Efficient allelic-drive in Drosophila

Abstract

Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.

Fig. 1

Super-Mendelian inheritance of a dominant Notch allele in Drosophila. a Scheme depicting a DsRed-marked y^<ccN> CopyCat element that carries two gRNAs: (1) gRNA-y (yellow), which drives copying of the y^<ccN> element at the yellow locus; and (2) gRNA-N+ (blue), which directs cleavage of the sensitive (S) wild-type Notch allele N^+S (scissors icon) to drive Super-Mendelian inheritance of the cleavage-insensitive (IS) N^Ax16 allele (N*, lock icon). Cas9 is provided in trans. b DNA sequence of the gRNA-N+ target site on the sensitive wild-type Notch allele (N⁺), and the cleavage-insensitive N^Ax16 allele (N*) is highlighted in blue, and PAM site in pink. The critical G → A substitution conferring cleavage insensitivity in N^Ax16 mutants is bold in red. The Cas9/gRNA cleavage site is indicated with a dashed line. c Wing phenotypes of wild-type (WT), N^Ax16 homozygous (N*/N*), N⁻ loss-of-function heterozygous (N⁻/N⁺), and N^Ax16/N⁻ heterozygous (N^*/N⁻) Drosophila adults. d Crossing scheme used to generate F1 “master females” and genotype classes of 104 isogenic lines from single F2 females (detailed analysis in Table S2). X donor chromosome carrying the DsRed marked y^<ccN> element (DR) and the N^Ax16 (N*) allele appears in light blue. WT (++) cut sensitive receiver chromosome is in dark blue. Third chromosome carrying a GFP-marked transgene expressing Cas9 (vasaCas9) is depicted in green, and wild-type (+) chromosomes appear in light gray. The multiply inverted FM7 balancer chromosome is depicted in dark gray. Red and blue arrowheads indicate copying of the y^<ccN> element and the N^Ax16 allele, respectively. e Percentage of transmission of y^<ccN> (DR, red circles) and N^Ax16 allele (N*, blue circles) in the presence or absence (gray circles) of Cas9. p Value intervals for this and all subsequent unpaired parametric t test analysis: ****p < 0.0001; ***p < 0.001; *p < 0.05. Bars denote mean value and standard deviation in this and all subsequent graphs. f Percentage conversion of receiver chromosomes in F2 progeny from F1 master females (y^<ccN> w^a N^Ax16/++; Cas9/+ ♀ X w⁻ ♂). Eye color was used to distinguish progeny receiving donor (w^a = orange eyes) vs. receiver (w⁻ = white eyes) chromosomes

Fig. 2

Drive-induced lethal events on the receiver chromosome. a The frequency of progeny inheriting the receiver chromosome from F1 master females (dark blue circles) compared to control F1 females lacking Cas9 (gray circles) in males vs. females. b Overnight embryo collections stained with an antibody against the pan-neural Elav protein. Embryos from wild-type females display normal central (CNS) and peripheral (PNS) nervous systems. Some embryos derived from F1 master females reveal a classic neurogenic phenotype in which nearly all cells derived from the ventral neuroectoderm develop as neurons. N⁻ control embryos collected from N^55e11/+ mothers. c Frequencies of embryos displaying neurogenic phenotypes in collections derived from control w⁻ mothers or from F1 master females crossed either to wild-type (WT; actually w⁻) ♂ or to N^Ax16 ♂. d Top panel: Sequence of a cleavage-insensitive N⁺ allele (N^+IS). Note the nucleotide change from C → A at position −4 relative to the PAM sequence for the N^+IS allele (which results in the phenotypically silent S → Y amino acid substitution. Middle panel: Crossing scheme in which F1 master females are mated to males carrying a WT N^+IS allele. Other diagram elements are the same as in Fig. 1d. Lower panel: percentage of adult female progeny displaying dominant N^−/+ heterozygous wing phenotype consisting of wing margin notches and thickened veins (see Fig. 1c). The presence of the N^−/+ phenotypic class was strictly Cas9 dependent. e Left panel: crossing scheme for testing inheritance potential of an NHEJ-induced N⁻ allele by progeny of y^<ccN> N⁻/Balancer females crossed to males carrying a vasaCas9 transgene on the third chromosome. Right panel: experimental results for three different NHEJ-induced N⁻ alleles (N⁻¹⁷, N⁻²⁰, and N⁻²¹) revealing that zero progeny were recovered from ccN N⁻ females carrying any of these three N⁻ alleles in the presence of zygotically provided Cas9

Fig. 3

Shadow-drive and co-drive analyses. a Three-generation crossing scheme for analyzing shadow-drive in the F3 generation. F2 ♀ derived from F1 master females were crossed to N^+IS ♂, and F3 progeny were scored for percentage conversion of F2 receiver chromosomes and generation of N⁻ alleles. b Percentage of F3 progeny demonstrating features of drive including (from left to right): percentage of receiver chromosomes converted to N^Ax16 allele (N*, blue circles; presence or absence of Cas9 refers to F1 generation); percentage of heterozygous N^−/+ females (N⁻, black circles); and percentage of individuals having copied DsRed marked y^<ccN> element to receiver chromosomes in either males or females (red circles). The small percentages shown in control crosses (gray circles), in which F1 females lacked source of Cas9, presumably reflect the low rates of recombination between w^a and N^Ax16 (0.5 cm) or the y^<ccN> element (1.5 cm). c Evidence for co-drive of N^Ax16 (N*) allele with DsRed (DR+) marked y^<ccN> element among individuals inheriting receiver chromosomes. d Chromosome pairing is required for efficient allelic-drive and co-drive. Top panel: Genetic crossing scheme depicting allelic-drive to a balancer chromosome (Basc) that sustains approximately normal chromosome pairing for the yellow locus but not for Notch. Lower panel: Experimental results showing approximately normal levels of DsRed conversion of the Basc chromosome but significantly reduced copying (~1/3) of the N^Ax16 allele. In addition, co-drive of the DsRed and N^Ax16 alleles was abolished by the Basc inversion

Fig. 4

Allelic-drive mediated by “copy-grafting.” a DNA sequence at the gRNA-N+ cleavage site for the wild-type reference allele (N^+S), the cleavage-insensitive wild-type (N^+IS), and the cleavage-sensitive N^AxE2 allele, with the C → T substitution (see Supplemental Fig. 6 for amino acid substitutions). b Copy-grafting scheme in which F1 master females carrying a wild-type cleavage insensitive wild-type Notch allele in trans to the sensitive N^AxE2 allele (N*^S) (y^<ccN> w^a N^+IS/y⁺ w⁺ N^AxE2; Cas9/+ ♀) are crossed to N^+IS ♂. F2 progeny were then scored based on inheritance of the y^<ccN> w^a N^+IS donor chromosome (light purple) or the w⁺ receiver chromosome (dark purple) based on their eye color phenotype (orange for donor and red for receiver). Red and blue arrowheads indicate copying of the y^<ccN> element and the N^+IS allele, respectively. c Percentage of F2 progeny demonstrating features of drive including: percentage of converted N^+IS receiver chromosomes (purple circles); heterozygous N^−/+ females (black circles); and copying of the DsRed marked y^<ccN> element to receiver chromosome (red circles). d Summary of different drive systems. MCR (full gene drive) elements have both Cas9 (blue) and a gRNA (yellow) inserted into the genome at the gRNA-directed cleavage site. CopyCat (split drive) elements carry only the gRNA (red) inserted at the gRNA-directed cleavage site, while Cas9 is provided from a Mendelian transgene at a different genomic location. Copy-cutting (special allelic-drive) is mediated by two gRNAs. One gRNA (yellow) propagates the CopyCat element while the second gRNA creating allelic-drive (dark blue) cuts a non-preferred allele, but not the favored allele (lock icon). This drive element could be either an MCR (including Cas9) or a CopyCat element (Cas9 provided from a separate genomic location, as depicted in this figure). Copy-grafting (general allelic-drive) is mediated by a gRNA (purple) that cuts the non-preferred, but not the favored, chromosome near the desired allelic variant, resulting in conversion of a short region of the receiving chromosome (indicated by purple highlight) with sequences from the donor chromosome (purple box) that encompass the favored allele (lock icon)