RT journal article
T1 PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines
A1 González-Romero, E.
A1 Martínez-Valiente, C.
A1 García-García, G.
A1 Rosal Vela, Antonio
A1 Millán, J.M.
A1 Sanz, M.A.
A1 Sanz, G.
A1 Liquori, A.
A1 Cervera, J.V.
A1 Vázquez-Manrique, R.P.
A2 BiomedicinaBiotecnología y Salud Pública
K1 fusion PCR
K1 hard-to-transfect cell lines
K1 leukemia
K1 sgRNA constructs
AB 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.
PB Multidisciplinary Digital Publishing Institute (MDPI)
SN 2072-6694
YR 2023
FD 2023
LK http://hdl.handle.net/10498/32128
UL http://hdl.handle.net/10498/32128
LA eng
DS Repositorio Institucional de la Universidad de Cádiz
RD 12-nov-2026