RESUMO
Efficient and precise genome editing requires a fast, quantitative, and inexpensive assay to assess genotype following editing. Here, we present ICE (Inference of CRISPR Edits), which enables robust analysis of CRISPR edits using Sanger data. ICE proposes potential outcomes for editing with guide RNAs, and then determines which are supported by the data via regression. The ICE algorithm is robust and reproducible, and it can be used to analyze CRISPR experiments within days after transfection. We also confirm that ICE produces accurate estimates of editing outcomes across a variety of benchmarks, and within the context of other existing Sanger analysis tools. The ICE tool is free to use and open source, and offers several improvements over current analysis tools, such as batch analysis and support for a variety of editing conditions. It is available online at ice.synthego.com, and the source code is available at github.com/synthego-open/ice.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , RNA Guia de Cinetoplastídeos/genética , SoftwareRESUMO
Following introduction of CRISPR-Cas9 components into a cell, genome editing occurs unabated until degradation of its component nucleic acids and proteins by cellular processes. This uncontrolled reaction can lead to unintended consequences including off-target editing and chromosomal translocations. To address this, we develop a method for light-induced degradation of sgRNA termed CRISPRoff. Here we show that light-induced inactivation of ribonucleoprotein attenuates genome editing within cells and allows for titratable levels of editing efficiency and spatial patterning via selective illumination.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Luz , Estabilidade de RNA/efeitos da radiação , RNA Guia de Cinetoplastídeos/metabolismo , Sistemas CRISPR-Cas/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Estudos de Viabilidade , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/efeitos da radiação , Ribonucleoproteínas/metabolismo , Translocação GenéticaRESUMO
The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies using purified, recombinant Kelch protein showed that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Two-dimensional electrophoresis demonstrated that Kelch is tyrosine phosphorylated in a src64-dependent pathway. Site-directed mutagenesis determined that tyrosine residue 627 is phosphorylated. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescued the actin disorganization phenotype of kelch mutant ring canals, but failed to produce wild-type ring canals. Electron microscopy demonstrated that phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopied src64 ring canals. KelY627A protein in ring canals also dramatically reduced the rate of actin monomer exchange. The phenotypes caused by src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of actin cross-linking by Kelch.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Proteínas de Insetos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Transdução de Sinais/fisiologia , Actinas/genética , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Reagentes de Ligações Cruzadas , Drosophila/metabolismo , Feminino , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica/métodos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Tirosina/genética , Tirosina/metabolismoRESUMO
Flytrap is a web-enabled relational database of transposable element insertions in Drosophila melanogaster. A green fluorescent protein (GFP) artificial exon carried by a transposable P-element is mobilized and inserted into a host gene intron creating a GFP fusion protein. The sequence of the tagged gene is determined by sequencing inverse-PCR products derived from genomic DNA. Flytrap contains two principle data types: micrographs of protein localization and a cellular component ontology, based on rules derived from the Gene Ontology consortium (http://www.geneontology.org), describing protein localization. Flytrap also has links to gene information contained in Flybase (http:// flybase.bio.indiana.edu). The system is designed to accept submissions of micrographs and descriptions from any type of tissue (e.g. wing imaginal disk, ovary) and at any stage of development. Insertion lines can be searched using a number of queries, including Berkeley Drosophila Genome Project (BDGP) numbers and protein localization. In addition, Flytrap provides online order forms linked to each insertion line so that users may request any line generated from this project. Flytrap may be accessed from the homepage at http://flytrap.med. yale.edu.