P3s cas9hc

The WT-Cas9-encoding plasmid (p3s-Cas9HC; Addgene plasmid #43945)^{37 (link)} and the sgRNA plasmid (pRG2; Addgene plasmid #104174)^{4 (link)} have been described previously. Plasmids encoding human codon-optimized eSpCas9 (1.1) and Cas9-HF1 (p3s-eCas9 (1.1), Addgene plasmid #104172; p3s-Cas9-HF1, Addgene plasmid #104173)^{4 (link)} were Gibson assembled into the p3s-Cas9HC plasmid backbone to change the location of the nuclear localization signal (NLS) from the N-terminus to the C-terminus. Human codon-optimized evoCas9, HypaCas9, and xCas9–3.7 (evoCas9, Addgene plasmid #107550, HypaCas9, Addgene plasmid #101178 and xCas9–3.7, Addgene plasmid #108379)^{8 (link),9 (link),19 (link)} constructs were created by Gibson assembly of sequences containing the necessary site mutations into the p3s-Cas9HC plasmid backbone. All constructs were confirmed by Sanger sequencing (Supplementary Data 1). Human codon-optimized WT-BE3 and Sniper-BE3 were made by exchanging WT-SpCas9 from CMV-BE3 (a kind gift from David Liu; Addgene plasmid # 73021)^{25 (link)} with WT-Cas9 and Sniper-Cas9 derived from p3s-Cas9HC.

The Sim1-Puro cell line was generated from the RW4 mouse ESC line (American Type Culture Collection, Manassas, VA). Approximately 1 × 10⁷ RW4 ESCs were resuspended in electroporation buffer with 10 μg of Sim1-Puro-pStartTK vector and 200–300 ng of a Cas9 guide RNA vector (deemed gSim1.MS8.mSim1.g6a, with guide RNA (Fig. 1c, Cas9 Guide RNA) targeting 5’-gtccatcattcgtgtcttcc cgg-3’ near the Sim1 start codon (Fig. 1c, Cas9 Target)) in the MLM3636 plasmid (Addgene plasmid #43860) and 200–300 ng of the Cas9 nuclease expression plasmid p3s-Cas9HC (Addgene plasmid #43945). Both Cas9 vectors were obtained from Genome Engineering Core, Washington University in St. Louis and originally gifted by Keith Joung and Jin-Soo Kim, respectively. Cells were electroporated using a Biorad Gene Pulser Xcell Eukaryotic System at 0.23 kV and 975 μF in a 0.4 cm cuvette (Bio-Rad, Hercules, CA). Following electroporation, cells were seeded on gelatin-coated 100 mm dishes for 24 hours then treated with G418 (200 μg/mL, Life Technologies) and 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil (150 nM, Movarek Biochemicals, Brea, CA) for positive and negative selection, respectively. After 14 days, surviving clones were picked and seeded into individual wells of a gelatin-coated 96 well plate.

Each type of plasmid used in the Sniper-screen contains replication origins and resistance markers that are compatible with each other. (Fig. 1b) The ccdB plasmid (p11-lacY-wtx1) was a kind gift from the Zhao lab^{9 (link)}. It was double-digested with SphI and XhoI enzymes (Enzynomics), which was ligated to oligos (Cosmogenetech) containing target sequences (Supplementary Table 1) with T4 DNA ligase (Enzynomics). The sgRNA vector was constructed (Supplementary Figure 19) with a temperature-sensitive Psc101 replication origin^{12 (link)} (from pgrg36, a kind gift from Nancy Craig), tetR (from the tn10 locus of ElectroTen-Blue Electroporation Competent Cells, Agilent), a Kanamycin resistance marker, the pltetO1 promoter and the sgRNA sequence containing two BsaI sites (synthesized at Bioneer). The components were PCR-amplified and Gibson assembled (NEBuilder HiFi DNA Assembly kit, NEB). The guide RNA sequences to EMX1 with various mismatches (Supplementary Table 1) were cloned into the vector after BsaI digestion. The Cas9 library plasmid (Supplementary Figure 19) was derived from human codon-optimized WT-Cas9 (p3s-Cas9HC; Addgene plasmid #43945)^{37 (link)}, dual CMV-pltetO1 (synthesized at Bioneer) and the p15a replication origin and chloramphenicol resistance marker (from the PBLC backbone, Bioneer). The components were Gibson assembled.