A5029678421- CRISPR and Genetic Engineering
- RNA and protein synthesis mechanisms
- Advanced biosensing and bioanalysis techniques
- Plant Virus Research Studies
- Chromosomal and Genetic Variations
- Insect symbiosis and bacterial influences
- Plant Reproductive Biology
- Innovation and Socioeconomic Development
- Photosynthetic Processes and Mechanisms
- Genomics and Phylogenetic Studies
- Evolution and Genetic Dynamics
- Genetics, Bioinformatics, and Biomedical Research
- RNA Interference and Gene Delivery
- RNA Research and Splicing
- RNA regulation and disease
- Mosquito-borne diseases and control
- Plant tissue culture and regeneration
- Sunflower and Safflower Cultivation
- Genetic Mapping and Diversity in Plants and Animals
- Bacterial Genetics and Biotechnology
- Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Pioneer Hi-Bred
2015-2023
Corteva (United States)
2018-2022
University of Wisconsin–Madison
2001
Abstract Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In past two decades, several break technologies developed. CRISPR–Cas9 has quickly become technology choice for editing due its simplicity, efficiency versatility. Currently, in plants primarily relies on delivering reagents form vectors. Here we report biolistic delivery pre-assembled Cas9–gRNA ribonucleoproteins into maize embryo cells regeneration with both...
Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing codon-optimized Streptococcus pyogenes Cas9 endonuclease single guide RNAs were cointroduced with or without repair templates into immature embryos by biolistic transformation targeting five different genomic regions: upstream the...
In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery extend application. Here, we present collection of 10 exceptionally (422-603 amino acids) CRISPR-Cas12f that recognize dsDNA in PAM dependent manner. Categorized as...
To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method simultaneous examination guide RNA and protospacer adjacent motif (PAM) requirements. The relies on cleavage plasmid libraries containing randomized PAM as function Cas9-guide complex concentration. Using this method, accurately reproduce canonical preferences Streptococcus pyogenes, thermophilus CRISPR3 (Sth3), CRISPR1 (Sth1). Additionally, sgRNA solutions novel Cas9 protein from Brevibacillus...
Abstract Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants used for applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free screens assess the protospacer adjacent motif (PAM) guide RNA (gRNA) requirements 79 proteins, thus identifying at least 7 distinct gRNA...
Abstract Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small 400-600 aa Cas12f. For in vivo genome editing applications, compact RNA-guided desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown cleave...
Abstract CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in complex plant genome. Our approach pairs computational prediction with genome-wide biochemical detection followed by validation maize plants. results reveal high frequency (up 90%) on-target editing no evidence cleavage activity when...
Abstract CRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome transcriptome of eukaryotic organisms. Most groups use class 2 effectors, such as Cas9 Cas12a, however, other may provide unique opportunities genome engineering. Indeed, multi-subunit composition 1 offers to expand number domains functionalities that be recruited a genomic target. Here we report DNA targeting in Zea mays using type I-E system from S. thermophilus . First, engineer its Cascade...
Modern maize hybrids often contain biotech and native traits. To-date all traits have been randomly inserted in the genome. Consequently, developing with multiple is expensive, time-consuming complex. Here we report using CRISPR-Cas9 to generate a Complex Trait Locus (CTL) facilitate trait stacking. A CTL consists of preselected sites positioned within small well-characterized chromosomal region where genes are inserted. We generated individual lines, each carrying site-specific insertion...
Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need emasculate female inbred during hybrid seed production, referred as Seed Production Technology, been described. The foundation of this system relied on classical methods identify genes critical anther pollen development. One these is P450 which expressed tapetum anthers....
Article21 October 2022Open Access Source DataTransparent process A new family of CRISPR-type V nucleases with C-rich PAM recognition Tomas Urbaitis CasZyme, Vilnius, Lithuania Institute Biotechnology, Vilnius University, Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, review & editing Search for more papers by this author Giedrius Gasiunas Corresponding Author [email protected]...
ABSTRACT In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery extend application. Here, we present collection of 10 exceptionally (422-603 amino acids) CRISPR-Cas that recognize dsDNA in PAM dependent manner. Categorized...
Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas)9 transactivating CRISPR RNAs (tracrRNAs) form distinct structures essential for target recognition and cleavage dictate exchangeability between orthologous proteins. As noncoding that are often apart from the array, their identification can be arduous. In this article, a new bioinformatic method detection of Cas9 tracrRNAs is presented. The approach utilizes covariance model based on both sequence homology...
Abstract (Note: a correction was made to the Index Reverse primer 20 Sep2023) CRISPR-Cas9 RNA-guided endonucleases are widely used in genome engineering, yet information on biochemical and cellular off-target cleavage activity is lacking. Here, we present method, based selective enrichment identification of adapter-tagged DNA ends by sequencing \(SITE-Seq). SITE-Seq can be identify sites within genomic sample. This protocol details preparation libraries for high throughput Next Generation...
ABSTRACT CRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted characterization 79 orthologs with at least 7 distinct classes gRNAs 50 different PAM sequence requirements. recognition spanned entire spectrum T-, A-, C-, G-rich nucleotides ranging from simple di-nucleotide...
Abstract Cas9 trans-activating CRISPR RNAs (tracrRNAs) form distinct structures essential for target recognition and cleavage dictate exchangeability between orthologous proteins. As non-coding that are often apart from the array, their identification can be arduous. In this paper, a new bioinformatic method detection of tracrRNAs is presented. The approach utilizes co-variance model (CM) based on both sequence homology predicted secondary structure to locate tracrRNAs. This predicts...
A sunflower ( Helianthus annuus L.) line that produces some plants with heads of vegetative tissue was studied. These completely sterile have a massive proliferation chaffy where disk florets, and later achenes, usually form. The resulting head is revolute, extremely contorted, more or less globular, splits in the center early development. Ray flowers are not present, nor usual florets. derived from PI 175728 introduced Turkey. In 1976 normal were selfed segregating nursery rows, 113 normal:...