Academic journal article Genetics

Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis Elegans

Academic journal article Genetics

Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis Elegans

Article excerpt

ABSTRACT Facilitated by recent advances using CRISPR/Cas9, genome editing technologies now permit custom genetic modifications in a wide variety of organisms. Ideally, modified animals could be both efficiently made and easily identified with minimal initial screening and without introducing exogenous sequence at the locus of interest or marker mutations elsewhere. To this end, we describe a coconversion strategy, using CRISPR/Cas9 in which screening for a dominant phenotypic oligonucleotide-templated conversion event at one locus can be used to enrich for custom modifications at another unlinked locus. After the desired mutation is identified among the F^sub 1^ progeny heterozygous for the dominant marker mutation, F^sub 2^ animals that have lost the marker mutation are picked to obtain the desired mutation in an unmarked genetic background. We have developed such a coconversion strategy for Caenorhabditis elegans, using a number of dominant phenotypic markers. Examining the coconversion at a second (unselected) locus of interest in the marked F^sub 1^ animals, we observed that 14-84% of screened animals showed homologous recombination. By reconstituting the unmarked background through segregation of the dominant marker mutation at each step, we show that custom modification events can be carried out recursively, enabling multiple mutant animals to be made. While our initial choice of a coconversion marker [rol-6(su1006)] was readily applicable in a single round of coconversion, the genetic properties of this locus were not optimal in that CRISPR-mediated deletion mutations at the unselected rol-6 locus can render a fraction of coconverted strains recalcitrant to further rounds of similar mutagenesis. An optimal marker in this sense would provide phenotypic distinctions between the desired mutant/+ class and alternative +/+, mutant/null, null/null, and null/+ genotypes. Reviewing dominant alleles from classical C. elegans genetics, we identified one mutation in dpy-10 and one mutation in sqt-1 that meet these criteria and demonstrate that these too can be used as effective conversion markers. Coconversion was observed using a variety of donor molecules at the second (unselected) locus, including oligonucleotides, PCR products, and plasmids. We note that the coconversion approach described here could be applied in any of the variety of systems where suitable coconversion markers can be identified from previous intensive genetic analyses of gain-of-function alleles.

TYPE II CRISPR/Cas9 bacterial immunity systems provide programmable DNA endonuclease activities that have recently revolutionized genome editing in a wide range of organisms (Wang et αi. 1999; Chiu et αi. 2013; Cho et αi 2013; Dicarlo et αi. 2013; Friedland et αi 2013; Gratz et αi. 2013; Hwang et αi. 2013; Jiang et αi. 2013; Katie and Großhans 2013; Li et αi. 2013; Lo et αi. 2013; Nekrasov et αi. 2013; Kim et αi. 2014; Zhao et αi. 2014). Recognition by the Cas9 protein entails two sequence elements in the target: a protospacer adjacent motif (PAM) (NGG for Strep- tococcus pyogenes Cas9) and a region of ~20 bp of comple- mentarity to its guide RNA (gRNA) (Jinek et αi. 2012). Following cleavage by CRISPR/Cas9 in vivo, the double- strand break site can be repaired to generate mutations, including insertions and deletions via endogenous pathways such as nonhomologous end joining (NHEJ) or targeted base mutations via homologous repair (HR) from a template or donor DNA. The ease of use of CRISPR/Cas9 for genome editing has led to its widespread adoption and promises to usher in a new era of biology.

In our application of CRISPR/Cas9 to edit the Caenorhab- ditis elegans genome, we sought a conversion system that met the following criteria: (1) It should be possible to make any mutation in a gene, without extraneous marker sequen- ces, and with no constraint on the genetic background; (2) edited animals should be efficiently made and easily identifi- able, so that multiple independent isolates are recovered with minimal downstream screening; and (3) the system should be fast, enabling isolation of the mutation in as few generations as possible, and require minimal plasmid con- struction. …

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