PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines

Area of Science:

• Hematology
• Molecular Biology
• Gene Editing

Background:

• Acute myeloid leukemia (AML) is a heterogeneous clonal disorder.
• Transfecting blood cells for genetic manipulation, including CRISPR/Cas9, is challenging.
• Existing methods often rely on viral vectors, limiting accessibility.

Purpose of the Study:

• To develop an accessible, non-viral strategy for generating CRISPR constructs.
• To enable efficient gene editing in hard-to-transfect leukemic cell lines.
• To validate a novel fusion PCR-based method for introducing specific gene mutations.

Main Methods:

• Generation of CRISPR constructs using fusion PCR.
• Introduction of PCR-generated constructs into leukemic cells (NB4, HL60) and HEK293 cells.
• Validation of gene modification by targeting MYBL2 and introducing IDH2 R172 mutation.
• Utilizing Cas9-expressing cell lines for efficient editing.

Main Results:

• Successful generation and delivery of PCR-based CRISPR constructs into leukemic cells.
• Demonstrated functional gene modification of MYBL2 and introduction of IDH2 R172 mutation.
• Achieved mutation introduction efficiency comparable to ribonucleoprotein strategies.
• Confirmed absence of detectable off-target mutations.

Conclusions:

• The fusion PCR strategy provides a simple, equipment-accessible method for creating CRISPR constructs.
• This non-viral approach is effective for gene editing in challenging leukemic cell models.
• The methodology offers a viable alternative to viral transduction for genetic modification in hematological research.