Therapeutic gene editing of IVS2-654C>T. (A) Schema of IVS2-654C>T mutation and therapeutic editing strategy. Cut site is shown at midpoint of expected Cas12a staggered cleavage. (B) Indicated donors and crRNAs used for therapeutic editing. Five days after RNP electroporation, amplicon deep sequencing was performed on the LbCas12a-treated cells. Following sequence analysis, alleles were classified as edited, unedited IVS2-654C>T, or unedited IVS2-654C. (C) Nucleotide quilt showing indels and substitutions at each position around IVS2-654 for indicated donors and LbCas12a RNP treatment groups. β⁺β⁰_#5 with sgAAVS1 shown as a representative example of an unedited IVS2-654C>T heterozygous donor with rs1609812-T/T. β⁺β⁰_#4 shown as a donor in which the IVS2-654C/rs1609812-C and IVS2-654C>T/rs1609812-T alleles could be distinguished. (D) RT-PCR from erythroid progeny with primers spanning the exon 2 to exon 3 junction, demonstrates abrogation of aberrant (A) and increase in normal (N) splicing after therapeutic editing. (E) RT-qPCR of globin genes shows increase in β-globin relative to α-globin expression in erythroid progeny after therapeutic editing. (F) Hemoglobin HPLC shows increase in the HbA fraction after therapeutic editing. (G-H) Flow cytometry shows increase in enucleation fraction and cell size of enucleated erythroid cells after therapeutic editing.