Table 2. Frequency of HDR knock-in events.

(A) No. of Fertile P	No. (%) of Broods from P Crosses Without e	No. (%) of Broods from P Crosses with e	No. of F1 Lines Analyzed
43	26 (61)	17 (40)	72
nos-Cas9 embryos were injected with a mix of gRNA-e, gRNA-lbk2, and a repair template. P indicates the number of injected, fertile flies used in Parental crosses. The rate of e mutations: of the 43 P, 61% produced no ebony offspring, and 40% produced ebony offspring, with individual broods ranging from 2 to 81% ebony. 72 crosses were set up from ebony F1 individuals, and the resulting balanced lines were analyzed by PCR for an HDR insert.

(B)% ebony in F1 Broods	No. of Lines Analyzed	No. (%) of Lines with Knock-In
1–50	43	1 (2)
>50	29	3 (10)
Top two jackpot broods	11	2 (18)

nos-Cas9 embryos were injected with a mix of gRNA-e, gRNA-lbk2, and a repair template. Each F1 brood is grouped by percentage of ebony: 1–50, >50% jackpot, and the top two jackpot broods (a subset of the >50% jackpot broods). We first tested lines from the two best jackpot broods and found an HDR knock-in rate of 18%. Our overall insertion rate in jackpot lines was 10% and only 2% for low ebony broods, suggesting enrichment between percentage of ebony in F1 broods and HDR knock-in events.