{'fraction_non_alphanumeric': 0.025953389830508475, 'fraction_numerical': 0.00847457627118644, 'mean_word_length': 5.581881533101045, '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}}
CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells

Background Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has become an increasingly vital tool for modifying gene expression in a variety of cell types. Lentiviral transduction and electroporation are the two main approaches used to deliver CRISPR/Cas9 into cells. However, the application of CRISPR/Cas9 in primary hematopoietic cells has been limited due to either low transduction efficiency in terms of viral-based delivery or difficult selection and enrichment of transfected and edited cells with respect to electroporation of CRISPR/Cas9 ribonucleoprotein (RNP). Methods In this study in vitro transcription was used to synthesize the guide RNA (gRNA), and plasmid pL-CRISPR.EFS.GFP was used as its DNA template. Then the in vitro transcribed gRNA was labeled with pCp-Cy5 via T4 ligase before incubating with Cas9 protein. Furthermore, CRISPR/Cas9 RNP was electroporated into primary CD34+ cells isolated from cord blood, and cell survival rate and transfection efficiency were calculated and compared to that of lentiviral transduction. Results Here, we show that electroporation of CRISPR/Cas9 RNP resulted in higher cell viability compared to electroporation of CRISPR/Cas9 all-in-one plasmid, providing important findings for further studies in hematology via CRISPR/Cas9 technology. Moreover, we established a method for labeling in vitro-transcribed gRNA with fluorophore and the sorted fluorescent cells displayed higher knockout efficiency than nonsorted transfected cells. Conclusions Electroporation of fluorescence labeled CRISPR/Cas9 RNP is a perspective approach of gene editing. Our study provides an efficient and time-saving approach for genome-editing in hematopoietic cells.