Cosmids

Plasmids used in this study are described in Supplementary Table 1. For expression of chimeric gRNAs targeting various protospacer sequences in the genome, oligonucleotides were annealed and cloned into BbsI-digested pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene plasmid #42230), for editing the LMNA locus, or pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) (Addgene plasmid # 42335), for editing the PML locus, which were gifts from Feng Zhang (2 (link)). The gRNA sequences were analyzed using the COSMID (CRISPR Off-target Sites with Mismatches, Insertions and Deletions) website (http://crispr.bme.gatech.edu/) to check the sgRNA against the GRCh38 (hg38) genome build for potential off-target sites (30 (link)).
pcDNA3-Clover was a gift from Michael Lin (Addgene plasmid # 40259) (31 (link)). piRFP670-N1 was a gift from Vladislav Verkhusha (Addgene plasmid # 45457) (32 (link)). Homology repair templates were generated by PCR using Phusion high-fidelity polymerase (New England BioLabs) and cloned into pCR2.1 using a TOPO-TA Cloning Kit (Life Technologies). Site-directed mutagenesis of the BRCA1 in pHA-BRCA1 (a gift from David Livingston) (33 (link)) was carried out using overlap extension PCR.

Replication-incompetent, E1/E3-deleted recombinant adenoviral vectors based on adenovirus type 26 and 35 were engineered using the AdVac® system. Rescue and manufacturing of the replication deficient adenoviral vectors were performed in the complementing cell line PER.C6® [24] (link), [42] (link).
The transgene sequences encoding the different glycoproteins (GP) of the Ebola and Marburg Filoviridae, were inserted in the E1-position of the Ad genome and the coding sequences were optimized for efficient expression in mammalian cells and were placed under transcriptional control of the human CMV promoter and the SV-40 polyadenylation sequence. This expression cassette was cloned into an E1-deleted-Adapter plasmid containing the left portion of the adenoviral genome including the left inverted terminal repeat (ITR) and packaging signal. Co-transfection of this Adapter plasmid with a cosmid containing the remainder of the Ad26/35 adenoviral sequence (lacking the E3-gene and including the Ad5 E4orf6) yielded the recombinant, E1/E3-deleted, replication-deficient vaccine vectors.
Once transfected, a single plaque for each of the rAd EBOV vectors was purified and expanded up to a production scale. A two step cesium chloride gradient ultracentrifugation procedure was used to purify the rAd vectors, which were stored as single use aliquots below −65°C. Virus particle titers were quantified by measurement of optical density at 260 nm [43] (link) and infectivity was assessed by TCID50 on the human helper cell line 911 [44] (link). Adenovirus-mediated GP expression was assessed by infection of A549 cells followed by analysis of culture lysates on western blot. These A549 cells were chosen as these cells lack E1A expression and therefore do not allow interfering adenoviral replication. These cells have been utilized in the past for evaluation of multiple Ad26 and Ad35 vectors where in vitro expression correlated with in vivo immunogenicity. The identity of the purified vectors was confirmed through PCR and the complete transgene sequence, including flanking regions, was verified by DNA sequencing. rAd26.empty and rAd35.empty vectors were produced similarly, but do not contain a transgene in the E1 region.

Vectors for complementation of Tn mutants were made in the integrating plasmid pMV306hyg (Table S5). We generated pMV306hyg by replacing the aph Kan resistance marker in pMV306 (81 (link)) with a Hyg resistance cassette. pMV306 without aph was PCR amplified with primers pMV306_F and pMV306_R (Table S6), digested with SbfI and AflII, and ligated to a Hyg resistance cassette that was removed from pJT6a (82 (link)) by restriction with SblI and AflII. Each gene was PCR amplified along with ~300 bases 5′ of the translation start site to include the native promoter (Table S6), cloned in pCR2.1 TOPO (Invitrogen) and sequenced, and then removed from pCR2.1 by restriction with XbaI and HindIII and ligated to XbaI- and HindIII-digested pMV306hyg. Cosmid 8C3.1 containing the genomic region rv1317 to rv1343 surrounding clpS was obtained from the lab of William R. Jacobs. The pMV361hyg-ppsD vector was generated by replacing the Kan resistance cassette in an existing pMV361-ppsD vector (60 (link)) with a Hyg resistance cassette by Gibson assembly. Primers pMV361.FOR/pMV361.REV and hyg_fwd_2/hyg_rev_2 were used to PCR amplify pMV361-ppsD without the Kan resistance cassette and the Hyg resistance cassette from pMV306hyg, respectively (Table S6). PCR products were assembled with New England Biolabs (NEB) HiFi Assembly master mix (New England Biolabs) followed by sequencing of ppsD and the Hyg resistance cassette. Tn mutants were electroporated with the corresponding complementation vector or cosmid as described previously (83 (link)). Transformants were selected on Middlebrook 7H10 agar containing Kan and Hyg. The presence of the complementing plasmid or cosmid was confirmed by PCR (Table S6).

The presence
of a chromosomal-targeting crRNA leads to Cas9-targeting and killing
of targeted bacteria. Therefore, cosmids encoding crRNA are not stably
maintained in targeted bacteria after transduction. Hence, the titers
of chromosomal-targeting, cas9-transducing units,
were routinely assessed on E. coli K-12
strain EMG2, which is not targeted by the crRNA used in this study.
However, P4 transducing units with chimeric P2-P1(S′) tail
fibers and P2-ϕV10 tail spikes can only infect S. flexneri and E. coli O157, respectively. In these bacteria, the chromosomal-targeting cas9 cosmid is inherently unstable after transduction. Hence,
the titers of chromosomal-targeting cas9-transducing
units with chimeric tail fibers were quantified by measuring the copy
number of cosmid DNA that were packaged into phage units by qPCR.^{78 (link),79 (link)} A LightCycler 96 (Roche) was used to perform qPCR with SYBR Green
PCR Master Mix (Thermo Fisher Scientific, 4309155). The sequences
of the primers used for qPCR (qPCR1 and qPCR2) are provided in Table S3. They bind and amplify the rep gene of the pBBR1 origin of replication. The rep gene is specific to the cosmid DNA and not present in the genome
of the bacterial strains used in this study. The 10 μL reactions
were prepared with 5 μL of SYBR Green PCR Master Mix, 1 μL
of DNAse I treated lysate, 0.3 μL (300 nM) of each primer, and
3.4 μL of nuclease-free water. PCR cycling conditions were 95
°C for 10 min; 45 cycles of 95 °C for 20 s, 60 °C for
20 s, 72 °C for 20 s; and melting at 95 °C for 10 s, 65
°C for 60 s, 97 °C for 1 s. The nontargeting Cas9 P4 cosmid
DNA was used to construct a standard curve for quantification of the
transducing units in the lysate. The concentration of P4 cosmid DNA
was measured using Qubit dsDNA HS assay kit (Thermo Fisher Scientific)
and was serially diluted 10-fold from 8.02 × 10⁸ to
8.02 × 10² copy number/μL to construct the standard
curve. The lysate produced using pACK57 cosmid, lacking the target
sequence, was used as a negative control. The results were analyzed
using a LightCycler 96 SW 1.1. See Figure S2 for the standard curve and the titer of the transducing units (copy
number/mL).

The P4 cosmids were constructed using Gibson assembly and the primers
listed in Table S3. The P4 cosmid original
to this study will be made available via the Addgene plasmid repository.
Molecular cloning of spacer sequences was based on a protocol established
by Jiang et al.^{31 (link)} The crRNA guide sequence
of our cas9 construct contains two BsaI recognition
motifs, which allow digestion of the cosmid backbone and annealing
of the spacer sequence. A pair of oligonucleotides that contain the
spacer sequence was designed to have 5′ overhangs, which allowed
annealing of the oligonucleotide pair into the CRISPR array. The chromosomal-targeting
spacer sequences were designed using CHOPCHOP.^{44 (link)} The primer pairs were phosphorylated by incubating 1 μL
of each primer (100 μM) with 5 μL of 10× T4 ligase
buffer (New England Biolabs), 1 μL of T4 polynucleotide kinase
(PNK) (New England Biolabs), and 42 μL of water at 37 °C
for 30 min, followed by heat inactivation at 65 °C for 20 min.
The primers were then annealed by incubating at 95 °C for 5 min
and cooled to room temperature slowly in the heat block for a minimum
of 30 min. The P4 cosmid DNA was digested with the BsaI restriction enzyme (New England Biolabs) according to the manufacturer’s
instruction and gel purified. The annealed primers were ligated to
the digested P4 cosmid by incubating 50 μg of cosmid with 2
μL of annealed primers (diluted to 50–100 μg/μL),
2 μL of 10× T4 ligase buffer (New England Biolabs), 1 μL
of T4 ligase (New England Biolabs), and water added to the final volume
of 20 μL. The reaction was incubated at room temperature for
2 h and heat-inactivated at 65 °C for 20 min. Two microliters
of the reaction was used for transformation for plasmid propagation.
The DNA sequence of P4 cosmid is available in Table S6.