Autologous engineered cell therapies such as autologous chimeric antigen receptor T-cell (CAR-T) therapies have revolutionized the treatment of hematologic cancers, however they are limited by manufacturing time and variability, the requirement for lymphodepletion, and side effects related to cytokine release. Allogeneic cell therapies derived from gene-edited induced pluripotent stem cells (iPSCs) are being developed to address the challenges associated with autologous engineered cell therapies. These “off-the-shelf” cell therapies contain specific edits designed to reduce immune rejection and to confer enhanced therapeutic properties and greater safety. However, efficient, footprint-free, biallelic targeting of defined loci in iPSCs remains technically challenging with current gene-editing approaches. We demonstrate efficient targeting of defined loci in iPSCs using novel messenger RNA (mRNA)-encoded gene-editing endonucleases comprising DNA-binding domains containing novel linker regions. We targeted exon 3 of beta-2 microglobulin (B2M), a key component of MHC class I molecules, and confirmed targeted editing in 10/12 lines, with 6/12 lines containing a desired biallelic deletion. Gene knockout in iPSCs was confirmed via RT-PCR and immunofluorescence in the context of B2M upregulation following exposure to interferon-γ. We show differentiation of B2M-/- iPSCs into CD34+ hematopoietic progenitor cells using both 2D and 3D directed-differentiation protocols. This mRNA gene editing platform could serve an important tool for the development of minimally-immunogenic cell lines for future allogeneic cell therapies.