Patent ID: 12252690

The sequences listed in this table (SEQ ID NO 01-SEQ ID NO 18) refer to the DNA sequences encoding the targeting sequences of the respective gRNAs.

METHODS

Hybridoma Cell Culture Conditions

The WT hybridoma cell line (Wen1.3) was obtained as a gift from Prof. Annette Oxenius (ETH Zurich). All hybridoma cell lines were cultivated in high-glucose Dulbecco's Modified Eagle Medium [(DMEM), Thermo Fisher Scientific (Thermo), 11960-044] supplemented with 10% heat inactivated fetal bovine serum [(FBS), Thermo, 10082-147)], 100 U/ml Penicillin/Streptomycin (Thermo, 15140-122), 2 mM Glutamine (Sigma-Aldrich, G7513), 10 mM HEPES buffer (Thermo, 15630-056) and 50 μM 2-mercaptoethanol (Sigma-Aldrich, M3148). All hybridoma cells were maintained in incubators at a temperature of 37° C. and 5% CO2. Hybridomas were typically maintained in 10 ml of culture in T-25 flasks (Thermo, NC-156367), and split every 48/72 hours.

Cloning and Assembly of CRISPR-Cas9 Targeting Constructs

Unless otherwise noted, cloning of CRISPR-Cas9 plasmids and HDR donor constructs was done by Gibson assembly and cloning with the Gibson Assembly® Master Mix (NEB, E2611S) (Gibson et al., Nat Methods 2009, 6:343-345). When necessary, fragments for the Gibson assembly cloning were amplified with the KAPA HiFi HotStart Ready Mix [KAPA Biosystems (KAPA), KK2602]. All gRNAs were obtained from Integrated DNA Technologies (IDT) as single-stranded 5′-phosphorylated oligonucleotides purified by standard desalting. The basis for CRISPR-Cas9 experiments relied on the plasmid pSpCas9(BB)-2A-GFP (pX458), obtained as a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al., Nat Protoc 2013, 8:2281-2308). An alternate version of pX458 was generated by replacing the GFP (eGFP variant) with BPF (TagBFP variant) (pX458.2 or pSpCas9(BB)-2A-BFP). For cloning gRNAs, both versions of pX458 were digested with Bbsl [New England BioLabs (NEB), R0539S], gRNA oligonucleotides were ligated into plasmids with DNA T4 ligase (NEB, M0202S). The gene or mRuby (mRuby2 variant) was derived from the plasmid pcDNA3-mRuby2, a gift from Michael Lin (Addgene plasmid #40260) (Lam et al., Nat Methods 2012, 9:1005-1012; Jinek et al., eLife 2013, 2:e00471-e00471). The HDR donors (mRuby and the antibody constructs) were cloned in the pUC57(Kan)-HDR plasmid, obtained from Genewiz. The vector was designed with homology arms according to the annotated mouse genomic sequence (GRCm38). The 2A antibody constructs were obtained as synthetic gene fragments (gBlocks, IDT). The HDR donor vectors were linearized by PCR with the KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2602). All plasmid and linear versions of HDR donors, as well as pX458 and pX458-BFP, were ethanol precipitated as a final purification step.

Hybridoma Transfection With CRISPR-Cas9 Constructs

Hybridoma cells were transfected with the 4D-Nucleofector™System (Lonza) using the SF Cell Line 4D-Nucleofector® X Kit L (Lonza, V4XC-2024) with the program CQ-104. Cells were prepared as follows: 106cells were isolated and centrifuged at 90×G for 5 minutes, washed with 1 ml of Opti-MEM® I Reduced Serum Medium (Thermo, 31985-062), and centrifuged again with the same parameters. The cells were finally re-suspended in 100 μl of total volume of nucleofection mix, containing the vector(s) diluted in SF buffer (per kit manufacturer guidelines). For the exchange of VHlocus, 5 μg of pX458 (or pX458-BFP) with gRNA-E (targeting VH) or gRNA-J (targeting mRuby), and 5 μg of the circular or linearized HDR donor constructs were nucleofected into cells. For VLdeletion, 5 μg each of pX458 with gRNA-F and gRNA-H were co-transfected into cells. Following transfection, the cells were typically cultured in 1 ml of growth media in 24-well plates (Thermo, NC-142475). When a significant cell expansion was observed, cells were supplemented 24 hours later with 0.5-1.0 ml of fresh growth media. After sorting, typically 48 hours after transfection, cells were recovered in 24-well plates, and progressively moved into 6-well plates (Thermo, NC-140675) and T-25 flasks, following expansion. After replacing the VHwith mRuby, cells were single-cell sorted in U-bottom 96-well plates (Sigma-Aldrich, M0812) in a recovery volume of 100 μl. The clones were eventually expanded in 24-well plates, 6-well plates and T-25 flasks.

Genomic and Transcript Analysis of CRISPR-Cas9 Targeting

Genomic DNA of hybridoma cell lines were recovered from typically 106 cells, which were washed with PBS by centrifugation (250×G, 5 minutes) and re-suspended in QuickExtract™ DNA Extraction Solution (Epicentre, QE09050). Cells were then incubated at 68 C for 15 minutes and 95 C for 8 minutes. For transcript analysis, total RNA was isolated from 106—5×106 cells. The cells were lysed with TRIzol® reagent (Thermo, 15596-026) and total RNA was extracted with the Direct-zol™ RNA MiniPrep kit (Zymo Research, R2052). Maxima Reverse Transcriptase (Thermo, EP0742) was used for cDNA synthesis from total RNA (Taq DNA Polymerase with ThermoPol® Buffer, NEB, M0267S). Both genomic DNA and cDNA were used as templates for downstream PCR reactions.

The gRNAs targeting WT IgH and IgK loci and mRuby were initially tested for their activity by 30 induction of NHEJ. The targeted fragment was amplified by PCR with KAPA2G Fast ReadyMix (KAPA, KK5121) and the PCR product digested with the Surveyor nuclease for the detection of mismatches (Surveyor® Mutation Detection Kit, IDT, 706020). For HDR evaluation, PCR was performed on genomic and cDNA using primers binding inside and outside homology arms, followed by fragment size analysis on DNA agarose gels. Selected PCR products were subjected to Sanger sequencing.

Flow Cytometry Analysis and Sorting of Hybridomas

Flow cytometry-based analysis and cell isolation were performed using the BD LSR Fortessa™ and BD FACS Aria™ III (BD Biosciences), respectively. At 24 hours post-transfection, approximately 100 μl of cells were harvested, centrifuged at 250×G for 5 minutes, resuspended in PBS and analyzed for expression of Cas9 (via 2A-GFP/-BFP). 48 hours post-transfection, all transfected cells were harvested and resuspended in Sorting Buffer (SB): PBS supplemented with 2 mM EDTA and 0.1% BSA). When labeling was required, cells were washed with PBS, incubated with the labeling antibody or antigen for 30 minutes on ice, protected from light, washed again with PBS and analyzed or sorted. The labeling reagents and working concentrations are described in table 3 below. For cell numbers different from 106, the antibody/antigen and incubation volume were adjusted proportionally.

TABLE 3Flow cytometry labeling reagents with their working concentrationsTargetWorkingDilutionIncubatn.antigenconc.from stockvolumeFluorophoreProduct IDIgG2C3.3μg/ml1:150100 μlAllophycocyanin115-135-208(APC)(JacksonImmunoResearch)IgG2C1.6mg/ml1:100100 μlAlexaFluor ®115-547-188488(JacksonImmunoResearch)IgK2.5μg/ml1:80100 μlBrilliant Violet409511421 ™(BioLegend)Hen egg0.1μg/ml1:62.5100 μlAlexaFluor ®62971-10G-Flysozyme647(Sigma- Aldrich)
Measurement of Antibody Secretion by ELISA

Sandwich ELISAs were used to measure the secretion of IgG from hybridoma cell lines. Plates were coated with capture polyclonal antibodies specific for Vklight chains (goat anti-mouse, Jackson ImmunoResearch, 115-005-174) concentrated at 4 μg/ml in PBS (Thermo, 10010-015). Plates were then blocked with PBS supplemented with 2% w/v milk (AppliChem, A0830) and 0.05% v/v Tween®-20 (AppliChem, A1389) (PBSMT). Supernatants from cell culture (106cells/sample, volume normalized to least concentrated samples) were then serially diluted (at 1:3 ratio) in PBS supplemented with 2% w/v milk (PBSM). As a positive control, a purified mouse IgG2b, K isotype control (BioLegend, 401202) was used at a starting concentration of 5 ng/μl (diluted in hybridoma growth media) and serially diluted as the supernatants. After blocking, supernatants and positive controls were incubated for 1 hour at RT or O/N at 4° C., followed by 3 washing steps with PBS supplemented with Tween-20 0.05% v/v (PBST). A secondary HRP-conjugated antibody specific for mouse Fc region was used (goat anti-mouse, Sigma-Aldrich, A2554), concentrated at 1.7 μg/ml in PBSM, followed by 3 wash steps with PBST. ELISA detection was performed using a 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo, 34028) as the HRP substrate. Absorbance at 450 nm was read with Infinite® 200 PRO NanoQuant (Tecan). For antigen specificity measurements, plates were coated with purified hen egg lysozyme (Sigma-Aldrich, 62971-10G-F) concentrated at 4 μg/ml in PBS. Blocking, washing, and supernatant incubation steps were made analogously to the previously described procedure, with the exception of serial dilutions of supernatants at 1:5 ratios. A secondary HRP-conjugated antibody was used specific for Vklight chain (rat anti-mouse, Abcam, AB99617) concentrated at 0.7 μg/ml. ELISA detection by HRP substrate and absorbance reading was performed as previously stated.

Targeting of the ROSA26 Locus

The mouse safe harbor locus ROSA26 was amplified and Sanger sequenced from Wen1.3 cells, and the sequence obtained was used to design DNA cassette homology arms. Guide RNA target sequences (gRNA-L to gRNA-P) were individually validated by Surveryor Assay (FIG.7). This locus was targeted for the creation of the PnP-mRuby-AID, PnP-IgG-AID and the PnP-mRuby-Cas9 cell lines.

For the targeting of the ROSA26 in the creation of the PnP-mRuby-AID and the PnP-mRuby-Cas9 cell lines, gRNA-O and gRNA-P were selected due to their high cleavage efficiency. Generation of the AID cell lines and induction by Doxycycline

Cloning of the donor cassette for the inducible AID (iSSHM) system was performed in three steps. (1) The Tet-One™ Inducible Expression System was purchased from Takara Clontech (634301); GFP-2A-AID was obtained as synthetic gene fragment (gBlocks, IDT) and cloned into the pTetOne vector by Gibson assembly cloning. (2) Homology arms (829 and 821 bp) for the hybridoma's ROSA26 locus were obtained by genomic DNA PCR and cloned in a pUC57(Kan) plasmid. (3) Finally, the previously cloned—see point (1)—Tet-One-GFP-2A-AID construct (containing, in the forward orientation: the human phosphoglycerate kinase 1 promoter (hPGK), the Tet-On 3G transactivator protein, and the SV40 poly-A signal; in the reverse orientation: the PTRE3GSInducible promoter, the GFP-2A-AID construct and the SV40 poly-A signal) was inserted between the homology arms through Gibson assembly cloning. The HDR donor was linearized by PCR with restriction digestion with the enzyme Ajul (Thermo, ER1951). gRNA-O was obtained and cloned as previously described in pX458-BFP. As an alternative NHEJ insertion design, the TetOne-GFP-2A-AID construct is linearized without homology arms. The cell lines engineered for introduction of the TetOne-iSSHM system are: PnP-mRuby-pA (PnP-mRuby with a bGH poly-A tail); PnP-HEL23-IgH−(PnP-HEL23 with a frameshift insertion in the HCDR3 knocking out antibody expression—see next sections). The workflow for these cells are shown inFIGS.9and10.

a. Cell Lines Generation

From the transfection stage, the cells were kept in Tet-free GM: regular growth media supplemented with Tet System Approved FBS, US-sourced (Takara Clontech, 631105). PnP-HEL23-IgH−and PnP-mRuby-pA cells were transfected with ˜2.5 μg px458-BFP with gRNA-O and 2.5 μg linearized pTetOne-HDR donor (see previous section). 48 hours after transfection the cells were sorted for BFP and grown for recovery. Once recovered, induction experiments were performed to verify the system's functionality: Doxycycline (Takara Clontech, 631311) was dissolved in nuclease-free H2O at 1 mg/ml, sterile filtered and diluted in Tet-free GM at need directly before use. Concentrations in the range between 1 ng/ml and 2 μg/ml were tested, with 1 μg/ml proving to be the most efficient. Cells were induced by incubation at 37 C for 24 or 48 hours; however, 24 hour incubation gave the best results and was selected as main condition to check for positive integration and induction efficiency.

The cells were sorted after 24 hours of induction: from the GFP positive population, single-cell clones were isolated, grown and characterized. An initial screening was performed to select the most positive clones after Dox induction: each sample was seeded at 1 μg Dox/105cells/1 ml culture and screened for GFP 24 hours later.

The best performing clones from the initial screening steps (9 for PnP-HEL23-IgH−, 4 for PnP-mRuby-pA) were used for genomic DNA extraction, locus-specific amplification and Sanger sequencing. According to the genomic sequence, one final clone was selected for each cell line.

b. Induction Optimization

After selection of the best clone for each transfected cell line, a second and tighter titration was performed with concentrations in the range between 500 ng/ml and 1.5 μg/ml, and induction measured at different time points (ideally: 24 hrs; 48 hrs; 72 hrs; 96 hrs. Due to Doxycycline having a half-life of 24 hours, it was replaced in culture every 48 hours, as recommended by the manufacturer (Clontech).

For each time point, induction was assessed by:FACS analysis (GFP)RT-PCR (mRNA/cDNA)Western Blot

Amplification of AID from cDNA was performed with KAPA HiFi HotStart Ready Mix. For Western Blot, M-PER™ Mammalian Protein Extraction Reagent (Thermo, 78501), supplemented with Halt™ Protease Inhibitor Cocktail (Thermo, 78430) was used to obtain lysates from cultured hybridomas, typically from 106cells. Anti-Human/Mouse Activation-Induced Cytidine Deaminase (AID) Purified (eBioscience, 14-5959-80) was used as primary antibody for AID detection via WB.

c. iSSHM (AID Activity) Assessment

For the PnP-mRuby-pA-AID cell line, hypermutation activity was first evaluated by FACS analysis and detection of decreasing mRuby fluorescence. For a more thorough evaluation, the mRuby gene was amplified from cDNA and analyzed by sanger or next-generation sequencing (NGS) using the method of molecular amplification fingerprinting (FIG.12).

For PnP-HEL23-IgH−—AID cell line, restored antibody expression and/or antigen-specificity was evaluated by flow cytometry after labelling cells (see previous sections) using anti-IgG2C and anti-IgK (any positivity arisen through random mutations) and HEL-647 (re-gained HEL positivity).

For more definitive assessment and optimization of the system, the iSSHM workflow was repeated for a cell line (obtained by either of the two starting platforms) expressing a functional antibody. To obtain such a situation. PnP-mRuby-pA cells were transfected to exchange mRuby with a sAb donor; PnP-HEL23-IgH−—AID cells were transfected to exchange the knocked-out HEL23 sAb with a functional one with the same or another specificity; in an alternative setting, the HEL23 frame was restored by HDR with a 120 ssODN containing a codon-mutated version of the original HCDR3. In case of no previously tested binders, antibodies with a known and testable antigen were typically chosen to evaluate affinity maturation.

Once obtained a PnP-sAb-AID cell line, AID was induced like previously described (ON). After a 48-96 hours induction, Doxycycline was retracted from the system (OFF). To assess affinity maturation, FACS labelling was performed as previously described, but with decreasing antigen concentrations (typically 1-0.001 μg/ml, decreasing ten-fold for each round of analysis/affinity maturation cycle). Positive cells were sorted and underwent a further iSSHM cycle; the cycle was repeated as needed. For each stage of affinity maturation, after Doxycycline retraction from the system, the effective switch OFF was evaluated by monitoring GFP fluorescence by FACS. Once in the OFF state, VLand VHregions were amplified from cDNA and iSSHM was evaluated by NGS using the method of molecular amplification fingerprinting.

Generation of the PnP-mRuby2-Cas9 Cell Line

Cloning of the donor cassette for constitutive expression of the Cas9 protein was performed in three steps. (1) The pSpCas9(BB)-2A-Puro vector (pX459) and MDH1-PGK-GFP_2.0 vector were obtained from Addgene (plasmid #48139, #11375, respectively). The Cas9-2A-Puro and GFP gene fragments were obtained from their respective vectors through PCR (KAPA HiFi HotStart ReadyMix) and assembled together with Gibson assembly cloning. (2) Homology arms (1,000 and 976 bp) for the hybridoma's ROSA26 locus were obtained by genomic DNA PCR and assembled with the pUC57(Kan) plasmid backbone through Gibson assembly cloning. (3) Finally, the previously assembled fragments—see points (1 and 2)—were assembled through Gibson assembly cloning. The HDR donor was linearized by restriction digestion with the Xhol and Mlul restriction endonucleases followed by gel electrophoresis purification. gRNA-P was cloned as previously described in pX458 (BFP). For the alternative NHEJ insertion design, the Cas9-2A-Puro-GFP construct was linearized without homology arms.

Following transfection with the Cas9-2A-Puro-GFP donor, GFP+cells were sorted and expanded. Cells were then selected for stable integration of the donor construct by culturing in regular growth media supplemented with 2.5 μg/ml of Puromycin (Thermo, A1113802) for up to one week before single-cell isolation, growth and PCR characterization. After identification of a single clone with correct integration of the Cas9-2A-Puro-GFP cassette, Cas9 activity within the cell was validated through transfection of a guide RNA targeting GFP. Cas9 cleavage activity was validated by T7E1 assay and Sanger sequencing of PCR amplicons (FIG.8). GFP knock out effectiveness was confirmed by flow cytometry.

The cell lines engineered for constitutive Cas9 expression are: PnP-mRuby-pA; PnP-HEL23-IgH−. A more comprehensive cell line was designed to incorporate both the constitutive Cas9 and the inducible AID; due to the constructs bearing homology with two different regions of the ROSA26 locus, it was possible to incorporate them in tandem like shown inFIG.8A. This allowed us to merge the high HDR efficiency achieved by constitutively expressing Cas9 with the iSSHM workflow.

Cas9 Cells—In Vitro Transcription of Guide RNA or Synthetic Oligonucleotides (IDT)

The PnP-mRuby-Cas9 cell line, constitutively expressing Cas9, was transfected with the 10 appropriate HDR donor and already transcribed guide RNAs. The latter were obtained as oligonucleotides from IDT or in vitro transcribed. In the case of in vitro transcription, the previously described guide-DNA oligodeoxynucleotides (see Cloning and assembly CRISPR-Cas9 targeting constructs section) served as templates for the MEGAscript® T7 transcription kit (Thermo, AM1334). An adapted protocol was as described previously (https://www.protocols.io/view/In-vitro-transcription-of-guide-RNAs-d4w8xd?step=3 accessed Feb. 21, 2017).