Genome editing. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations

Abstract

An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype, whereas individuals homozygous for two copies of the allele will display a mutant phenotype. We have developed a method called the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome-editing system for generating autocatalytic mutations, to produce homozygous loss-of-function mutations. In Drosophila, we found that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome, thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms.

Figure 1

Scheme outlining the Mutagenic Chain Reaction (MCR). A plasmid consisting of a core cassette carrying a Cas9 transgene, a gRNA targeting a genomic sequence of interest, and flanking homology arms corresponding to genomic sequences abutting the target cleavage site (A) inserts the core Cas9/gRNA cassette into the targeted locus via HDR (B,C). In turn, the inserted cassette expresses both Cas9 and the gRNA leading to cleavage (D) and HDR-mediated insertion of the cassette into the second allele, thereby rendering the mutation homozygous (E,F). HA1 and HA 2 denote the two homology arms that directly flank the gRNA-directed cut site.

Figure 2

Experimental demonstration of MCR in Drosophila. A) Mendelian male inheritance of an X-linked trait. B) Theoretical MCR-based inheritance results in the initially heterozygous allele converting the second allele generating homozygous female progeny. C) Diagram of y-MCR construct. Two y locus homology arms flanking the vasa-Cas9 and y-gRNA transgenes are indicated and the locations of the PCR primers used for analysis of the genomic insertion site (listed in the Methods section). D) PCR analysis of a y+ MCR-derived F2♂ (lanes 1–3, see Fig. S1 for sequence), y^MCR F1♀ (lanes 4–6) and ♂ (lanes 7–9) showing junctional bands corresponding to y-MCR insertion into the chromosomal y locus (lanes 2–3,5–6,8–9) and an approximately wild-type size band from the y locus (lanes 1,4,7). Although the y^MCR F1♂ (carrying a single X-chromosome) displays only MCR derived PCR products (lanes 8–9), y^MCR F1♀s generate both MCR and non-insertional amplification products. E) Summary of F2 progeny obtained from crosses described in detail in Table S1. F) Low magnification view of F2 progeny flies from an y^MCR X y+. Nearly all female progeny display a y- phenotype. G) High magnification view of a full body y^MCR F1♀. H) A rare 50% left-right mosaic female. I) A y+ control fly.