| Open AccessEffective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements
The use of CRISPRa in bacteria lacks predictive rules for identifying effective gRNA target sites. Here the authors define features of bacterial promoters that impose stringent requirements on effective sites.
- Jason Fontana
- , Chen Dong
- & Jesse G. Zalatan
-
Article
| Open AccessThe mechanism of a high-affinity allosteric inhibitor of the serotonin transporter
The serotonin transporter (SERT) terminates serotonin signaling and its activity is modulated by antidepressants. Here authors reveal the mechanistic details underlying the coupling between the two binding sites in SERT and a high-affinity ligand for the allosteric site.
- Per Plenge
- , Ara M. Abramyan
- & Claus J. Loland
-
Article
| Open AccessA severe leakage of intermediates to shunt products in acarbose biosynthesis
Biosynthetic mechanism for the type 2 diabetes treatment drug acarbose is not fully revealed. Here, the authors show that shunt pathways and inefficient amino-deoxyhexose biosynthesis lead to 1-epi-valienol and valienol accumulation, and minimizing the flux to these shunt products can increase acarbose titer in Actinoplanes species.
- Qinqin Zhao
- , Yuchang Luo
- & Zixin Deng
-
Article
| Open AccessSmall molecule regulated sgRNAs enable control of genome editing in E. coli by Cas9
Bacteria lack the same suite of CRISPR tools that have been developed for mammalian cells. Here, the authors link aptamers to sgRNAs to allow small molecule control of gene editing in E. coli.
- Roman S. Iwasaki
- , Bagdeser A. Ozdilek
- & Robert T. Batey
-
Review Article
| Open AccessMultiplexed CRISPR technologies for gene editing and transcriptional regulation
Multiplexed CRISPR technologies have recently emerged as powerful approaches for genetic editing and transcriptional regulation. Here the authors review this emerging technology and discuss challenges and considerations for future studies.
- Nicholas S. McCarty
- , Alicia E. Graham
- & Rodrigo Ledesma-Amaro
-
Article
| Open AccessA toxin-antidote CRISPR gene drive system for regional population modification
CRISPR homing gene drives are highly invasive and can fail due to the rapid evolution of resistance. Here the authors present TARE drive, inspired by naturally occurring selfish genetic elements, which is less vulnerable to resistance and can potentially be confined to a target population.
- Jackson Champer
- , Esther Lee
- & Philipp W. Messer