Synthesized oligonucleotides

Synthesized oligonucleotides (Twist Bioscience) were dissolved in Buffer EB (Qiagen). 16 ng/uL single-stranded pooled oligos were amplified with NEBNext High-Fidelity 2X PCR Master Mix (NEB) with the following PCR protocol: Denaturation 98°C for 30s, 8 cycles of 98°C for 10s, 63°C for 10s, 72°C for 15s, final elongation 72°C for 3 min 40 μg pTF vector containing sgRNA-E + F scaffold with U6 promoter and Cas9-P2A-Zeocin with EFS-NS promoter is digested with Esp3l (Thermo Scientific) in FastDigest Buffer (10X, Thermo Scientific) and 1 mM DTT in 100 uL (Chen et al., 2013 (link)). The digested vector is also dephosphorylated in FastAP Thermosensitive Alkaline Phosphatase (1U/μL, Thermo Scientific) in FastDigest Buffer (10X) in 200 μL volume. Purified pooled oligos were cloned into digested pTF vector using 2X Gibson Assembly Master Mix with 10 times molar ratio of pooled oligos to digested vector and transformed to Endura Electro Competent Cells (Lucigen) and plated on LB + Amp plates (Figure S1D). 565 E.coli colonies per guide ratio (>500× coverage) was used to process the 4 Maxi-Prep (Qiagen) reactions. After bacterial transformation, the plasmid library was sequenced to verify uniform sgRNA coverage and minimal bias (Figure S1E).