High-throughput single-end sequencing and CRISPR/Cas9 genotyping

Genomic DNA from individual embryos/larvae was extracted according to the “still quick, less dirty” protocol⁴⁶. The regions containing the tyr target or tyr off-target were amplified in a standard PCR (PCR parameters: 2 min at 95 °C followed by 25 cycles with 30 s at 95 °C, 30 s at 59 °C, and 30 s at 72 °C followed by 7 min at 72 °C) using tyr-P5-tail and tyr-P7-tail or tyr-off-P5-tail and tyr-off-P7-tail (Supplementary Table ³), respectively. After amplification, PCR products were purified using NuceloSpin Gel and PCR Clean-up (Macherey Nagel, 740609.250) following the manufacturer’s instructions. In order to sequence the CRISPR PCR products, an 8-cycle index PCR with Illumina TruSeq-primer was run with 2 ng starting material. Libraries were quantified, and 75 bp single-end reads were sequenced on the Illumina NextSeq 500 platform to a minimum depth of 4 million reads. We used the tool CRISPResso2²⁴ to trim sequencing adapters, align sequencing reads to the amplified PCR sequence, quantify the number of insertions, substitutions and deletions in the reads, and finally visualize the results. Default parameters were applied with the exception of --trim_sequences and --trimmomatic_options_string to trim sequencing adapters, --quantification_window_size 10, and --exclude_bp_from_left 0 --exclude_bp_from_right 0.