Figure 1. Heritable Cas9-induced nonhomologous recombination in C elegans:A) Overview of the workflow used to induce nonhomologous recombination. A cartoon depiction of the genome composition of the QX2501 NIL used in this study is shown, where yellow represents QX1211 and purple represents XZ1516. Genomic coordinates (with XZ1516 as the reference genome) of the three gRNAs used in this study are shown below the genome representation of the NIL. Genomic coordinates of two indel variants used to differentiate between the parental genotypes are shown in red and labeled A and B. Young adult QX1211/QX2501 heterozygotes are injected with a gRNA that targets Cas9 to the desired location, along with a co-injection marker (
dpy-10 gRNA + ssODN repair template). The progeny of the injected animals that express the co-injection phenotype are singled to wells of a 96-well plate and allowed to self. A subset of the worms is genotyped with the A and B primer pairs. Individuals displaying loss of heterozygosity (LOH) such that both parental genotypes are still represented (i.e. individuals heterozygous at one marker and homozygous on the other, or homozygous at both markers) are marked with a green plus sign and recovered to agar plates for further characterization.B) Overview of the Cas9-induced nonhomologous recombination results. The table shows the PCR genotypes of rol/dpy progeny in this study. Left-most column depicts the expected strain genotypes based on PCR genotyping of the A and B indel variants. Within this column, recombinant genotypes are indicated by "LOH" in the upper-right corner. Dotted red lines indicate the marker where the LOH event occurred. The next three columns-one for each guide-show the number of genotyped rol/dpy progeny with the corresponding genotype. Black dots in the upper left corner correspond to the strains with whole-genome sequence data. The right-most column shows the F2 progeny genotypes when QX2501/QX1211 heterozygous animals were allowed to self without injection The last row of the table shows the total number of recombinants identified over the total number of F2 individuals genotyped for each gRNA.