sgRNA design and library construction

All sgRNAs were cloned into the 2mU6>sgRNA(F+E) scaffold as per Stolfi, et al.⁴⁹ 2mU6>sgRNA(F+E) plasmids were digested with BsaI-HF (NEB R3535, discontinued) in rCutsmart buffer for optimal positivity rates. Oligos ordered from Sigma at 50 mM concentration in water, were annealed by combining 10 uL of forward and reverse oligos and incubating in a thermocycler with the following protocol: 1) 95 °C for 30 seconds (melt) 2) 72 °C for 2 minutes, 3) 37 °C for 2 minutes, 4) 25 °C for 2 minutes. Annealed oligos were diluted 1:50 ul in TE buffer, and ligated into an opened vector using Instant Sticky-end Ligase Mix (NEB M0370S) with 0.5 μL vector, 2.5 μL diluted oligo, 3 μL ligase mix. 3 μL of ligation mix were transformed into 15 μL Stellar Competent cells (Takara 636763) and plated on AMP LB selection plates followed by overnight incubation at 37 °C. 4 colonies per plate were selected for colony PCR using the U6-F-910 forward primer (5’-caattgccccaagctctcttc-3’) and the guide RNA specific sgRNA-R oligo (same as the R for annealing diluted to 10 μM). Positive colonies were grown overnight in 4 ml LB amp overnight, miniprepped, and sent for sequencing using the seqU6-F primer (5’-ggatcgcgcgagccc-3’). Sequence results were checked to assure no mutations were present in the guide RNA sequence and correct clones were frozen down in Hogness Freezing Medium (Bioworld 30629174-1) and stored at −70 °C.