F0-injected zebrafish are grown to adulthood and outcrossed to a wild-type zebrafish. 12 embryos from this outcross are sacrificed for genomic DNA extraction and PCR analysis of the region encompassing the target site. PCR products are directly run on a 15% polyacrylamide gel. This gel represents an outcross in which the F0-injected founder is carrying a single 10-bp deletion at gad2 exon 1 that is transmittable at a frequency of ∼33% to the F1 generation. The second pair of bands that are noted with an asterisk are heteroduplexes. These heteroduplexes are seen with all heterozygous samples for all alleles and are not a reflection of nonspecific primer binding.

(A) PAGE results showing 12 individual embryos from an outcross in which the F0-injected founder transmits four different germline mutations in gad1a exon 5 at a frequency of ∼67%. The first lane is a known heterozygous zebrafish for a different allele that serves as a positive control. The starred samples were sequenced and determined to have the following types of mutations: embryo numbers 3, 4, 10, 12 have a 14-bp insertion; embryos 5 and 11 have a 10-bp deletion; embryo number 7 has a 9-bp deletion; embryo number 8 has a 2-bp insertion. (B) PAGE results from an incross of a line of fish (gav2501) that are heterozygous for a CRISPR-Cas9-induced 10-bp deletion at gad2 exon 1. Fish number 1 is a homozygous mutant, fish numbers 2 and 3 are wild-type and fish number 4 is a heterozygous mutant. In both gels, the second pair of bands that are noted with an asterisk are heteroduplexes.