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. Author manuscript; available in PMC: 2021 Nov 29.
Published in final edited form as: Nat Genet. 2021 May 6;53(6):869–880. doi: 10.1038/s41588-021-00861-8

Extended Data Fig. 1. Establishment of an ABEmax-based system to perturb regulatory sequences.

Extended Data Fig. 1

(a) ABEmax-Cas9 protein levels measured by Western blot analysis in wild-type HUDEP-2 cells (WT) and HUDEP-2 cells infected with different dosages of ABEmax lentivirus. β-Actin was used as a loading control. The result is representative of three independent experiments (Image was cropped from source data Fig. 2).

(b) HUDEP-2 cells with different levels of ABEmax expression were transduced with the same amount of gRNA targeting the HBG promoter. The graphs show the hemoglobin (Hb) protein content, as measured by isoelectric focusing high-performance liquid chromatography (IE-HPLC) in HUDEP-2 cells after 5 additional days of induced erythroid maturation. The result is representative of three independent experiments.

(c) Jitter plots showing the percentage of adenosine-to-inosine RNA modification by ABEmax in wild-type HUDEP-2 cells (WT), HUDEP-2 cells stably expressing an ABE (ABEmax), and HEK293T cells. The y-axis represents the efficiency of A-to-I RNA editing. n = total number of modified adenines observed.

(d) Targeted deep-sequencing analysis of the BCL11A CRE after editing with ABEmax and BCL11A_ENH gRNA. The mutations are indicated in bold. The red arrowhead indicates the targeted nucleotide.

(e) Western blot analysis with the indicated antibodies in undifferentiated (Day 0) and differentiated (Day 5) HUDEP-2 cells transduced with non-targeting control gRNA (Ctrl) or with BCL11A-ENH gRNAs. The result is representative of three independent experiments. (Image was cropped from source data Fig. 3)