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 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.

Automated summary

We developed a PCR-based alternative strategy for generating CRISPR constructs and successfully edited two genes (IDH2 and MYBL2) in hard-to-transfect leukemic cells. The efficiency of our methodology was similar to that of ribonucleoprotein strategies, without any off-target events. Our strategy provides a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction.