Rapid self-selecting and clone-free integration of transgenes into engineered CRISPR safe harbor locations in Caenorhabditis elegans
| Autor: | Megan J. Moerdyk-Schauwecker, Zachary C Stevenson, Brennen Jamison, Patrick C. Phillips |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Transposable element
clone free Transgene ved/biology.organism_classification_rank.species Computational biology Investigations QH426-470 Insert (molecular biology) Animals Genetically Modified 03 medical and health sciences 0302 clinical medicine Genome editing C . elegans Extrachromosomal DNA Genetics Animals CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Transgenes Guide RNA crispr Caenorhabditis elegans Model organism Molecular Biology Genetics (clinical) 030304 developmental biology Gene Editing 0303 health sciences biology ved/biology c. elegans transformation biology.organism_classification safe harbor CRISPR-Cas Systems 030217 neurology & neurosurgery |
| Zdroj: | G3: Genes, Genomes, Genetics, Vol 10, Iss 10, Pp 3775-3782 (2020) G3: Genes|Genomes|Genetics |
| Popis: | Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert transgenes into the model nematode Caenorhabditis elegans has focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require laborious screening processes that often result in false positives from heritable extrachromosomal arrays or rely on co-CRISPR markers to identify likely edited individuals. As a result, verification of transgene insertion requires anti-array selection screening methods or extensive PCR genotyping respectively. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods, and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native homology-based formation of extra-chromosomal arrays to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|