Fig. 2: The double gene drive system driven by Cas9 and Cas12a displays simultaneous super-Mendelian inheritance rates of two independent genetic elements.

a Schematic of the double CopyCat gene-drive system. The DsRed-marked Cas12a and Cas9 represent the nuclease sources as static transgenes. Two independent CopyCat gene-drive elements marked with GFP (w2-GD and e1-GD) are driven by the Cas9 or Cas12a nucleases, respectively. To track their inheritance, we marked all four transgenes without overlapping fluorescent markers in any body parts. b Cross scheme of males expressing Cas12a and Cas9 crossed to virgin females carrying two CopyCat constructs (e1 and w2 gene drives). Collected F1 virgin females (Cas12a-Cas9-DsRed; gene drives-GFP) were crossed to ebony/ white mutant males to score germline transmission rates by screening the GFP markers in the F2 progeny. The donor chromosomes in w2-GD and e1-GD were marked by Cas12a and Cas9 transgenes with different fluorescent markers, respectively. A dark-gray half arrow indicates the male Y chromosome. The green triangles in the F1 female indicate potential gene drive copying onto the wildtype allele. c Possible outcomes in F2 progeny (Top panel: w2-GD in Chromosome. X; Bottom panel: e1-GD in Chromosome. III). The outcomes were classified into four categories based on the phenotypic readouts in F2 progenies: 1) Original gene drive from donor chromosome, represented as individuals with w- or e-; GFP+ phenotypic markers; 2) Allelic conversion or HDR events, represented as individuals with w- or e-; GFP+ and marked with DsRed+ by Cas elements; 3) Indel/NHEJ events, represented as individuals with w- or e-; GFP- and marked with DsRed+ by Cas elements; and 4) wildtype represented as w+ or e + ; GFP- and marked with DsRed+ by Cas elements. d Evaluation of gene drive parameters in the germline of F1 females by phenotypically scoring the F2 progeny. The inheritance, HDR/conversion, indels, and wildtype averages are represented on the top of the graph along with the number of F1 germline scores (n). Each dot represents the data from a single F1 female. Source data is provided as a Source Data File 3.