Gel purified

Standard molecular biology techniques (106 ) were used for PCR, cloning, and plasmid construction. Nonpolar deletions of genes or regions of interest were constructed via gene replacement with the aacCI Gm-resistance (Gm^r) cassette, which comprises 228 bp of sequence upstream of the initiation codon and the complete aminoglycoside-(3)-acetyltransferase coding sequence. The Gm^r cassette was fused to flanking H. influenzae fragments by overlap extension PCR (OE-PCR) (107 (link)) via tails added to the amplification primers. PCR products used as templates in splicing reactions were gel purified (Qiagen). The primers used in the study are listed in Table S1. For complementation of H. influenzae mutants, DNA fragments were amplified by PCR and cloned between adjacent restriction sites of the chromosomal delivery vector pXT10, as previously described (49 (link)). Typically, pXT10 was digested with SapI or the isoschizomer BspQI, and inserts were digested with EarI or BspQI. Ligation reactions were dialyzed and electroporated into E. coli DH5α. Following purification, plasmids were linearized by digestion with ApaLI prior to transformation and selection for double crossover homologous recombination. Competent cell preparation and transformation was accomplished as previously described (108 (link)). The plasmids used in the study are listed in Table 1.

Genomic DNA from F₁ animals was extracted from ear clips using a DNA Extract All Reagents Kit (Applied Biosystems). Potential off-target sites predicted by the WTSI Genome Editing (WGE) webtool for sgRNA_U1 and sgRNA_D1 (design 1), and containing ≤3 mismatches (Additional file 1: Table S2) were PCR amplified using High fidelity Expand Long Range dNTPack (Roche) and the corresponding genotyping primers (Additional file 1: Table S3). PCR amplicons were gel-purified (QIAGEN) and analysed by Sanger sequencing.

All Env expression plasmids were made using the pLEXm vector, which is designed for protein expression in mammalian cells [38] (link). The human tissue plasminogen activator leader sequence was used to replace the native HIV-1 gp140 leader, a modification which has been shown to increase gp140 expression [39] (link). The 3′ primer incorporated a XhoI site and a His₆ tag sequence followed by a stop codon. PCR products were digested with EcoRI (New England Biolabs, Beverly, MA; NEB) and XhoI (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid. Ligation reactions were performed at room temperature (RT) for 1 hr using T4 DNA ligase (NEB) prior to transformation into DH5 alpha Escherichia coli cells (Invitrogen, Carlsbad, CA). Following DNA extraction, plasmids were sequence-verified.
Gp140-encoded regions of env were amplified, incorporating the full native gp120 and gp41 mature protein encoding regions with the furin site replaced with SEKS and ending with amino-acid position 668, located at the membrane proximal region of gp41, followed by a His₆ tag. All PCR reactions were performed with KOD DNA Polymerase (Novagen, Madison, WI), according to the manufacturer’s instructions. Primer details can be found in S1 Table.