CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells

被引:4
作者
Cheng, Qinquan [1 ]
Xia, Jing [1 ]
Wang, Kaimin [1 ]
Zhang, Yue [2 ]
Chen, Yan [1 ]
Zhong, Qi [1 ]
Wang, Xue [3 ]
Wu, Qi [1 ,4 ,5 ]
机构
[1] Tongji Univ, Dept Clin Lab, Shanghai Peoples Hosp 10, Shanghai, Peoples R China
[2] Tongji Univ, Dept Cent Lab, Shanghai Peoples Hosp 10, Shanghai, Peoples R China
[3] Shanghai Jiao Tong Univ, Dept Dermatol, Shanghai Peoples Hosp 9, Sch Med, Shanghai, Peoples R China
[4] Guizhou Med Univ, Key Lab Endem & Ethn Dis, Minist Educ, Guiyang, Peoples R China
[5] Shanghai Tenth Peoples Hosp, Dept Clin Lab, Chong Ming Branch, Shanghai, Peoples R China
关键词
CRISPR/Cas9; ribonucleoprotein (RNP); guide RNA (gRNA); electroporation; acute myeloid cells; CAS9; RIBONUCLEOPROTEIN; NANOPARTICLE DELIVERY;
D O I
10.21037/atm-22-3279
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
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 hematopoictic cells.
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页数:9
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