Both A-toG and CtoT base editing in human cells with dual base editor ( A & C-BEmax )

Biyun Zhu Institute of Biomedical Sciences and School of Life Sciences, East China Normal University Xiaohui Zhang Institute of Biomedical Sciences and School of Life Sciences, East China Normal University Liang Chen Institute of Biomedical Sciences and School of Life Sciences, East China Normal University Liren Wang Institute of Biomedical Sciences and School of Life Sciences, East China Normal University Dali Li (  dalidalili@yahoo.com ) Institute of Biomedical Sciences and School of Life Sciences, East China Normal University https://orcid.org/0000-0002-0046-8493


Introduction
Base editors, including cytidine(CBE) and adenosine(ABE) editors, have been developed through fusion of nucleotide deaminases to nuclease impaired Cas9 variants 1-4 . The commonly used base editors BE3 and ABE7.10 typically generate C•G to T•A or A•T to G•C conversions within a ~5-nucleotide editing window on the target DNA respectively, without inducing DNA double strand breaks 1, 2 . These advantegies make them promising gene-editing tools in disease modeling 3,4 , genetic breeding 5 , gene therapy 6 and other areas.Many efforts have been made to improve the performance of the base editors. Through recruitment of multiple cytidine deaminase domains to mutant Cas9 protein (CRISPR-X 7 or BE-Plus 8 ), using activation-induced cytidine deaminase (AID) instead of APOBEC1 9 , or fusing deaminase with circularly permuted Cas9 10 , CBEs with wider editing window were created to increase the genomic diversi cation which has multiple use, such as directed protein evolution. However, CBE or ABE could only catalyze single type of nucleotide, either C•G to T•A or A•T to G•C, which limits the diversity of the products.
Through fusion of cytidine and adenosine deaminases to Cas9n (SpCas9 D10A), a dual base editor, A&C-BEmax has been developed. A&C-BEmax can generate simultaneous A-to-G and C-to-T base conversions in the same allele with increased CBE editing window and activity. As A&C-BEmax generates more mutant allele types suggesting increased mutation spectra of the products, it exhibits a broad potential for investigation and modi cation of both coding and non-coding sequences at a single base resolution. As an example, we have demonstrated that A&C-BEmax could generate various point mutations, including simultaneous C-to-T and A-to-G conversions, in the promoter of HBG1/2 in HUDEP-2 cells with a single sgRNA for the potential treatment of β-hemoglobinopathy. This protocol below contains the construction of the A&C-BEmax and generation of site-speci c point mutations in HUDEP-2 cells.  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.

Reagents
A: SgRNA plasmids cloning 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.
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 veri cation of the correct clones.
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 ×10 5 HEK293T cells into 24-well plates allowing approximately 80% con uency 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 previously 12 .

D: Base editing e ciency evaluation
The DNA fragment containing the target-HBG site1 is obtained by PCR using KOD-Plus-Neo DNA Polymerase and site-speci c primers containing an adaptor sequence (Forward 5′-ggagtgagtacggtgtgc-3′; Backward 5′-gagttggatgctggatgg-3′) at the 5′ end. Successfully edited results can be rst discriminated by sanger sequencing chromatography. Detailed e ciency results are obtained from high-throughput deep sequencing data analyzed by BE-Analyzer 14 or CRISPResso2 15 .
Day3-18: Cell sorting and culturing 48h after lentiviral transduction, 1 μg/mL ( nal 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 nal concentration) for another week. Harvest at least 8x10 4 HUDEP-2 cells for genomic DNA extraction using Blood/Cell/Tissue DNA Isolation Kit. DNA fragment containing the target site is pcr ampli ed 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 e ciency result i.e. indels, can be quantitated via deep sequencing as mentioned above. C: HUDEP-2 cell differentiation 1. Erythroid Differentiation.
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.

RT-qPCR analysis of globin and erythroid markers
After 8 days of differentiation, the cell precipitation changes from white to dark red , then harvest 1x10 5 cells for total mRNA isolation by Trizol reagent according to the standard protocol 18 .
Isolated mRNA is reversely transcribed using HiScript II Q RT SuperMix (Vazyme) kit according to the manufacturer's protocol 19 .
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 brochure 19 . 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.