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

Qinquan Cheng; Jing Xia; Kaimin Wang; Yue Zhang; Yan Chen; Qi Zhong; Xue Wang; Qi Wu

doi:10.21037/atm-22-3279

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

Ann Transl Med. 2022 Aug;10(16):862. doi: 10.21037/atm-22-3279.

Authors

Qinquan Cheng^#¹, Jing Xia^#¹, Kaimin Wang^#¹, Yue Zhang², Yan Chen¹, Qi Zhong¹, Xue Wang³, Qi Wu^{1

4

5}

Affiliations

¹ Department of Clinical Laboratory, Shanghai Tenth People's Hospital of Tongji University, Shanghai, China.
² Department of Central Laboratory, Shanghai Tenth People's Hospital of Tongji University, Shanghai, China.
³ Department of Dermatology, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China.
⁴ Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education, Guizhou Medical University, Guiyang, China.
⁵ Department of Clinical Laboratory, Shanghai Tenth People's Hospital Chong Ming Branch, Shanghai, China.

^# Contributed equally.

Abstract

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.

Keywords: CRISPR/Cas9; acute myeloid cells; electroporation; guide RNA (gRNA); ribonucleoprotein (RNP).