Poster #053, Sanford Burnham Prebys
Assessing CRISPR-Cas12a Genome Editing of CD33 and CD45 in Acute Myeloid Leukemia Cell Lines
Mentors: Neha Niranjan, BS; Ani Deshpande, PhD
CRISPR-based genome editing has rapidly transformed biomedical research and is increasingly being explored in the development of targeted therapies for cancer. Cas9 is the most widely used CRISPR nuclease, cleaving DNA through the use of guide RNA. More recently, Cas12 has emerged as a promising alternative because of its distinct DNA cleavage mechanism, broad targeting range, and potentially improved efficiency compared to Cas9. To evaluate the performance of Cas12, we targeted the genes encoding the cell surface proteins CD33 and CD45 in the U937 and P31 acute myeloid leukemia (AML) cell lines. Single guide RNAs targeting each gene were designed and cloned into lentiviral vector pRDA_052. Lentiviral particles carrying the sgRNAs were generated by transfection and transduced into the Cas12 expressing AML cells. Genome editing efficiency was evaluated by using PCR across their cut sites and TIDE/ICE analysis. The overall goal of this work is to create a stable Cas12 system that expands the range of targetable genomic loci, overcomes the limitations of Cas9, and allows evaluation of sgRNA knockout efficiency and potential off target effects.