Genemorph 2

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).

A library of IpaC mutants with missense mutations in the coiled-coil domain and flanking regions was generated using error-prone PCR with the GeneMorphII (Agilent) domain mutagenesis kit. The library was cloned under the control of the ara promoter and was expressed in S. flexneri 2457T ΔipaC pTSAR. The resulting strains were arrayed and were used to infect MEFs seeded at 2 x 10⁴ cells per well in a 96-well plate. The bacteria were pelleted onto the cells at 800 x g for 10 minutes at 25°C. Following an additional 50 min incubation at 37°C, the cells were washed and fixed with 3.7% paraformaldehyde. Fixed cells were stained with Hoechst and were imaged using a Cell Discover 7 automated microscope at the Harvard Center for Biological Imaging. Images were manually screened to identify IpaC variants that supported fewer GFP-positive bacteria associating with cells. 137 clones meeting these criteria were identified; for each, the ipaC-containing plasmid DNA was isolated, and ipaC was sequenced. 131 of the ipaC mutants contained non-sense or frameshift mutations. Six contained one or two missense mutations.

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).

The BG505 SOSIP whole-gene saturation mutagenesis and “rare” amino acid libraries were synthesized by Integrated DNA Technologies and GenScript, respectively. Libraries for germline targeting were created by error-prone PCR (GeneMorph II, Agilent), site-directed mutagenesis (QuikChange, Agilent) or two-step assembly PCR of degenerate primers with the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and cloned into a modified version of the gateway cloning entry vector pENTR/D-TOPO (Ota et al., 2012 (link)) with the circular polymerase extension cloning (CPEC) method (Quan and Tian, 2014 (link)) or Gibson Assembly (New England Biolabs), according to the manufacturer’s instructions. All libraries were then transferred to the lentiviral vector pLenti CMVTRE3G puro Dest (Ota et al., 2012 (link)) with the LR Clonase II enzyme mix (Thermo Scientific).

A mutagenic PhaG library was constructed by error-prone PCR using GeneMorph II from Agilent (Senta Clara, CA) with a low mutation frequency (0–4.5 mutations/kb). The plasmid backbone (pBTRCK) was PCR-amplified using a high-fidelity DNA polymerase Q5 from New England Biolabs (Ipswich, MA). The library was assembled using an isothermal assembly method^{50 (link)}. Primers used in the creation of the library contained the start and stop codons in order to prevent mutation of them.
The sequence of improved PhaG variants was obtained by Sanger sequencing of colony PCR products made by high-fidelity Q5 DNA polymerase. To re-introduce single point mutations, we amplified ^PkPhaG with mutagenic primers and subcloned the fragments into pTRC99A-’^PkphaG’-^TdTER by an isothermal assembly method^{51 (link)}. The resulting plasmids were transformed into E. coli CM23 harboring pBTRCK-^MatesB* and pACYC-^PaphaJ3 plasmids.