Cre-lox is an essential tool for creating conditional gene knockouts in human induced pluripotent stem cells (hiPSCs) for disease modeling and drug screening. Traditionally, both loxP sites are inserted into specific regions of the genome through homology-directed repair (HDR) prompted by delivery of CRISPR-Cas9 and a single donor template. Here, loxP sites were sequentially inserted to flank exons 45-55 in the dystrophin (DMD) gene. Deletion of this mutational hotspot could treat approximately 47% of all Duchenne muscular dystrophy patients1. The efficiency of loxP insertion and HDR is dependent on the size of the donor template, and larger cassettes are inherently more challenging to insert. Donor sequences for the Cre-lox system generally include both loxP sites, an antibiotic selection cassette, and the DNA sequence to be deleted, resulting in a large cassette. Hotta et al, used the ExPERTTM platform to deliver two small single-stranded oligodeoxynucleotides (ssODNs) encoding 34 bp loxP sites into hiPSCs2. MaxCyte electroporation of ssODNs showed high knockin efficiencies without antibiotic selection which led to the precise deletion of a large 342kb DNA genomic region.