The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations

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Science  24 Apr 2015:
Vol. 348, Issue 6233, pp. 442-444
DOI: 10.1126/science.aaa5945
  • Fig. 1 Scheme outlining the mutagenic chain reaction (MCR).

    (A to C) 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) and (C)]. (D to F) 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) and (F)]. HA1 and HA2 denote the two homology arms that directly flank the gRNA-directed cut site.

  • Fig. 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, thereby 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, as are the locations of the PCR primers used for analysis of the genomic insertion site (see supplementary materials). (D) PCR analysis of a y+ MCR-derived F2♂ (lanes 1 to 3; see fig. S1 for sequence), yMCR F1♀ (lanes 4 to 6), and yMCR F1♂ (lanes 7 to 9) showing junctional bands corresponding to y-MCR insertion into the chromosomal y locus (lanes 2, 3, 5, 6, 8, and 9) and the presence (lanes 1 and 4) or absence (lane 7) of a PCR band derived from the y locus. Although the yMCR F1♂ (carrying a single X chromosome) displays only MCR-derived PCR products (lanes 8 and 9), yMCR F1♀s generate both MCR and noninsertional amplification products. (E) Summary of F2 progeny obtained from crosses described in table S1. (F) Low-magnification view of F2 progeny flies from an yMCR × ♂ x y+♀ cross. Nearly all female progeny display a y– phenotype. (G) High-magnification view of a full-bodied yMCR F1♀. (H) A rare 50% left-right mosaic female. (I) A y+ control fly.

Supplementary Materials

  • The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations

    Valentino M. Gantz and Ethan Bier

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text
    • Fig. S1
    • Table S1

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