pha-1 co-conversion: "To find a needle, remove the haystack."
In the background are 504 restriction digests of PCR products used to identify a single 2xFLAG knock-in by direct screening. The center images depict the power of lethal mutation co-selection. Only animals rescued for the conditional lethal mutation survive the selection; from a representative co-selection experiment, five knock-ins were recovered from seven rescued F1 animals (gel image under worm).
Genome editing with the CRISPR/Cas9 system has revolutionized genetics in a wide range of model organisms. A major challenge has been to recover rare editing events quickly and easily. In C. elegans. a range of approaches have been developed to circumvent these challenges. Dug resistance cassettes, GFP, or unc-119 markers can be used to enrich for rare knock-ins (Dickinson et al., 2013; Chen et al., 2013; Tzur et al., 2013). Recently co-CRISPR (Kim et al., 2014) and co-conversion (Arribere et al., 2014) approaches where mutation or knock-in at one locus that produce visible phenotypes enriched for edits at other genomic loci, obviating the need for selection markers.
Recently, I developed an alternate co-conversion marker: a temperature-sensitive lethal allele of pha-1. Oligo-templated repair of a CRISPR/Cas9 DNA double strand break at the pha-1 locus enriches for CRISPR/Cas9-catalyzed modifications at other loci and allows recovery of edits with minimal handling. Other than the injected parental animal, only pha-1(ts) rescued F1 are alive on a plate. Inactivation of non-homologous end-joining further increases knock-in efficiency. Homology arms of 35-80 bp are sufficient for efficient editing, and DSBs up to 54 bp away from the insertion point resulted in knock-ins. This method is described in detail in a publication (Ward, Genetics, 2015). Here, I provide a detailed guide to experimental design and practical considerations. A link to a printable PDF document is also provided below.
Recently, I developed an alternate co-conversion marker: a temperature-sensitive lethal allele of pha-1. Oligo-templated repair of a CRISPR/Cas9 DNA double strand break at the pha-1 locus enriches for CRISPR/Cas9-catalyzed modifications at other loci and allows recovery of edits with minimal handling. Other than the injected parental animal, only pha-1(ts) rescued F1 are alive on a plate. Inactivation of non-homologous end-joining further increases knock-in efficiency. Homology arms of 35-80 bp are sufficient for efficient editing, and DSBs up to 54 bp away from the insertion point resulted in knock-ins. This method is described in detail in a publication (Ward, Genetics, 2015). Here, I provide a detailed guide to experimental design and practical considerations. A link to a printable PDF document is also provided below.
