Highlights • ZFNs, TALENs, and CRISPR/Cas-based RNA-guided DNA endonucleases are programmable site-specific nucleases. • Site-specific nucleases induce DNA DSBs that stimulate NHEJ and HDR at ...targeted genomic loci. • We discuss the therapeutic potential of site-specific nuclease technologies and discuss future prospects for the field.
Life-changing biology Woolfson, Adrian
Science (American Association for the Advancement of Science),
03/2022, Volume:
375, Issue:
6584
Journal Article
Peer reviewed
Genetically engineered genomes will disrupt the bioeconomy and rewrite human nature—are we ready?
The number of phalanges and joints are key features of digit 'identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their ...associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, here we show that functional 5'Hoxd-Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. We propose that 5'Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation.
Genome editing technology, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, ...has enabled far more efficient genetic engineering even in non-human primates. This biotechnology is more likely to develop into medicine for preventing a genetic disease if corrective genome editing is integrated into assisted reproductive technology, represented by in vitro fertilization. Although rapid advances in genome editing are expected to make germline gene correction feasible in a clinical setting, there are many issues that still need to be addressed before this could occur. We herein examine current status of genome editing in mammalian embryonic stem cells and zygotes and discuss potential issues in the international regulatory landscape regarding human germline gene modification. Moreover, we address some ethical and social issues that would be raised when each country considers whether genome editing-mediated germline gene correction for preventive medicine should be permitted.
Highlights • The development of genome editing is framed by earlier DNA-modifying technologies. • The various aspects underlying genome editing are reviewed and integrated. • The main genome editing ...approaches and their collateral effects are discussed. • The efforts seeking improved genome editing end-points are highlighted.
Like other abiotic stresses, salt stress has become a major factor that restricts the growth, distribution and yield of crops. Research has shown that increasing the nitrogen content in soil can ...improve the salt tolerance of plants and nitrate transporter (NRT) is the primary nitrogen transporter in plants. Suaeda salsa (L.) Pall is a strong halophyte that can grow normally at a salt concentration of 200 mM. The salt stress transcriptome database of S. salsa was found to contain four putative genes that were homologous to NRT, including SsNRT1.1A, SsNRT1.1B, SsNRT1.1C and SsNRT1.1D. The cDNA of SsNRT1.1s was predicted to contain open reading frames of 1791, 1782, 1755 and 1746 bp, respectively. Sequence alignment and structural analysis showed that the SsNRT1.1 amino acids were inducible by salt and have conserved MFS and PTR2 domains. Subcellular localization showed they are on the endoplasmic reticulum. Overexpression of SsNRT1.1 genes in transgenic Arabidopsis improves its salt tolerance and SsNRT1.1C was more effective than others. We constructed a salt-stressed yeast cDNA library and used yeast two-hybrid and BiFC technology to find out that SsHINT1 and SsNRT1.1C have a protein interaction relationship. Overexpression of SsHINT1 in transgenic Arabidopsis also improves salt tolerance and the expressions of Nasup.+ and Ksup.+ were increased and reduced, respectively. But the Ksup.+/Liratio was up-regulated 11.1-fold compared with the wild type. Thus, these results provide evidence that SsNRT1.1C through protein interactions with SsHINT1 increases the Ksup.+/Nasup.+ ratio to improve salt tolerance and this signaling may be controlled by the salt overly sensitive (SOS) pathway.
The NAC transcription factor family is well known to play vital roles in plant development and stress responses. For this research, a salt-inducible NAC gene, PsnNAC090 (Po-tri.016G076100.1), was ...successfully isolated from Populus simonii × Populus nigra. PsnNAC090 contains the same motifs at the N-terminal end of the highly conserved NAM structural domain. The promoter region of this gene is rich in phytohormone-related and stress response elements. Transient transformation of the gene in the epidermal cells of both tobacco and onion showed that the protein was targeted to the whole cell including the cell membrane, cytoplasm and nucleus. A yeast two-hybrid assay demonstrated that PsnNAC090 has transcriptional activation activity with the activation structural domain located at 167–256aa. A yeast one-hybrid experiment showed that PsnNAC090 protein can bind to ABA-responsive elements (ABREs). The spatial and temporal expression patterns of PsnNAC090 under salt and osmotic stresses indicated that the gene was tissue-specific, with the highest expression level in the roots of Populus simonii × Populus nigra. We successfully obtained a total of six transgenic tobacco lines overexpressing PsnNAC090. The physiological indicators including peroxidase (POD) activity, superoxide dismutase (SOD) activity, chlorophyll content, proline content, malondialdehyde (MDA) content and hydrogen peroxide (Hsub.2Osub.2) content were measured in three transgenic tobacco lines under NaCl and polyethylene glycol (PEG) 6000 stresses. The findings reveal that PsnNAC090 improves salt and osmotic tolerance by enhancing reactive oxygen species (ROS) scavenging and reducing membrane lipid peroxide content in transgenic tobacco. All the results suggest that the PsnNAC090 gene is a potential candidate gene playing an important role in stress response.
Brassinosteroids (BRs) are important hormones that play crucial roles in plant growth, reproduction, and responses to abiotic and biotic stresses. CYP85A1 is a castasterone (CS) synthase that ...catalyzes C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to CS, after which CS is converted into brassinolide (BL) in a reaction catalyzed by CYP85A2. Here, we report the functional characteristics of rice (Oryza sativa L.) OsCYP85A1. Constitutive expression of OsCYP85A1 driven by the cauliflower mosaic virus 35S promoter increased endogenous BR levels and significantly promoted growth and biomass production in three groups of transgenic Populus tomentosa lines. The plant height and stem diameter of the transgenic poplar plants were increased by 17.6% and 33.6%, respectively, in comparison with control plants. Simultaneously, we showed that expression of OsCYP85A1 enhanced xylem formation in transgenic poplar without affecting cell wall thickness or the composition of cellulose. Our findings suggest that OsCYP85A1 represents a potential target candidate gene for engineering fast-growing trees with improved wood production.
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