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PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines
SIMPLE SUMMARY: We used PCR-generated small CRISPR constructs to edit two genes (IDH2 and MYBL2) in hard-to-transfect hemopoietic cells, which are central to the progression of the devastating disease known as acute myeloid leukemia LMA (AML). MYBL2 is a transcription factor; when AML patients show...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10487029/ https://www.ncbi.nlm.nih.gov/pubmed/37686539 http://dx.doi.org/10.3390/cancers15174263 |
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author | González-Romero, Elisa Martínez-Valiente, Cristina García-García, Gema Rosal-Vela, Antonio Millán, José María Sanz, Miguel Ángel Sanz, Guillermo Liquori, Alessandro Cervera, José Vicente Vázquez-Manrique, Rafael P. |
author_facet | González-Romero, Elisa Martínez-Valiente, Cristina García-García, Gema Rosal-Vela, Antonio Millán, José María Sanz, Miguel Ángel Sanz, Guillermo Liquori, Alessandro Cervera, José Vicente Vázquez-Manrique, Rafael P. |
author_sort | González-Romero, Elisa |
collection | PubMed |
description | SIMPLE SUMMARY: We used PCR-generated small CRISPR constructs to edit two genes (IDH2 and MYBL2) in hard-to-transfect hemopoietic cells, which are central to the progression of the devastating disease known as acute myeloid leukemia LMA (AML). MYBL2 is a transcription factor; when AML patients show an altered expression of this factor, an adverse prognostic value is involved. IDH2 is particularly interesting because it encodes isocitrate dehydrogenase 2, an enzyme of the citric acid cycle, which, when mutated, produces a different phenotype in AML patients. Hence, our system provides a way to produce CRISPR constructs to easily target genes within mammalian cells, and it provides a model which can be used to study AML mechanisms in vitro. ABSTRACT: Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction. |
format | Online Article Text |
id | pubmed-10487029 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-104870292023-09-09 PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines González-Romero, Elisa Martínez-Valiente, Cristina García-García, Gema Rosal-Vela, Antonio Millán, José María Sanz, Miguel Ángel Sanz, Guillermo Liquori, Alessandro Cervera, José Vicente Vázquez-Manrique, Rafael P. Cancers (Basel) Article SIMPLE SUMMARY: We used PCR-generated small CRISPR constructs to edit two genes (IDH2 and MYBL2) in hard-to-transfect hemopoietic cells, which are central to the progression of the devastating disease known as acute myeloid leukemia LMA (AML). MYBL2 is a transcription factor; when AML patients show an altered expression of this factor, an adverse prognostic value is involved. IDH2 is particularly interesting because it encodes isocitrate dehydrogenase 2, an enzyme of the citric acid cycle, which, when mutated, produces a different phenotype in AML patients. Hence, our system provides a way to produce CRISPR constructs to easily target genes within mammalian cells, and it provides a model which can be used to study AML mechanisms in vitro. ABSTRACT: Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction. MDPI 2023-08-25 /pmc/articles/PMC10487029/ /pubmed/37686539 http://dx.doi.org/10.3390/cancers15174263 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article González-Romero, Elisa Martínez-Valiente, Cristina García-García, Gema Rosal-Vela, Antonio Millán, José María Sanz, Miguel Ángel Sanz, Guillermo Liquori, Alessandro Cervera, José Vicente Vázquez-Manrique, Rafael P. PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines |
title | PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines |
title_full | PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines |
title_fullStr | PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines |
title_full_unstemmed | PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines |
title_short | PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines |
title_sort | pcr-based strategy for introducing crispr/cas9 machinery into hematopoietic cell lines |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10487029/ https://www.ncbi.nlm.nih.gov/pubmed/37686539 http://dx.doi.org/10.3390/cancers15174263 |
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