Bibliografías temáticas / CRISPR, Cas9, genome editing, gRNA

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Literatura académica sobre el tema "CRISPR, Cas9, genome editing, gRNA"

Autor: Grafiati
Publicado: 11 de marzo de 2023

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Artículos de revistas sobre el tema "CRISPR, Cas9, genome editing, gRNA"

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Jo, Areum, Sangwoo Ham, Gum Hwa Lee, Yun-Il Lee, SangSeong Kim, Yun-Song Lee, Joo-Ho Shin y Yunjong Lee. "Efficient Mitochondrial Genome Editing by CRISPR/Cas9". BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/305716.

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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.
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Xie, Kabin, Bastian Minkenberg y Yinong Yang. "Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system". Proceedings of the National Academy of Sciences 112, n.º 11 (2 de marzo de 2015): 3570–75. http://dx.doi.org/10.1073/pnas.1420294112.

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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.
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Mekler, Vladimir, Konstantin Kuznedelov y Konstantin Severinov. "Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences". Journal of Biological Chemistry 295, n.º 19 (1 de abril de 2020): 6509–17. http://dx.doi.org/10.1074/jbc.ra119.012239.

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The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.
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Bruegmann, Tobias, Khira Deecke y Matthias Fladung. "Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars". International Journal of Molecular Sciences 20, n.º 15 (24 de julio de 2019): 3623. http://dx.doi.org/10.3390/ijms20153623.

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CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.
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Wardhani, Bantari W. K., Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy y Mitsuyasu Kato. "TMEPAI genome editing in triple negative breast cancer cells". Medical Journal of Indonesia 26, n.º 1 (16 de mayo de 2017): 14–8. http://dx.doi.org/10.13181/mji.v26i1.1871.

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Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.
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Kong, Qihui, Jie Li, Shoudong Wang, Xianzhong Feng y Huixia Shou. "Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean". Plants 12, n.º 5 (23 de febrero de 2023): 1017. http://dx.doi.org/10.3390/plants12051017.

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The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43–97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3′ terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.
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Jameel, Mohd Rizwan. "From design to validation of CRISPR/gRNA primers towards genome editing". Bioinformation 18, n.º 5 (31 de mayo de 2022): 471–77. http://dx.doi.org/10.6026/97320630018471.

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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated system) is used to edit specific genomic sequences with precision and efficacy. There are many online platforms/software for the design of gRNAs and related primers. However, there are concerns in design regarding off-site deletions besides knocking out sequences in the target genes. Nonetheless, a well known robust platform for CRISPR/gRNA primers design is CRISPRdirect. We demonstrate the use of this tool in the design of CRISPR/gRNA primers for soluble starch synthases (SSS) II-1, 2, and 3 genes in the Oryza sativa genome followed by the PCR-mediated amplification of SSS genes with corresponding confirmation towards genome editing having improved phenotype features.
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Jung, Soo Bin, Chae young Lee, Kwang-Ho Lee, Kyu Heo y Si Ho Choi. "A cleavage-based surrogate reporter for the evaluation of CRISPR–Cas9 cleavage efficiency". Nucleic Acids Research 49, n.º 15 (4 de junio de 2021): e85-e85. http://dx.doi.org/10.1093/nar/gkab467.

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Abstract CRISPR–Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR–Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.
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Foreman, Hui-Chen Chang, Varvara Kirillov, Gabrielle Paniccia, Demetra Catalano, Trevor Andrunik, Swati Gupta, Laurie T. Krug y Yue Zhang. "RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production". PLOS ONE 16, n.º 6 (4 de junio de 2021): e0252313. http://dx.doi.org/10.1371/journal.pone.0252313.

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Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
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Yoo, Byung-Chun, Narendra S. Yadav, Emil M. Orozco y Hajime Sakai. "Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts". PeerJ 8 (6 de enero de 2020): e8362. http://dx.doi.org/10.7717/peerj.8362.

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We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated “Edit Plasmids,” were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.
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Tesis sobre el tema "CRISPR, Cas9, genome editing, gRNA"

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Roidos, Paris. "Genome editing with the CRISPR Cas9 system". Thesis, KTH, Skolan för bioteknologi (BIO), 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-163694.

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Ran, Fei Ann. "CRISPR-Cas: Development and applications for mammalian genome editing". Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11610.

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The ability to introduce targeted modifications into genomes and engineer model organisms holds enormous promise for biomedical and technological applications, and has driven the development of tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). To facilitate genome engineering in mammalian cells, we have engineered the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 programmable nuclease systems from Streptococcus pyogenes SF370 (SpCas9) and S. thermophilus LMD-9 (St1Cas9) for mouse and human cell gene editing through heterologous expression of the minimal protein and RNA components. We have demonstrated that Cas9 nucleases can be guided by several short RNAs (sgRNAs) to introduce double stranded breaks (DSB) in the mammalian genome and induce efficient, multiplexed gene modification through non-homologous end-joining-mediated indels or homology-directed repair. Furthermore, we have engineered SpCas9 into a nicking enzyme (SpCas9n) to facilitate recombination while minimizing mutagenic DNA repair processes, and show that SpCas9n can be guided by pairs of appropriately offset sgRNAs to induce DSBs with high efficiency and specificity. In collaboration with Drs. Osamu Nureki and Hiroshi Nishimasu at the University of Tokyo, we further report the crystal structure of SpCas9 in complex with the sgRNA and target DNA, and elucidate the structure-function relationship of the ribonucleoprotein complex. Finally, through a metagenomic screen of orthologs, we have identified an additional small Cas9 from Staphylococcus aureus subsp. aureus (SaCas9) that cleaves mammalian endogenous DNA with high efficiency. SaCas9 can be packaged into adeno-associated virus for effective gene modification in vivo. Together, these technologies open up exciting possibilities for applications across basic science, biotechnology, and medicine.
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Hirosawa, Moe. "Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch". Kyoto University, 2019. http://hdl.handle.net/2433/242421.

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Castanon, velasco Oscar. "Targeting the transposable elements of the genome to enable large-scale genome editing and bio-containment technologies". Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLX006.

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Les nucléases programmables et site-spécifiques comme CRISPR-Cas9 sont des signes avant-coureurs d’une nouvelle révolution en génie génétique et portent en germe un espoir de modification radicale de la santé humaine. Le « multiplexing » ou la capacité d’introduire plusieurs modifications simultanées dans le génome sera particulièrement utile en recherche tant fondamentale qu’appliquée. Ce nouvel outil sera susceptible de sonder les fonctions physiopathologiques de circuits génétiques complexes et de développer de meilleures thérapies cellulaires ou traitements antiviraux. En repoussant les limites du génie génétique, il sera possible d’envisager la réécriture et la conception de génomes mammifères. Le développement de notre capacité à modifier profondément le génome pourrait permettre la création de cellules résistantes aux cancers, aux virus ou même au vieillissement ; le développement de cellules ou tissus transplantables compatibles entre donneurs et receveurs ; et pourrait même rendre possible la résurrection d’espèces animales éteintes. Dans ce projet de recherche doctoral, nous présentons l’état de l’art du génie génétique « multiplex », les limites actuelles et les perspectives d’améliorations. Nous tirons profit de ces connaissances ainsi que de l’abondance des éléments transposables de notre ADN afin de construire une plateforme d’optimisation et de développement de nouveaux outils de génie génétique qui autorisent l’édition génomique à grande échelle. Nous démontrons que ces technologies permettent la production de modifications à l’échelle du génome allant jusqu’à 3 ordres de grandeur supplémentaires que précédemment, ouvrant la voie au développement de la réécriture des génomes de mammifères. En outre, l’observation de la toxicité engendrée par la multitude de coupures double-brins dans le génome nous a amenés à développer un bio-interrupteur susceptible d’éviter les effets secondaires des thérapies cellulaires actuelles ou futures. Enfin, en conclusion, nous exposons les potentielles inquiétudes et menaces qu’apporte le domaine génie génétiques et apportons des pistes de réflexions pour diminuer les risques identifiés
Programmable and site-specific nucleases such as CRISPR-Cas9 have started a genome editing revolution, holding hopes to transform human health. Multiplexing or the ability to simultaneously introduce many distinct modifications in the genome will be required for basic and applied research. It will help to probe the physio-pathological functions of complex genetic circuits and to develop improved cell therapies or anti-viral treatments. By pushing the boundaries of genome engineering, we may reach a point where writing whole mammalian genomes will be possible. Such a feat may lead to the generation of virus-, cancer- or aging- free cell lines, universal donor cell therapies or may even open the way to de-extinction. In this doctoral research project, I outline the current state-of-the-art of multiplexed genome editing, the current limits and where such technologies could be headed in the future. We leveraged this knowledge as well as the abundant transposable elements present in our DNA to build an optimization pipeline and develop a new set of tools that enable large-scale genome editing. We achieved a high level of genome modifications up to three orders of magnitude greater than previously recorded, therefore paving the way to mammalian genome writing. In addition, through the observation of the cytotoxicity generated by multiple double-strand breaks within the genome, we developed a bio-safety switch that could potentially prevent the adverse effects of current and future cell therapies. Finally, I lay out the potential concerns and threats that such an advance in genome editing technology may be bringing and point out possible solutions to mitigate the risks
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Valladares, Rodrigo y Hanna Briheim. "Metoder och tillämpningar av CRISPR-Cas9 i cancerforskning. : Samt hur CRISPR-Cas9 kan implementeras i skolundervisningen". Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-166140.

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CRISPR-Cas9 är ett effektivt genredigeringsverktyg som har upptäckts på senare år. Verktyget härstammar från ett adaptivt immunförsvar hos prokaryoter. Tekniken används för att modifiera DNA hos växter, djur och människor på ett enkelt och billigt sätt. CRISPR-Cas9 har visat sig ha stor potential vid bekämpning av olika sjukdomar däribland cancer som idag är ett globalt hälsoproblem. Inom cancerforskningen ses CRISPR-Cas9 som ett lovande verktyg vid cancerterapi och läkemedelsutveckling. I denna studie sammanställer vi aktuella metoder och användningsområden med CRISPR-Cas9 inom cancerforskning. Dessutom undersöker vi hur denna form av genteknik kan lyftas upp och tillämpas i biologiundervisningen.
CRISPR-Cas9 has recently emerged as an effective genome editing tool. The tool derives from an adaptive immune system in prokaryotes. The technology is used for modification of DNA in plants, animals and humans in a simple and inexpensive way. CRISPR-Cas9 has shown great potential in fighting different diseases like cancer which today is a global health issue. It is seen as a promising tool for cancer research when it comes to cancer therapy and drug development. Here we summarize current methods and applications of CRISPR-Cas9 for cancer research. Furthermore, we explore the possibilities of introducing and applying this kind of genetic engineering in biology teaching.

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Toffessi, Tcheuyap Vanina. "Development of von Willebrand Factor Zebrafish Mutant Using CRISPR/Cas9 Mediated Genome Editing". Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc984227/.

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von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.
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7

Canver, Matthew. "Elucidation of Mechanisms of Fetal Hemoglobin Regulation by CRISPR/Cas9 Mediated Genome Editing". Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493407.

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Despite nearly complete understanding of the genetics of the β-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Fetal hemoglobin (HbF) reinduction represents a “silver bullet” for therapy of the β-globin disorders. Recent development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease system has allowed for facile manipulation of the genome for the study of genes and genetic elements. Here we developed CRISPR/Cas9-based methodology to reliably engender targeted genomic deletions ranging from 1.3 kilobases to over 1 megabase, which suggested an inverse relationship between deletion size and deletion frequency. Targeted deletion methods and Cas9-mediated in situ saturating mutagenesis were applied to the enhancer of the HbF repressor BCL11A, which revealed discrete vulnerabilities. This finding is consistent with emerging evidence in the field that large enhancers are comprised of constituent parts with some harboring the majority of the activity. The identified “Achilles heel” of the enhancer represents a promising therapeutic target. We further enhanced the resolution of the in situ saturating mutagenesis technique by using multiple Cas9 nucleases and variant-aware library design to identify functional sequences within the HBS1L-MYB intergenic region, a locus associated with elevated HbF levels. These data demonstrate the robustness of CRISPR/Cas9 mediated in situ saturating mutagenesis and targeted deletion to interrogate functional sequence within regulatory DNA. Harnessing the power of genome editing may usher in a second generation form of gene therapy for the β-globin disorders.
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8

Antoniani, Chiara. "A genome editing approach to induce fetal hemoglobin expression for the treatment of β-hemoglobinopathies". Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB077.

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Les β-hémoglobinopathies (β-thalassémies et drépanocytose) sont des anémies génétiques qui touchent des milliers de nouveaux nés chaque année dans le monde. Ces maladies sont causées par des mutations affectant l'expression de l'hémoglobine chez l'adulte. Le seul traitement disponible est la transfusion sanguine à vie, associée à une chélation du fer. Pour les patients les plus touchés, la greffe de cellule souche hématopoïétique (CSH) demeure le seul traitement curatif. Néanmoins, la transplantation autologue de cellules souches génétiquement corrigées représente une alternative thérapeutique pour les patients dépourvus de donneur compatible. Certaines délétions naturelles comprenant les gènes de la β- et δ- globine dans le locus de l'hémoglobine sont corrélées à une persistance de l'expression de l'hémoglobine fœtale (HPFH) à l'âge adulte. Ainsi il a été démontré que un taux élevé d'hémoglobine fœtale (HbF) améliore l'évolution clinique de ces deux pathologies. Afin d'identifier les régions régulatrices potentielles de la γ-globine, nous avons combiné les données issues d'analyses de mutations rencontrées chez des patients HPFH avec les sites d'hybridation de facteur de transcription. Sur la base de cette analyse, en ayant recours à la technologie CRISPR/CAS9, nous avons développé un protocole permettant de générer: (i) la délétion d'un potentiel suppresseur de l'HbF situé entre les gènes des globines δ et γ, ciblé par le répresseur de l’HbF BCL11A chez les érythroblastes adultes; (ii) la plus courte délétion associée à des taux élevés d’HbF (délétion Corfu) chez les patients β-thalassemiques; (iii) une délétion de 13.6-kb rencontrée fréquemment chez les patients HPFH et incluant les gènes des globines β et δ ainsi que le potentiel suppresseur de l'HbF. Notre travail a montré que la délétion de la région génomique de 13.6-kb entraîne une forte production de HbF et une réduction concomitante de l'expression de la β-globine soit dans des lignées cellulaires érythroïdes humaines soit dans des érythroblastes primaires dérivées des cellules souches et progéniteurs hématopoïétiques (CSPH). Par ailleurs, nous avons montré que la génération de cette délétion sur des CSPHs issus de patients drépanocytaires entraîne une augmentation de la transcription de la γ-globine dans une proportion significative d'érythroblastes, conduisant à une amélioration du phénotype drépanocytaire. Enfin, nous avons exploré le mécanisme menant à la réactivation de l'expression de la γ-globine. Nous avons évalué des changements dans la conformation de la chromatine et des modifications épigénétiques dans le locus de la β-globine lors de la délétion ou de l'inversion de la région de 13.6 kb. Dans l'ensemble, cette étude contribue à la connaissance des mécanismes favorisant l'échange de l'hémoglobine fœtale à l'adulte et fournit des indices pour une approche d'édition du génome dans le traitement de la β-thalassémies et de la drépanocytose
Β-hemoglobinopathies (β-thalassemias and sickle cell disease) are genetic anemias affecting thousands of newborns annually worldwide. β-thalassemias and sickle cell disease (SCD) are caused by mutations affecting the adult hemoglobin expression and are currently treated by red blood cell transfusion and iron chelation regiments. For patients affected by severe β-hemoglobinopathies, allogenic hematopoietic stem cell (HSCs) transplantation is the only definitive therapy. However, transplantation of autologous, genetically corrected HSCs represents an alternative therapy for patients lacking a suitable HSC donor. Naturally occurring large deletions encompassing β- and δ-globin genes in the β-globin gene cluster, defined as Hereditary Persistence of Fetal Hemoglobin (HPFH) traits, lead to increased fetal hemoglobin (HbF) expression ameliorating both thalassemic and SCD clinical phenotypes. In this study, we integrated transcription factor binding site analysis and HPFH genetic data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy disrupting: (i) a putative δγ-intergenic HbF silencer targeted by the HbF repressor BCL11A in adult erythroblasts; (ii) the shortest deletion associated with elevated HbF levels (“Corfu” deletion) in β-thalassemic patients, encompassing the putative δγ-intergenic HbF silencer; (iii) a 13.6-kb genomic region including the δ- and β-globin genes and the putative intergenic HbF silencer. Targeting the 13.6-kb region, but not the Corfu and the putative δγ-intergenic regions, caused a robust HbF re-activation and a concomitant reduction in β-globin expression in an adult erythroid cell line and in healthy donor hematopoietic stem/progenitor cells (HSPC)-derived erythroblasts. We provided a proof of principle of this potential therapeutic strategy: disruption of the 13.6-kb region in HSPCs from SCD donors favored the β-to-γ globin switching in a significant proportion of HSPC-derived erythroblasts, leading to the amelioration of the SCD cell phenotype. Finally, we dissected the mechanisms leading to HbF de-repression demonstrating changes in the chromatin conformation and epigenetic modifications within the β-globin locus upon deletion or inversion of the 13.6-kb region. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult hemoglobin switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia
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Lin, ChieYu. "Characterization and Optimization of the CRISPR/Cas System for Applications in Genome Engineering". Thesis, Harvard University, 2014. http://etds.lib.harvard.edu/hms/admin/view/61.

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The ability to precisely manipulate the genome in a targeted manner is fundamental to driving both basic science research and development of medical therapeutics. Until recently, this has been primarily achieved through coupling of a nuclease domain with customizable protein modules that recognize DNA in a sequence-specific manner such as zinc finger or transcription activator-like effector domains. Though these approaches have allowed unprecedented precision in manipulating the genome, in practice they have been limited by the reproducibility, predictability, and specificity of targeted cleavage, all of which are partially attributable to the nature of protein-mediated DNA sequence recognition. It has been recently shown that the microbial CRISPR-Cas system can be adapted for eukaryotic genome editing. Cas9, an RNA-guided DNA endonuclease, is directed by a 20-nt guide sequence via Watson-Crick base-pairing to its genomic target. Cas9 subsequently induces a double-stranded DNA break that results in targeted gene disruption through non-homologous end-joining repair or gene replacement via homologous recombination. Finally, the RNA guide and protein nuclease dual component system allows simultaneous delivery of multiple guide RNAs (sgRNA) to achieve multiplex genome editing with ease and efficiency. The potential effects of off-target genomic modification represent a significant caveat to genome editing approaches in both research and therapeutic applications. Prior work from our lab and others has shown that Cas9 can tolerate some degree of mismatch with the guide RNA to target DNA base pairing. To increase substrate specificity, we devised a technique that uses a Cas9 nickase mutant with appropriately paired guide RNAs to efficiently inducing double-stranded breaks via simultaneous nicks on both strands of target DNA. As single-stranded nicks are repaired with high fidelity, targeted genome modification only occurs when the two opposite-strand nicks are closely spaced. This double nickase approach allows for marked reduction of off-target genome modification while maintaining robust on-target cleavage efficiency, making a significant step towards addressing one of the primary concerns regarding the use of genome editing technologies. The ability to multiplex genome engineering by simply co-delivering multiple sgRNAs is a versatile property unique to the CRISPR-Cas system. While co-transfection of multiple guides is readily feasible in tissue culture, many in vivo and therapeutic applications would benefit from a compact, single vector system that would allow robust and reproducible multiplex editing. To achieve this, we first generated and functionally validated alternate sgRNA architectures to characterize the structure-function relationship of the Cas9 protein with the sgRNA in DNA recognition and cleavage. We then applied this knowledge towards the development and optimization of a tandem synthetic guide RNA (tsgRNA) scaffold that allows for a single promoter to drive expression of a single RNA transcript encoding two sgRNAs, which are subsequently processed into individual active sgRNAs.
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Hsu, Patrick David. "Development of the CRISPR nuclease Cas9 for high precision mammalian genome engineering". Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13068392.

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Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. To facilitate successful and specific Cas9 targeting, we first optimize the guide RNAs (sgRNA) to significantly enhance gene editing efficiency and consistency. We also systematically characterize Cas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target mutagenesis. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that Cas9-mediated cleavage is unaffected by DNA methylation and that the dosage of Cas9 and sgRNA can be titrated to minimize off-target modification. Additionally, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. We next demonstrate that Cas9 nickase mutants can be used with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs can reduce off-target activity by over 1,500-fold in human cells. In collaboration with researchers at the University of Tokyo, we further identified a PAM-interacting domain of the Cas9 nuclease that dictates Cas9 target recognition specificity. Finally, we present protocols that provide experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks. Taken together, this work enables a variety of genome engineering applications from basic biology to biotechnology and medicine.
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Libros sobre el tema "CRISPR, Cas9, genome editing, gRNA"

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María Vaschetto, Luis. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases. New York: CRC Press, 2021. http://dx.doi.org/10.1201/9781003088516.

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Kozubek, James. Modern Prometheus: Editing the Human Genome with Crispr-Cas9. Cambridge University Press, 2016.

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Ravindhran, Ramalingam, Govindan Ganesan y Vinoth A. Crop Genome Editing Using CRISPR/Cas9: Theory and Practice. Elsevier Science & Technology, 2019.

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Modern Prometheus: Editing the Human Genome with Crispr-Cas9. Cambridge University Press, 2018.

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Kozubek, Jim. Modern Prometheus: Editing the Human Genome with Crispr-Cas9. Cambridge University Press, 2018.

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Ravindhran, Ramalingam, Govindan Ganesan y Vinoth A. Crop Genome Editing Using CRISPR/Cas9: Theory and Practice. Elsevier Science & Technology Books, 2019.

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Kozubek, James. Modern Prometheus: Editing the Human Genome with Crispr-Cas9. Cambridge University Press, 2019.

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Vaschetto, Luis María. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases. Taylor & Francis Group, 2021.

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Vaschetto, Luis M. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases. CRC Press LLC, 2022.

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Vaschetto, Luis M. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases. Taylor & Francis Group, 2022.

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Capítulos de libros sobre el tema "CRISPR, Cas9, genome editing, gRNA"

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Hoof, Jakob B., Christina S. Nødvig y Uffe H. Mortensen. "Genome Editing: CRISPR-Cas9". En Methods in Molecular Biology, 119–32. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7804-5_11.

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Seruggia, Davide y Lluis Montoliu. "CRISPR/Cas9 Approaches to Investigate the Noncoding Genome". En Genome Editing, 31–43. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-34148-4_2.

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Patial, Meghna, Kiran Devi y Rohit Joshi. "CRISPR/Cas9-Mediated Targeted Mutagenesis in Medicinal Plants". En Genome Editing, 55–70. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-08072-2_3.

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Bansal, Monika y Shabir Hussain Wani. "Virus-Mediated Delivery of CRISPR/CAS9 System in Plants". En Genome Editing, 197–203. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-08072-2_10.

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Gurumurthy, Channabasavaiah B., Rolen M. Quadros, Masahiro Sato, Tomoji Mashimo, K. C. Kent Lloyd y Masato Ohtsuka. "CRISPR/Cas9 and the Paradigm Shift in Mouse Genome Manipulation Technologies". En Genome Editing, 65–77. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-34148-4_4.

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Katam, Ramesh, Fatemeh Hasanvand, Vinson Teniyah, Jessi Noel y Virginia Gottschalk. "Biosafety Issue Related to Genome Editing in Plants Using CRISPR-Cas9". En Genome Editing, 289–317. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-08072-2_16.

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Saeed, Fozia, Tariq Shah, Sherien Bukhat, Fazal Munsif, Ijaz Ahmad, Hamad Khan y Aziz Khan. "Genome Engineering as a Tool for Enhancing Crop Traits: Lessons from CRISPR/Cas9". En Genome Editing, 3–25. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-08072-2_1.

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Nadakuduti, Satya Swathi, Colby G. Starker, Daniel F. Voytas, C. Robin Buell y David S. Douches. "Genome Editing in Potato with CRISPR/Cas9". En Methods in Molecular Biology, 183–201. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-8991-1_14.

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Liu, Junqi, Samatha Gunapati, Nicole T. Mihelich, Adrian O. Stec, Jean-Michel Michno y Robert M. Stupar. "Genome Editing in Soybean with CRISPR/Cas9". En Methods in Molecular Biology, 217–34. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-8991-1_16.

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Velusamy, Thilaga, Anjali Gowripalan y David C. Tscharke. "CRISPR/Cas9-Based Genome Editing of HSV". En Methods in Molecular Biology, 169–83. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9814-2_9.

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Actas de conferencias sobre el tema "CRISPR, Cas9, genome editing, gRNA"

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Shukla, Jayanti, Bhairvi Pant, Neema Tufchi, Gracy Chand Paul, Kumud Pant, Manu Pant y Somya Sihna. "CRISPR Cas9 genome editing approach for gRNA of the genes associated with Schizophrenia: A computational approach". En 2022 2nd International Conference on Innovative Sustainable Computational Technologies (CISCT). IEEE, 2022. http://dx.doi.org/10.1109/cisct55310.2022.10046633.

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Stacey, Minviluz. "Utility of CRISPR/Cas in accelerating gene discovery in soybean". En 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/rzne1660.

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The use of CRISPR/Cas9 has been successfully applied in various plant species to induce targeted genome editing, including soybean. Soybean is recalcitrant to transformation and thus, plants with stable CRISPR gene edits are costly and take a long time to produce. Moreover, soybean is allotetraploid and editing paralogous genes are often necessary to obtain observable phenotype(s). For each gene target, we designed two gRNAs to increase editing efficiency and allow rapid genotyping by PCR. We also tested the CRISPR reagents in transient hairy root transformation to determine if the Cas9 and gRNAs would perform properly in transgenic soybean plants. Our results showed that we can indeed obtain highly efficient, cost-effective CRISPR/Cas editing in soybean to generate novel genotypes for gene discovery and downstream field propagation and breeding efforts. Examples of CRISPR-edited genes and their associated seed traits will be discussed.
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METLEVA, Anastasia S. y Konstantin V. BESPOMESTNYKH. "Selection of Grna for Genomic Editing of the Bovine Leucosis Virus Susceptible Alleles of the 2 Exon of the Bola-DRB3 Gene by CRISPR/Cas9". En IV International Scientific and Practical Conference "Modern S&T Equipments and Problems in Agriculture". Sibac, 2020. http://dx.doi.org/10.32743/kuz.mepa.2020.148-157.

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Tedesco, Donato, Paul Diehl, Mikhail Makhanov, Sylvain Baron, Dmitry Suchkov y Alex Chenchik. "Abstract C161: CRISPR/Cas9 genome-wide gRNA library screening platform". En Abstracts: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; November 5-9, 2015; Boston, MA. American Association for Cancer Research, 2015. http://dx.doi.org/10.1158/1535-7163.targ-15-c161.

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Tedesco, Donato, Paul Diehl, Mikhail Makhanov, Sylvain Baron, Dmitry Suchkov, Costa Frangou y Alex Chenchik. "Abstract 4354: CRISPR/Cas9 genome-wide gRNA library for target identification". En Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-4354.

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Rastogi, Khushboo. "Rice Biofortification through CRISPR/Cas9-Multiplex Genome Editing". En ASPB PLANT BIOLOGY 2020. USA: ASPB, 2020. http://dx.doi.org/10.46678/pb.20.1383191.

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Shima, K., T. Suzuki, Y. Ma, C. Mayhew, A. Sallese, B. C. Carey, P. Arumugam y B. C. Trapnell. "CRISPR/Cas9 Genome Editing Therapy for Hereditary Pulmonary Alveolar Proteinosis". En American Thoracic Society 2019 International Conference, May 17-22, 2019 - Dallas, TX. American Thoracic Society, 2019. http://dx.doi.org/10.1164/ajrccm-conference.2019.199.1_meetingabstracts.a4004.

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Кершанская, О. И., Г. Л. Есенбаева, Д. С. Нелидова, З. Н. Садуллаева y С. Н. Нелидов. "PERSPECTIVES OF BREEDING DEVELOPMENT IN BARLEY THROUGH CRISPR/CAS9 GENOME EDITING". En Материалы I Всероссийской научно-практической конференции с международным участием «Геномика и современные биотехнологии в размножении, селекции и сохранении растений». Crossref, 2020. http://dx.doi.org/10.47882/genbio.2020.48.47.015.

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Zheng, Qi, Ling-Jie Kong, Huanyu Jin, Jinling Li y Ruby Yanru Chen-Tsai. "Abstract 663: Factors affecting genome editing using CRISPR/Cas9 in mouse model". En Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-663.

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"CRISPR/Cas9 – mediated genome editing of bread wheat to modulate heading time". En Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-135.

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Informes sobre el tema "CRISPR, Cas9, genome editing, gRNA"

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Paran, Ilan y Allen Van Deynze. Regulation of pepper fruit color, chloroplasts development and their importance in fruit quality. United States Department of Agriculture, enero de 2014. http://dx.doi.org/10.32747/2014.7598173.bard.

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Pepper exhibits large natural variation in chlorophyll content in the immature fruit. To dissect the genetic and molecular basis of this variation, we conducted QTL mapping for chlorophyll content in a cross between light and dark green-fruited parents, PI 152225 and 1154. Two major QTLs, pc1 and pc10, that control chlorophyll content by modulation of chloroplast compartment size in a fruit-specific manner were detected in chromosomes 1 and 10, respectively. The pepper homolog of GOLDEN2- LIKE transcription factor (CaGLK2) was found as underlying pc10, similar to its effect on tomato fruit chloroplast development. A candidate gene for pc1was found as controlling chlorophyll content in pepper by the modulation of chloroplast size and number. Fine mapping of pc1 aided by bulked DNA and RNA-seq analyses enabled the identification of a zinc finger transcription factor LOL1 (LSD-One-Like 1) as a candidate gene underlying pc1. LOL1 is a positive regulator of oxidative stress- induced cell death in Arabidopsis. However, over expression of the rice ortholog resulted in an increase of chlorophyll content. Interestingly, CaAPRR2 that is linked to the QTL and was found to affect immature pepper fruit color in a previous study, did not have a significant effect on chlorophyll content in the present study. Verification of the candidate's function was done by generating CRISPR/Cas9 knockout mutants of the orthologues tomato gene, while its knockout experiment in pepper by genome editing is under progress. Phenotypic similarity as a consequence of disrupting the transcription factor in both pepper and tomato indicated its functional conservation in controlling chlorophyll content in the Solanaceae. A limited sequence diversity study indicated that null mutations in CaLOL1 and its putative interactorCaMIP1 are present in C. chinensebut not in C. annuum. Combinations of mutations in CaLOL1, CaMIP1, CaGLK2 and CaAPRR2 are required for the creation of the extreme variation in chlorophyll content in Capsicum.
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