Xl1 red competent cells

SpCas9 mutant libraries were constructed using three independent protocols. For the first library, the Cas9 library plasmid was transformed into XL1-red competent cells (Agilent), which were grown according to instructions in the vendor's manual. For the second and third libraries, error-prone PCR was performed on whole WT-SpCas9 from Cas9 library plasmid sequences using Genemorph II (Agilent) and Diversify PCR random mutagenesis (Clontech) kits under low error rate (0–5 mutations per kb) conditions with primers designed for Gibson Assembly (Supplementary Table 3); PCR products were subsequently gel purified (4.3 kb). The purified randomly mutagenized library and the backbone of the Cas9 library plasmid (double-digested with BamHI and XbaI, followed by gel purification of the 3 kb fragment) were Gibson assembled. The assembled libraries were transformed into Endura™ electrocompetent cells (Lucigen) and incubated on chloramphenicol LB plates (12.5 μg/mL) at 37 °C overnight. A total of 3 × 10⁶ colonies were obtained for each library, resulting in a library complexity of 10⁷ overall. Pooled library plasmids were purified using a midi prep kit (NucleoBond Xtra Midi EF, Macherey-Nagel).

Sniper1 mutant libraries were constructed using three independent protocols for mutagenesis from XL1-red competent cells (Agilent), Genemorph II (Agilent) and Diversify polymerase chain reaction (PCR) random mutagenesis (Clontech) kits. All reaction conditions have been described previously^{7 (link)}. The assembled libraries were transformed into Endura electrocompetent cells (Lucigen) and incubated on LB plates containing chloramphenicol (12.5 μg ml⁻¹) at 37 °C overnight. A total of 3 × 10⁶ colonies were obtained for each library, resulting in an overall library complexity of 10⁷. Pooled library plasmids were purified using a midi prep kit (NucleoBond Xtra Midi EF; Macherey-Nagel).