Cas9 protein

Target gRNA sequences are listed in the Supplementary file 1. Axolotl matings and microinjections were carried out as previously described (Flowers and Crews, 2015 (link)) with the following modifications for haploid mutagenesis: Haploids were allowed to develop for 7 hr until they reached a two-cell stage. For multiplex mutagenesis, each blastomere was injected with an equal volume of injection mix for a total of 1000 pg of cas9 mRNA and 5 pg per gRNA (five total). Each gRNA was injected in two separate pools of gRNAs into embryos from two separate matings. Control embryos were injected as described, but with 50 pg of gRNA. The data described represents the results from ten independent in vitro fertilization, injection, and grafting experiments to produce experimental animals, and three additional experiments to produce control animals.
For tail regeneration experiments, embryos were produced by a single mating and injected with 1250 pg of gRNA coupled with 1250 pg of Cas9 protein (PNA Bio) as described previously within 2 hr of being laid (Fei et al., 2018 (link)). Successful mutagenesis of tyrosinase was assessed by loss of pigmentation. Mutations of catalase and fetuin-b were confirmed by fragment analysis PCR (Figure 6—source data 1).

Fly maintenance and crosses were performed under standard conditions at 25 °C. Rainbow Transgenics (Camarillo, CA, USA) carried out embryo injections for the SGyA, SGyB, and SGyC plasmids. Embryos were injected with a premixed solution of the construct, Cas9 protein (PNA Bio Inc., Newbury Park, CA, USA) and in vitro transcribed sgRNAs into the Nos-Cas9−attp2 strain (y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=nos-Cas9.R}attP2; BDSC #78782). Injecting the premix into a Cas9 line was performed to increase the chances of HDR insertion in the pole cells of embryos. A transformant was obtained for SGyA. No transformants were obtained for SGyB and SGyC. The genetic source of Nanos-SpCas9 (marked with vermillion [v]) was removed from the SGyA population by outcrossing these transformants to WT ♀’s. The resulting progeny were then scored to collect transgenic ♂’s (marked with tdTomato) without the v marker. These collected ♂’s lacking v were then outcrossed to WT ♀’s for multiple generations to ensure Nanos-SpCas9 was indeed not present. A stock was established by outcrossing these ♂’s to virgin w[1118] ♀’s. The SGyA transgenic line (w[*]/TI{Disc\RFP[tdTom.3xP3]=vas-Cas9.T2A.GFP,attP}SGyA) is available from the Bloomington Stock Center (#91386).

Zebrafish embryos were collected after natural spawning and injected at the single cell stage with one nanoliter of injection mix (4.5 μl gRNA (1μg/μl), 2.5 μl Cas9 protein (2μg/μl, PNA Bio), 2 μl 1M KCl and 1 μl 0.5% phenol red dye). To screen for genomic lesions, genomic DNA was extracted from a pool of 15 embryos at 2.5 days post fertilization (dpf).

Light chain single injection: Two sgRNAs, donor DNA and Cas9 protein (PNA BIO INC) were mixed together for the generation KI mice by CRISPR/Cas9 method (Lin et al, 2018). Heavy and Light chains double injection: Briefly, we prepared the injection mix of Cas9 protein (100 ng/μl, IDT), sgRNA (50 ng/μl, Synthego) and circular DNA donor (10 ng/μl) in injection buffer as previous papers described (Yang et al, 2013; Yang et al, 2014) and performed microinjecting 200 zygotes. After culturing the injected zygotes, we transferred around 30 of the zygotes into the oviducts of 5–7 (average 6) of pseudo‐pregnant foster mothers.

To establish genetically diverse immunocompromised host strains of mice, we engineered Rag1 knockout in selected stains using either CRISPR/Cas9 or Base Editing technology (BE4max) (Koblan et al., 2018 (link)), according to the scheme depicted in Fig. S1A. Both methods used the same sgRNA (5′-acagtcaggtctacttccca-3′, Synthego) generating a Q192X early termination modification when coupled with a base editor (BE; Trilink, custom mRNA) or an NHEJ-induced frameshift mutation when used with Cas9. As a backup for the BE-derived mutants, a second sgRNA was included (5′-tcatgcaaggcaggggctcc-3′, Synthego), designed to generate another early termination (Q456X) allele. Delivery of reagent into wild-type zygotes was achieved using either microinjection (BE targeting: 100 ng/µl BE4max mRNA, 50 ng/µl each sgRNA) or electroporation [CRISPR/Cas9 targeting: Cas9 protein (PNA Bio): 250 ng/µl, 100 ng/µl sgRNA]. Mutant alleles were identified and characterized by PCR and sequencing (o2686, 5′-ATCTGTGGGAATCGTTTCAAGA-3′; o2687, 5′-AGAAGGACTTTCTCGGCATTCC-3′). Candidate founders were backcrossed to wild-type cohorts from the host strain at least once before inbreeding was initiated. Homozygotes were subsequently confirmed for the lack of B- and T-lymphocytes (CD19 and TCR beta markers, respectively) using flow cytometry (Fig. S1B).