CRISPR Targeting of Zebrafish rp1l1

CRISPR sites were identified in the first coding exon of rp1l1 (Gene ID: 101882236) using Geneious R9 (Biomatters Inc., San Diego, CA, USA). Previously, there were two rp1l1 homologues annotated in zebrafish: rp1l1a and rp1l1b. However, rp1l1b did not resemble rp1l1 genes in other organisms (including humans, mice, cows, pigs, dogs, chickens, and Xenopus), as it was missing defining features that identify rp1l1 and was almost twice the size. In addition, in the current zebrafish genome (z10), rp1l1b is no longer annotated. Therefore, it appears that zebrafish have one rp1l1 homolog, formerly annotated as rp1l1a, which we targeted for our experiments. The guide RNA (gRNA; sequence: 5′-CATCTTGACGCCTTTGAACT-3′) was synthesized as previously described [18 (link)], using the mMessage mMachine SP6 Transcription Kit (Invitrogen, Waltham, MA, USA). Embryos were injected at the single-cell stage using a glass needle mounted on a micromanipulator. The injection mix consisted of 1 μL (>1500 ng/μL) of gRNA, 2 μL of Cas9 nuclease, S. pyogenes (New England Biolabs, Ipswich, MA, USA), 0.5 μL of Cas9 buffer (New England Biolabs, Ipswich, MA, USA), and 1.5 μL of 1.5 M KCl.