Cas9 enzyme

All gRNAs were individually injected into NHGRI-1 embryos to estimate the frequency of indels. gRNAs were prepared following the manufacturer’s protocol (IDT). Briefly, 2.5 μl of 100 μM crRNA, 2.5 μl of 100 μM tracrRNA, and 5 μl of Nuclease-free Duplex Buffer using an annealing program consisting of 5 min at 95 °C, a ramp from 95 °C to 50 °C with a − 0.1 °C/s change, 10 min (min) at 50 °C, and a ramp from 50 °C to 4 °C with a − 1 °C/s change. Ribonucleoprotein injection mix was prepared with 1.30 μl of Cas9 enzyme (20 μM, New England BioLabs), 1.60 μl of prepared gRNAs, 2.5 μl of 4x Injection Buffer (containing 0.2% phenol red, 800 mM KCl, 4 mM MgCl₂, 4 mM TCEP, 120 mM HEPES, pH 7.0), and 4.6 μl of Nuclease-free water. Microinjections directly into the yolks of NHGRI-1 embryos at the one-cell stage were performed as described previously [59 (link)], using needles from a micropipette puller (Model P-97, Sutter Instruments) and an air injector (Pneumatic MPPI-2 Pressure Injector). Embryos were collected and ~1 nl of ribonucleoprotein mix was injected per embryo, after previous calibration with a microruler. Twenty injected embryos per Petri dish were grown up to 5 dpf at 28 °C.

We performed RNA-seq of Cas9 injected NHGRI-1 larvae. One-cell stage NHGRI-1 embryos were injected with either Cas9 enzyme or Cas9 mRNA. Injection mix for Cas9 enzyme included Cas9 enzyme (20 μM, New England BioLabs), 2.5 μl of 4x Injection Buffer (0.2% phenol red, 800 mM KCl, 4 mM MgCl₂, 4 mM TCEP, 120 mM HEPES, pH 7.0), and Nuclease-free water. Cas9 mRNA was obtained from plasmid pT3TS-nCas9n (Addgene, plasmid #46757) [5 (link)], using the MEGAshotscript T3 transcription kit (Thermo Fisher) following manufacturer’s guidelines of 3.5 h 56 °C incubation with T3. mRNA was purified with the MEGAclear transcription clean-up kit (Thermo Fisher) and concentration of mRNA obtained using a NanoDrop (Thermo Fisher). The injection mix of Cas9 mRNA contained 100 ng/μl of mRNA, 4x Injection Buffer (0.2% phenol red, 800 mM KCl, 4 mM MgCl₂, 4 mM TCEP, 120 mM HEPES, pH 7.0), and Nuclease-free water. Additionally, uninjected batch-siblings and uninjected siblings from an additional batch were used as controls. All embryos were grown at 28 °C in a density of < 50 embryos per dish. At 5 dpf, three pools of five larvae were collected for each group (Cas9 enzyme, Cas9 mRNA, and uninjected) for RNA extraction using the RNeasy kit (Qiagen) with genomic DNA eliminator columns for DNA removal. Whole RNA samples were subjected to RNA-seq using the poly-A selection method (Genewiz, San Diego, CA).

DNA cleavage assay was carried out based on [3 (link)] with commercially available Cas9 enzyme (NEB). PCR amplified genomic fragments for each sgRNA-1 off-target site (OT#1_F 5’-AGAGGCAAGTAAAGGTCAAGTAGG-3’, OT#1_R 5’-TCACATTGCAATGATGAGCACTTT-3’; OT#2_F 5’-CCAGCTCATGTTGAAAAGACACAT-3’, OT#2_R 5’-CCCCCACAGATGAAATGAAAAGAC-3’; OT#3_F 5’-TACCCAAAAATTGTAAGCCAGCAG-3’, OT#3_R 5’-AGATCTGATCCGGTTTCAAAGTGA-3’) were cloned into pGEM-T easy vector (Promega). The plasmids were pre-linearized with BsaI (NEB) about 2kb from the sgRNA target site. 3nM of the pre-linearized plasmids were incubated for one 1h with 30nM sgRNA-1 and 30nM Cas9 protein (NEB) and supplemented with Cas9 nuclease buffer (NEB) in a 30μl reaction volume at 37°C. Gel electrophoresis was performed on 1.5% agarose gel in 1x TAE buffer (40mM Tris, 20mM acetic acid, 1mM EDTA).