Targeted genome engineering via zinc finger nucleases

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants

Marine Copepods, The Wildebeest of the Ocean

Copepods are amongst the most abundant animals on our planet. Who knew?! These small (typically 1–10 mm) crustaceans are found in all of the world’s oceans and play an important role in regulating Earth’s climate. Like wildebeest in the Serengeti graze on grasslands and are food for lions, herbivorous copepods represent a vital link in oceanic food chains between microscopic algae and higher predators, such as fish, birds, and whales. A group of copepods called Calanus are particularly important in the Northern Hemisphere. These tiny-but-mighty animals also share the wildebeest’s need to make a large annual migration—but in their case, they sink thousands of meters downwards to spend the winter in the deep, dark ocean. Understanding the lives of marine copepods, and how their populations will respond to climate change, is crucial for predicting the future health of the marine environment and how it helps our planet

Paper RFLP Section of the SEP Elephant Project

The Elephant Project is an SEP curriculum that integrates Internet research, DNA analysis, and bioethical issues as students track down the origins of a piece of confiscated ivory. Students discover how modern biomolecular research tools can help conserve a species, in this case the African elephant

Expression of Gene Product 126 from Phage Gizmo and Creation of a Substrate

Endonucleases are enzymes which cleave DNA at specific nucleotide sequences. Homing Endonucleases (HEase) are a group of endonucleases that reside as open reading frames within self-splicing introns or inteins. After expression of the gene containing the HEase the HEase goes on to cleave a target site in a homolog of the hosting gene to induce homologous recombination turning the vacant homolog into a homing endonuclease gene (HEG). It is predicted that gene product (gp126) from the mycobacteriophage Gizmo encodes a HEase. To test whether the gene encodes a HEase the phage sequence of gp126 Gizmo was transferred into the his-tag containing plasmid (PET14b). The protein has been expressed and purified. To test the activity of the HEase a substrate is being created that will allow the predicted function to be directly tested in an enzyme assay

Prediction of Talen Binding Site for Genome Editing in Stem Cells

Transcription activator-like effector (TALE) nucleases (TALENs) have currently risen as a genome editing tool in numerous live forms .Transcription activator-like effector nucleases (TALENs), comprising of an DNA binding domain and a FokI cleavage domain TALENs can behaves as adaptable molecular DNA scissors creating double-strand breaks (DSBs) at desired position of a genome, expanding the effectiveness of genomic modification. The TALE central repeat domains’s flexible nature empower to tailor DNA recognition specificity easily and target basically any desired DNA sequence. In this study we understood the structural basis of interaction between TALEN and DNA and the target binding site of TALEN was predicted. It was found that NH RVD binds Guanine more specifically than NN RVD and accordingly script of TALE NT TOOL 2.0(https://tale-nt.cac.cornell.edu/node/add/talen) was modified. Adh2 and adh1c gene were tested in both the existing and modified script of TALE NT tool 2.0. Experiment shows that a 63 aa CTS in TALEN scaffold ,could lead to effective gene modification in cells of human, when separated by 14-32 bp spacers and accordingly script of TALE NT was changed with the spacer length 15-30.The results of adh2 gene and adh1c gene were verified by online available tools of TALEN LIBRARY RESEARCH (http://www.talenlibrary.net/) and we got affirmative results. Thus, the modified script is expected to increase the efficiency of DNA targeting

CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca.

Broomrapes (Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 (CCD8). Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P. aegyptiaca. Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several CCD8Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the CCD8Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient CCD8Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the CCD8Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks

Incisions for excision

Excison implies two incisions. Plumbers and surgeons know this principle, and long ago it was put into practice by the evolutionarily ancient DNA-repair machinery. Dissection of the first incision made by the eukaryotic nucleotide-excision repair pathway has now been described by O'Donovan et al., and dissection of the second by Bardwell et al. When put together the two processes enable the replacement of a damaged piece of DNA by a new one. [...]One might predict that a category of patients will be found that are deficient in nucleotide-excision repair and also have symptoms of a recombination defect. If these striking multiple engagements reflect a general evolutionary strategy of function sharing, then intimate connections between nucleotide-excision repair and cell-cycle control or chromatin dynamics are bound to show up as well

Incisions for excision

Excison implies two incisions. Plumbers and surgeons know this principle, and long ago it was put into practice by the evolutionarily ancient DNA-repair machinery. Dissection of the first incision made by the eukaryotic nucleotide-excision repair pathway has now been described by O'Donovan et al., and dissection of the second by Bardwell et al. When put together the two processes enable the replacement of a damaged piece of DNA by a new one. [...]One might predict that a category of patients will be found that are deficient in nucleotide-excision repair and also have symptoms of a recombination defect. If these striking multiple engagements reflect a general evolutionary strategy of function sharing, then intimate connections between nucleotide-excision repair and cell-cycle control or chromatin dynamics are bound to show up as well

What history tells us XXXX. The success story of the expression “genome editing”

International audienceThe expression « genome editing » designates the possibilities opened by the CRISPR-Cas9 enzyme to cut and modify genemes at precise positions. This expression has been rapidly adopted by biologists, but also journalists and laymen. I descibe steps in the recent emergence of this expression, as well as the reasons of its success.L’expression « édition du génome » désigne la possibilité ouverte par l’enzyme CRISPR-Cas9 de couper et de modifier le génome à des sites précis. Cette expression est utilisée aussi bien par les biologistes que par les journalistes et le grand public. Nous montrons dans cet article que cet usage est récent, nous décrivons les étapes qui ont conduit à sa diffusion et les raisons de son succès