pcDNA3.1(−)-BE3 was synthesized by Guangzhou IGE biotechnology LTD. YEE-BE3 (W90Y/R126E/R132E triple mutant) was from Addgene (#851777). The pcDNA3.1(−)-BE3 was used for expression in human cells and in vitro transcription. pUC19-SpCas9 gRNA expression vector was cloned by amplifying the U6-gRNA fragment from pX330 (Addgene, #42230), and then inserting this fragment into pUC19 vector. Sequences for cloning the gRNA-1, gRNA-2, and gRNA-3 into the pUC19-SpCas9 gRNA expression vector were listed in Table S2. gRNAs was cloned into pDR274 (Addgene, #42250) for in vitro transcription. Sequence for cloning gRNA-1 into pDR274 was listed in Table S3. Target region, spanning HBB −28 sites, was amplified using HBB-FP and HBB-RP primers (Table S4). And then the PCR product was digested with NotI and AscI. This digested PCR product was then cloned into pENTR/D-TOPO vector (Invitrogen), resulting in pENTR/D-TOPO-HBB. −28 A>G mutation was then induced into this vector by quick change PCR using HBB-78-QC-FP and HBB-78-QC-RP primers (Table S4). And then gateway cloning was carried out to clone the −28 (A>G) mutant HBB fragment into pLenti-EF1a-DEST-SFB vector, resulting pLenti-EF1a-DEST-HBB-SFB vector.