Engen spy cas9 nls

For editing, the guide RNA (gRNA) sequence targeting the region surrounding the NAPB mutation was selected using CRISPR-Cas9 gRNA design tool (Integrated DNA technologies). Single guide RNA (sgRNA) was synthesized using EnGen sgRNA Synthesis Kit (NEB, E3322) according to the manufacturer’s instructions. Nucleofection was carried out using the Amaxa nucleofection system (P3 primary cell 4D-nucleofector kit, Cat#V4XP-3032) according to the manufacturer’s instructions. Briefly, RNP complex were generated by mixing 1 μg of sgRNA with 2 μM of EnGen SpyCas9 NLS (NEB, M0646) at room temperature for 15–20 min. Approximately 2.5–3 × 10⁵ iPSCs were electroporated using CB150 nucleofection program and plated onto Matrigel-coated plates. Approximately 2 μg of knock-in oligo (Supplementary Table S1) was added to the nucleofection mix just before nucleofection. The cleavage efficiency of sgRNA was evaluated using T7E1 cleavage assay. After 48 h the cells were diluted and plated as single cells on Matrigel-coated plates for 10–15 days to make colonies. Genomic DNA (gDNA) was extracted using quick extract genomic DNA extraction buffer (epicenter). The region of NAPB targeted by sgRNA was amplified with specific primers (Supplementary Table S1) using PCR-Master mix (Thermo Fisher Scientific) and knock-in was confirmed by sanger sequencing of the PCR product.

gRNAs were formed from chemically synthesized Alt-R®-modified crRNAs from Integrated DNA Technologies (IDT). Each crRNA was suspended in duplex buffer to 100 μM concentration, then a crRNA:tracrRNA duplex was formed by combining 3 μl crRNA, 3 μl 100 μM tracrRNA, and 19 μl duplex buffer at 95 °C for five minutes, then cooled to room temperature and stored at −20 °C. To make gRNA:Cas9 RNP complexes, a mix was formed as follows: 1.5 μl each gRNA, 0.75 μl 2 M KCl, 1.25 μl EnGen Spy Cas9 NLS (NEB). The mix was incubated at 37 °C for 5 min, then brought to room temperature. One nanoliter of the gRNA:Cas9 complex was injected into embryos at the 1-cell stage. The gRNAs used are listed in Supplementary Data 9.

The CDS sequence and genomic sequence of BmTrpA1 (LOC101744290) were downloaded from the NCBI website, and sgRNA target sites were designed using the online software CRISPRdirect (http://crispr.dbcls.jp/, accessed on 19 April 2021) (shown in Table 1). According to the instructions of the EnGen^® sgRNA Synthesis Kit for S. pyogenes (NEB #E3322), the sgRNA was synthesized and mixed with the Cas9 protein EnGen^® Spy Cas9 NLS (NEB #M0646) at a molar ratio of 1:1 and used for microinjection of the non-diapause eggs after incubation at 37 °C for 5 min [22 (link)]. After injection, the silkworm eggs were transferred to an incubator with the temperature set to 25 °C, in darkness, and with a relative humidity of 80%. After hatching, the larvae were fed fresh mulberry leaves at 25 °C ± 1 °C.
Snap Gene^®3.2.1 software was used to derive the corresponding amino acid sequence and alignment. The three-dimensional structure of the protein was obtained by the homology modeling method and predicted by the online software Swiss-Model (http://swissmodel.expasy.org/, accessed on 11 July 2023) [23 (link),24 (link)].

Each mix was prepared on ice the hour prior to injection. For calculating the amount to mix/pipette, we generated and used a template available in Supplementary Table S03. We used buffers and Cas9 protein from New England Biolabs (NEB, EnGen^® Spy Cas9 NLS, # M0646). We prepared a 10 µL mix that we stored on ice in a carrier box that was transported to the fish facility prior to setting up the injections.

The CHSE-214 wild-type (Wt) Sc 11 (NC) was nucleofected with CRISPR RNPs for genome editing using the 4D Nucleofector (Lonza). NC cells were passaged 1 day before nucleofection and seeded at 4 × 10⁶ cells per 75 -cm² flask. RNP solution was prepared by mixing 1 μg of sgRNA and 2 μg of recombinant Cas9 (EnGen^® Spy Cas9 NLS, New England Biolabs) with nucleofector solution SE (Lonza) to a final volume of 10 μl, followed by 10 min of incubation at room temperature for complexing. NC cells (4 × 10⁵) were trypsinized, centrifuged at 300xg for 10 min, resuspended in 10 μl of nucleofector solution SE, mixed with the RNP solution, and added to a well in a 16-well Nucleocuvette strip. After nucleofection with program DS-137, the sample was incubated with 80 μl of OptiMEM (Gibco) for 10 min at room temperature and seeded in 12-well plates in growth medium. Transfection controls with pmaxGFP (Lonza) were evaluated after 2 days of incubation at 20°C.

The CRISPR mutagenesis protocol was based on Davis et al. [51 (link)]. Single guide RNA targeting genes of interest was designed using Benchling [Biology Software (2021) Retrieved from https://benchling.com] based on Cas9 cutting efficiency [52 (link)]. The sgRNA template was synthesized as a primer (S1 Table, SWC4_sg132 or SWR1_sg1979) and served for sgRNA synthesis using the EnGen sgRNA Synthesis Kit (NEB, USA) following the manufacturer’s instructions. The sgRNA was then purified using the RNA Clean & Concentrator-25 Kit (Zymo) following the manufacturer’s instructions. RNA concentration and quality were assessed by NanoDrop spectrophotometer. For each CRISPR reaction: 2.5 μL concentrated sgRNA, 2 μL of EnGen Spy Cas9 NLS (NEB) in the presence of buffer 3.1 (NEB) in a final volume of 5 μL were incubated at 37°C for 30 min and kept on ice until use.