Back to Search
Start Over
Rapid and precise engineering of the Caenorhabditis elegans genome with lethal mutation co-conversion and inactivation of NHEJ repair.
- Source :
-
Genetics [Genetics] 2015 Feb; Vol. 199 (2), pp. 363-77. Date of Electronic Publication: 2014 Dec 09. - Publication Year :
- 2015
-
Abstract
- As in other organisms, CRISPR/Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks (DSBs). Here, I describe a methodology in C. elegans to efficiently knock in epitope tags in 8-9 days, using a temperature-sensitive lethal mutation in the pha-1 gene as a co-conversion marker. I demonstrate that 60mer oligos with 29 bp of homology drive efficient knock-in of point mutations, and that disabling nonhomologous end joining by RNAi inactivation of the cku-80 gene significantly improves knock-in efficiency. Homology arms of 35-80 bp are sufficient for efficient editing and DSBs up to 54 bp away from the insertion site produced knock-ins. These findings will likely be applicable for a range of genome editing approaches in C. elegans, which will improve editing efficiency and minimize screening efforts.<br /> (Copyright © 2015 by the Genetics Society of America.)
- Subjects :
- Animals
DNA Breaks, Double-Stranded
Gene Conversion
Gene Deletion
Gene Knock-In Techniques
Gene Targeting methods
Genes, Helminth
Male
RNA Editing
RNA Interference
RNA, Helminth
Recombinational DNA Repair
Caenorhabditis elegans genetics
DNA End-Joining Repair
Genes, Lethal
Genetic Engineering methods
Genome, Helminth
Mutation
Subjects
Details
- Language :
- English
- ISSN :
- 1943-2631
- Volume :
- 199
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Genetics
- Publication Type :
- Academic Journal
- Accession number :
- 25491644
- Full Text :
- https://doi.org/10.1534/genetics.114.172361