HDR enhancement with 53BP1 inhibition is most effective with i53 mRNA. (A) Depiction of CYBB c.676 C>T mutation (top; red arrowhead) and successful repair with a 100-nucleotide ODN donor or TI of CYBB E7-13pA, CYBB E7-13pA-WPRE, or CYBB E7-13pA-SFFV-GFP delivered by AAV. (B) Percentages of GFP⁺ in HD HSPCs following TI with increasing i53 concentration (0-300 µg/mL); (n = 1; 8 replicates). (C) Percentages of GFP⁺ cells GE with Cas9 RNA or RNP and i53 or e18 enhancer (n = 4 experiments; 2 donors). (D) TI rates after GE with Cas9 RNA or RNP and increasing AAV donor (multiplicity of infection 1 = 10³/cell) ±i53 (150 µg/mL; n = 3 [1 HD, 2 X-CGD patients]). (E) TI rates after GE in the presence of i53, BRCA2, or RAD51 at indicated concentrations (n = 2 experiments). *P < .05, **P < .01, ****P < .0001, paired Student t test.