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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 CR...

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Autores principales: Cheng, Qinquan, Xia, Jing, Wang, Kaimin, Zhang, Yue, Chen, Yan, Zhong, Qi, Wang, Xue, Wu, Qi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: AME Publishing Company 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9469150/
https://www.ncbi.nlm.nih.gov/pubmed/36111017
http://dx.doi.org/10.21037/atm-22-3279
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author Cheng, Qinquan
Xia, Jing
Wang, Kaimin
Zhang, Yue
Chen, Yan
Zhong, Qi
Wang, Xue
Wu, Qi
author_facet Cheng, Qinquan
Xia, Jing
Wang, Kaimin
Zhang, Yue
Chen, Yan
Zhong, Qi
Wang, Xue
Wu, Qi
author_sort Cheng, Qinquan
collection PubMed
description 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.
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spelling pubmed-94691502022-09-14 CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells Cheng, Qinquan Xia, Jing Wang, Kaimin Zhang, Yue Chen, Yan Zhong, Qi Wang, Xue Wu, Qi Ann Transl Med Original Article 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. AME Publishing Company 2022-08 /pmc/articles/PMC9469150/ /pubmed/36111017 http://dx.doi.org/10.21037/atm-22-3279 Text en 2022 Annals of Translational Medicine. All rights reserved. https://creativecommons.org/licenses/by-nc-nd/4.0/Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0 (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Original Article
Cheng, Qinquan
Xia, Jing
Wang, Kaimin
Zhang, Yue
Chen, Yan
Zhong, Qi
Wang, Xue
Wu, Qi
CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells
title CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells
title_full CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells
title_fullStr CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells
title_full_unstemmed CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells
title_short CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells
title_sort crispr/cas9 ribonucleoprotein (rnp) complex enables higher viability of transfected cells in genome editing of acute myeloid cells
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9469150/
https://www.ncbi.nlm.nih.gov/pubmed/36111017
http://dx.doi.org/10.21037/atm-22-3279
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