Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells
Background The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigated the potential off-target structural variations associated with CRISPR/Cas9, ABE and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. Results The results showed that both Cas9 and ABE generated off-target structural variations (SVs) in mouse embryos, while CBE induced rare SVs. In addition, off-target large deletions were detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activated the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulated cell cycle-related genes and arrested in G0 phase. A percentage of 16.59% and 4.29% aneuploid cells were still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE were universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs were significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). Conclusions This study raises urgent need for minimizing the off-target SVs of CRISPR/Cas9 and ABE. Overall design: In this study, two target genes, PDCD1 and B2M, were edited. The same single guide RNA (sgRNA) used by Gaudelli et al. [31] to target B2M was also used in our study, successfully targeting both by ABE and Cas9 through splice site disruption. For PDCD1, we used the same sgRNA as Nahmad et al. [4] did for Cas9 and Gaudelli et al. [31] did for ABE. For high-fidelity ABE, the same sgRNA targeting PDCD1 was used. For Huh7 ,, the same sgRNA targeting B2M was used. Prior to the experiment, T Cell Medium (Lonza X vivo5 + 5% human AB serum + NAC + 10ng/ml IL7 and 10ng/ml IL15) was prepared and allowed to equilibrate at room temperature for 20 minutes within a biological safety cabinet. Cryopreserved PBMCs were then retrieved from a liquid nitrogen tank, thawed in a 37°C water bath, and transferred into the cabinet after removing any remaining ice clumps. The outer surface of the container was sterilized with 5% ethanol, dried, and transferred into the cabinet. The cells were then transferred into 9 mL of T cell medium and centrifuged at 400g for 5 minutes at room temperature. After discarding the supernatant, the cells were resuspended in 5 mL of medium and counted using a ThermoFisher Attune NxT flow cytometer. Subsequently, the cells were seeded at a density of 1e6 cells/mL in plates or culture flasks. After 6 hours of PBMC revival, CD3/28 dynabeads were added for activation. Following 3 days of cell activation, the activation beads were removed using a magnet and the culture was continued for 24 hours. For electroporation, 24-well plates were prepared by adding 900 µL of PBMC complete medium per well and incubating at 37°C for 10 minutes. The Lonza 4D Nucleofector System with the P3 Primary Cell 4D kit was used, following the kit instructions for electroporation. Each electroporation reaction consisted of 1E6 cells, 1.2 pmol of EPIREG mRNA, and 40 pmol of sgRNA for each target gene. Immediately after electroporation, 100 µL of cell-free medium was added to each well, followed by a 1-hour incubation at 37°C. The cells were then transferred to 24-well plates for further culture. To assess changes in the protein levels of PDCD1 and B2M on T cell surfaces using flow cytometry, approximately 1e5 cells were transferred to 1.5 mL Eppendorf tubes and centrifuged at 400g for 5 minutes at room temperature. The supernatant was discarded and 50 µL of the corresponding dilution ratio of antibodies was added. The tubes were incubated at 4°C in the dark for 30 minutes, followed by the addition of 500 µL of MACS buffer to each tube. After centrifuging at 400g for 5 minutes at room temperature, the supernatant was discarded and 300 µL of MACS buffer was added to each tube, gently mixed by pipetting. The samples were then analyzed using a ThermoFisher Attune NxT flow cytometer. The 10x Genomics® Cell Preparation Guide was followed for washing, counting, and concentrating cells from both abundant and limited cell suspensions (greater than or less than 100,000 total cells, respectively) in preparation for use in 10x Genomics Single Cell Protocols. The cell suspension was loaded into Chromium microfluidic chips with 3' (v2 or v3, depending on the project) chemistry and barcoded with a 10× Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries were constructed using reagents from a Chromium Single Cell 3' v2 (v2 or v3, depending on the project) reagent kit (10X Genomics) according to the manufacturer's instructions. Sequencing was performed with Illumina (HiSeq 2000 or NovaSeq, depending on the project) according to the manufacturer's instructions (Illumina). The same library construction process was used in Huh7 cells.