Base Editing Mediated Generation of Point Mutations Into Human Pluripotent Stem Cells for Modeling Disease

Abstract

Human pluripotent stem cells (hPSCs) are a powerful platform for disease modeling and drug discovery. However, the introduction of known pathogenic mutations into hPSCs is a time-consuming and labor-intensive process. Base editing is a newly developed technology that enables facile introduction of point mutations into specific loci within the genome of living cells. Here, we design an all-in-one episomal vector that expresses a single guide RNA (sgRNA) with an adenine base editor (ABE) or a cytosine base editor (CBE). Both ABE and CBE can efficiently introduce mutations into cells, A-to-G and C-to-T, respectively. We introduce disease-specific mutations of long QT syndrome into hPSCs to model LQT1, LQT2, and LQT3. Electrophysiological analysis of hPSC-derived cardiomyocytes (hPSC-CMs) using multi-electrode arrays (MEAs) reveals that edited hPSC-CMs display significant increases in duration of the action potential. Finally, we introduce the novel Brugada syndrome-associated mutation into hPSCs, demonstrating that this mutation can cause abnormal electrophysiology. Our study demonstrates that episomal encoded base editors (epi-BEs) can efficiently generate mutation-specific disease hPSC models.

Keywords: Brugada syndrome; IPS; base editing; disease modeling; episomal vector; human pluripotent stem cell; long QT syndrome.

FIGURE 1

Efficient base editing with epi-ABEmax and epi-AncBE4max. (A) Schematic of the epi-ABEmax and epi-AncBE4max plasmids. The vector contains a mouse U6 promoter-driven gRNA scaffold, an EF1a promoter-driven base editor, an SV40 promoter-driven a blasticidin S deaminase (BSD), and oriP/EBNA1 elements for the plasmid replication in eukaryotes. (B) Procedure of base editing with epi-ABEmax and epi-AncBE4max. (C–F) Editing efficiency of epi-ABEmax in HEK293T, HeLa, H9, and iPSCs was measured at three-time points, n = 3 independent experiments. (G–J) Editing efficiency of epi-AncBE4max in HEK293T, HeLa, H9, and iPSCs was measured at three-time points, n = 3 independent experiments.

FIGURE 2

Base editing of L114P-KCNQ1 for LQT1 modeling. (A) L114P target site on KCNQ1. “CGG” PAM sequence is shown in orange; target nucleotide to be edited is indicated by a red triangle. (B) A heterozygous clone is confirmed by Sanger sequencing. The mutated nucleotide is shown in red; the mutated nucleotide is indicated by a red triangle. (C) Single trace of action potentials in WT-CMs and L114P-CMs. (D,E) Quantification of action potential at APD50 and APD90. n = 3 independent experiments, unpaired t-test, P < 0.0001. (F) Signals of field potential duration recorded by MEA. (G) Quantification of corrected field potential durations (FPDc). n = 3 independent experiments, unpaired t-test, P < 0.0001. (H) Representative traces of action potentials. The abnormal AP signals were labeled by black arrows. A value of P < 0.05 was considered to be statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 3

Base editing of Y616C-KCNH2 for LQT2 modeling. (A) Y616C target site on KCNH2. “AGC” PAM sequence is shown in orange; target nucleotide is indicated by a red triangle. (B) A heterozygous clone is confirmed by Sanger sequencing. The mutated nucleotide is shown in red; the mutated nucleotide is indicated by a red triangle. (C) Single trace of action potentials in WT-CMs and Y616C-CMs. (D,E) Quantification of action potential at APD50 and APD90. n = 3 independent experiments, unpaired t-test, P < 0.0001. (F) Signals of field potential duration recorded by MEA. (G) Quantification of corrected field potential durations (FPDc). n = 3 independent experiments, unpaired t-test, P < 0.0001. (H) Representative traces of action potentials. The abnormal AP signals were labeled by a black arrow. A value of P < 0.05 was considered to be statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 4

Base editing of E1784K-SCN5A for LQT3 modeling. (A) E1784K target site on SCN5A. PAM sequence is shown in orange; target nucleotide is indicated by a red triangle. (B) A heterozygous clone is confirmed by Sanger sequencing. The mutated nucleotide is shown in red; the mutated nucleotide is indicated by a red triangle. (C) Single trace of action potentials in WT-CMs and E1784K-CMs. (D,E) Quantification of action potential at APD50 and APD90. n = 3 independent experiments, unpaired t-test, P < 0.0001. (F) Signals of field potential duration recorded by MEA. (G) Quantification of corrected field potential durations (FPDc). n = 3 independent experiments, unpaired t-test, P < 0.0001. A value of P < 0.05 was considered to be statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 5

Evaluation of R1879W-SCN5A mutation. (A) Patient’s ECG results. The resting ECG showed an ST elevation in leads V1–V3 with a saddle-back pattern. (B) Sanger sequencing reveals that a C to T mutation occurs on SCN5A. (C) R1879W target site on SCN5A. PAM sequence is shown in orange; target nucleotide is indicated by a red triangle. (D) A heterozygous clone is confirmed by Sanger sequencing. The mutated nucleotide is shown in red; the mutated nucleotide is indicated by a red triangle. A neighbor nucleotide C is changed to T, but it does not change the amino acid. (E) Single trace of action potentials in WT-CMs and R1879W-CMs. (F,G) Quantification of action potential at APD50 and APD90. n = 3 independent experiments, unpaired t-test. (H) Quantification of corrected field potential duration (FPDc). n = 3 independent experiments, unpaired t-test. (I) Representative action potential traces with sustained triggered activity. Triggered beats were labeled by black arrows.