Part I: Base editing in HEK293T cells through transfection
In this section, we describe the construction of A&C-BEmax to achieve C•G to T•A and A•T to G•C substitutions in HEK293T cells, taking HBG site1 below for example.
A: SgRNA plasmids cloning
1. The oligonucleotide sequences are shown below:
HBG site1 Oligo-up CACCGTTGACCAATAGCCTTGACA
HBG site1 Oligo-dn AAACTGTCAAGGCTATTGGTCAAC
2. Generate oligonucleotide duplex:
15 μL Oligonucleotide-up (100 μM stock)
15 μL Oligonucleotide-dn (100 μM stock)
Mix the reaction thoroughly by pipetting. Denature the oligonucleotides at 95°C for 5min followed by slowly cooling to room temperature. This process takes about 2 hours. Dilute oligonucleotide duplex 1:100 with ultrapure water for subsequent use.
3. U6-sgRNA scaffold is amplified from PX458 (Addgene Plasmid #48138) and cloned into pcDNA 3.1(+) eGFP (Addgene Plasmid #78583) to generate U6-sgRNA(sp)-EF1α-GFP expression plasmids as described previously.11, 12
4. Digest the U6-sgRNA(sp)-EF1α-GFP vector backbone:
① 10× FastDigest Green Buffer 2 μI
② Fast Digest BbsI 1 μI
③ Plasmid vector 1 μg
④ ddH2O up to 20 μI
⑤ Incubate the reaction at 37 °C for 20 minutes.
5. Purify the linearized plasmid with the Agarose Gel DNA Recovery kit12.
6. Ligate the annealed oligonucleotide duplex into the U6-sgRNA(sp)-EF1α-GFP plasmid as follow:
① BbsI-linearized U6-sgRNA(sp)-EF1α-GFP vector 1 μI (~30 μg)
② Diluted oligonucleotide duplexes 1 μl
③ 10×T4 Buffer 1 μI
④ T4 DNA ligase 1 μI
⑤ ddH2O up to 10 μI
Mix by pipetting, centrifuge briefly, then incubate in a water bath at 16℃ for 30 minutes.
7. Mix the ligation mixture 10 μl (from step 6) with 30 μl DH5α competent cells, and incubate on ice for 10-30 minutes. Then heat shock at 42 °C water bath for 90s and then incubate on ice for 5 min. Add 1 ml LB medium (without antibiotics) and incubate at 37°C for 1 h with shaking, plate 100 μl of each transformation culture onto LB plates with corresponding antibiotics and incubate at 37 °C overnight (for about 12-16 hours). Inoculate two to four colonies into LB medium and incubate at 37 °C overnight with shaking. Extract plasmid DNA using TIANprep Mini Plasmid Kit the next day for verification of the correct clones.
B: Construction of the A&C-BEmax and Lenti A&C-BEmax plasmid
1. TadA-TadA* is amplified from pCMV-ABE7.10 (Addgene Plasmid #102919); AID is amplified from AID-BE3 (Addgene Plasmid #100803); two copies of uracil DNA glycosylase inhibitor (UGI) is amplified from pCMV_BE4max (Addgene Plasmid #112093) vector; the linker is synthesized by BioSune as depicted previously 13. Assemble the construct as the following order, N-AID-TadA/TadA*-nCas9-C to generate A&C-BEmax plasmid with ClonExpress MultiS One Step Cloning Kit (Vazyme, cat. no. C113-01).
2. PGK-puro-P2A-GFP is amplified from pLenti-FNLS-P2A-GFP-PGK-Puro (Addgene Plasmid #110869) and cloned into LentiCRISPR v2 vector (Addgene Plasmid #52961) to generate Lenti-P2A-GFP-PGK-puro plasmid. EF1a-AID-TadA-TadA*-nCas9 is amplified from A&C-BEmax and cloned into Lenti-P2A-GFP-PGK-puro vector to generate Lenti A&C-BEmax expression plasmid.
3. U6-sgRNA scaffold and EF1α promoter is amplified from PX458 (Addgene Plasmid #48138), UGI is amplified from pCMV_BE4max (Addgene Plasmid #112093) vector, assemble these fragment using the ClonExpress MultiS One Step Cloning Kit to generate Lenti U6-EF1α-UGI-P2A-GFP expression plasmid as above described.
4. Linearize the Lenti U6-EF1α-UGI-P2A-GFP vector by BbsI, ligate the annealed oligos (HBG site1-up/dn) into the linearized vector with T4 DNA ligase to generate Lenti U6-HBG site1-EF1α-UGI-P2A-GFP construct.
Critical step: The optimal amount of vector backbone and inserts can be roughly calculated using 0.02 × the number of base pairs of the DNA fragment.
C: Cell transfection and genomic DNA preparation
Seed 2 ×105 HEK293T cells into 24-well plates allowing approximately 80% confluency on the next day. On the second day, the cells are transfected with 750 ng of A&C-BEmax, 250 ng of sgRNA expression plasmid-HBG site1-U6-sg-EF1α-GFP using polyethyleneimine (1 μg plasmid : 3 μl PEI). Prepare transfection mixes in separate 1.5 ml tubes. Prepare A mix with 750ng A&C-BEmax and 250 ng of sgRNA expression plasmid into 50ul DMEM. Add 3ul PEI (Stock Concentration 1ug/ul) into 50 μl DMEM to get B mix.Vortex each mix well and incubate 5 minutes at room temperature. Combine A and B mix, vortex thoroughly and incubate for an additional 20 minutes at room temperature. Add the solution 100 μl to the HEK293T cells carefully. Three days later, harvest the cells and isolate genomic DNA using the Blood/Cell/Tissue DNA Isolation Kit as described previously12.
D: Base editing efficiency evaluation
The DNA fragment containing the target-HBG site1 is obtained by PCR using KOD–Plus-Neo DNA Polymerase and site-specific primers containing an adaptor sequence (Forward 5′-ggagtgagtacggtgtgc-3′; Backward 5′-gagttggatgctggatgg-3′) at the 5′ end. Successfully edited results can be first discriminated by sanger sequencing chromatography. Detailed efficiency results are obtained from high-throughput deep sequencing data analyzed by BE-Analyzer14or CRISPResso215.
Part II: Generation of HUDEP-2 cell Clones
A: HUDEP-2 cell culturing
Culturing HUDEP-2 cells as previously described16. HUDEP-2 cells are expanded in StemSpan SFEM (SFEM, Stemcell Technologies) supplemented with dexamethasone (DEX, 1µM, Sigma), human stem cell factor (SCF, 50 ng/ml, PeproTech), erythropoietin (EPO, 3 IU/ml, PeproTech), 1% L-glutamine (Life Technologies), and 2% penicillin/streptomycin (Gibco), Doxycycline hyclate (Sigma, 1 µg/ml). 5000–10,000 HUDEP-2 cells are seeded each well in a 12- or 24-well plate. All cell lines used are maintained at 37 °C, 5% CO2 in the incubator.
Critical step: The cells double every day and should always be maintained below 8x105 cells/ml. Never grow over 1x106 cells/ml to keep cell healthy.
B: Lentiviral infection of HUDEP-2 cells
1. Base editing in HUDEP-2 cells through lentiviral infection.
Day 1: Lentiviral infection
Detailed steps for virus packaging, purification and titration are described in a previous protocol 12. Two lentiviruses are prepared separately, Lenti A&C-BEmax and Lenti U6-HBG site1-EF1α-UGI-P2A-GFP. Supplement Lenti A&C-BEmax lentivirus into 2 x105 HUDEP-2 cell culture to reach an MOI of 40 along with Lenti U6-HBG site1-EF1α-UGI-P2A-GFP lentivirus reach an MOI of 1.
Day3-18: Cell sorting and culturing
48h after lentiviral transduction, 1 μg/mL (final concentration) puromycin is added into the expansion medium to enrich the transduced cells. After 3 days, most cells without resistance will die. Centrifuge the cell at 300 g for 5 min to remove cell debris. Maintain the cells in culture medium with puromycin (1 μg/mL final concentration) for another week.
Critical step: The volume of virus added is dependent on the amount of virus titer.
Day19-21: Genomic DNA preparation and evaluation of base editing results
① Harvest at least 8x104 HUDEP-2 cells for genomic DNA extraction using Blood/Cell/Tissue DNA Isolation Kit.
② The DNA fragment containing the target HBG site1 is amplified through PCR using HUDEP-HBG site1-F(Merlin)/R(Merlin) primers and KOD–Plus-Neo DNA Polymerase.
The primers sequences are as follows:
HUDEP-HBG site1-F(Merlin): AGTGTGTGGACTATTAGTCAA
HUDEP-HBG site1-R(Merlin): catggcgtctggactaggag
PCR products are subjected to sanger sequencing chromatography to estimate editing efficiency. Examples of sanger sequencing analysis are displayed in Figure 1.
2. Generating HUDEP-2 single cell clones (monoclonal cell population)
Single cell clones can be established from Lenti A&C-BEmax treated cells containing various genotypes to interrogate the relationships between the genotype and phenotype.
Day 21-22: Seed cell into 96-well
Seed HUDEP-2 cells into 96-well culture plates through limited dilution as previously described17. Diluted 30-50 HUDEP-2 cells into 12ml culture medium and seed 100ul of culture into one well of a 96-well plate17. The next day check the cell condition and label the wells that have only 1-3 cells for subsequent experiment.
Day 23-34: Cell clone expansion and evaluation of editing efficiency
① Expand the cell for about 10-11 days in a 96-well plate. Once cell clones have expanded to cover the bottom of the well, transfer the cells into larger size plates.
② Harvest at least 8x104 HUDEP-2 cells for genomic DNA extraction using Blood/Cell/Tissue DNA Isolation Kit. DNA fragment containing the target site is pcr amplified for sanger sequencing as described above to identify the genotypes of single clones. Examples of sanger sequencing analysis are displayed in Figure 2. Detailed editing efficiency result i.e. indels, can be quantitated via deep sequencing as mentioned above.
C: HUDEP-2 cell differentiation
1. Erythroid Differentiation.
HUDEP-2 cells are differentiated in a two-phase erythroid differentiation protocol. Phase 1 (day 1–4), cells are cultured in EDM-2 medium consisting of Iscove’s modified Dulbecco’s medium (IMDM; Gibco), 2%Human Serum AB (GEMINI Bio-products), 2 IU/ml Heparin, 10 μg/ml Recombinant Human Insulin, and 3 IU/ml erythropoietin (EPO, PeproTech), 330 μg/ml Holo-Transferrin Human (Sigma-Aldrich), 100 ng/ml SCF, 1 μg/mL doxycycline hyclate(DOX, Sigma), 2% penicillin–streptomycin (Gibco), 1% L-glutamine. In Phase 2 (day 4-8), cells are cultured in EDM-3 with the same ingredients as EDM-2 but is absent of DOX and SCF.
Critical step: The length of culturing for phase I or II or the whole process is variable and dependent on the stage of differentiation required.
2. RT-qPCR analysis of globin and erythroid markers
① After 8 days of differentiation, the cell precipitation changes from white to dark red , then harvest 1x105 cells for total mRNA isolation by Trizol reagent according to the standard protocol18.
② Isolated mRNA is reversely transcribed using HiScript II Q RT SuperMix (Vazyme) kit according to the manufacturer's protocol19.
③ Perform qPCR on the QuantiStudio 3 real-time PCR system (ABI) using Hieff® qPCR SYBR® Green Master Mix kit（Yeasen）to quantitate HBG and HBB expression level. Experimental operation and reagents shall be used according to the product brochure19 . Primer sequences used for RT-qPCR are as follow：
HBG: HBG- qPCR-F ggttatcaataagctcctagtcc
HBG- qPCR-R acaaccaggagccttccca
HBB: HBB- qPCR-F tgaggagaagtctgccgttac
HBB- qPCR-F accaccagcagcctgccca
Critical Step: Ensure that all apparatus and reagents to be used in the isolation procedure are RNase-free.
Avoid cross-contamination and aerosol contamination.