{'fraction_non_alphanumeric': 0.02737382378100941, 'fraction_numerical': 0.00427715996578272, 'mean_word_length': 5.573033707865169, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Efficient generation of site-specific point mutations in cell lines by hyper active CBEs (hyCBEs)


 Cytidine base editors (CBEs) are powerful genetic tools which catalyze cytidine to thymidine conversion at specific genomic loci. Further improvement of the editing range and efficiency is critical for their broader applications. In this protocol, we demonstrated that fusion of the single-strand DNA binding domain (ssDBD) from Rad51 protein between Cas9n and cytidine deaminases, serial hyper CBEs (hyCBEs) were generated with substantially increased activity and an expanded editing window toward the protospacer adjacent motif (PAM) in cell lines. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the hemoglobin gamma (HBG) gene promoter to mimic a naturally occurring genetic variant for amelioration of β-hemoglobinopathy, suggesting the therapeutic potential of the improved base editors. This step-by-step protocol is related to the publication “Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-strand DNA binding protein domain” in Nature Cell Biology.