Extending the capabilities of DNA writing tools to improve medicine

DNA gene editing tools, also referred to as DNA writers, have already improved human healthcare outcomes and may continue to be leveraged to further improve human health. Interesting but unsolved medical applications include using DNA writers to record cellular development, which underpins numerous diseases, and to cure mitochondrial genetic disease. Although gene editing techniques have previously been used to cure nuclear genomic diseases, delivery of DNA template and proteins into mitochondria hinders our ability to directly edit mitochondrial DNA. Here, we first show that the DNA writers Cas1 and Cas2 can be used to record the order of transcriptional events in cell populations and then develop a practical and accessible protocol allowing others to also implement the technique. Next, we show that combining Cas9 with a retron reverse transcription enables researchers to achieve precise edits, including exon-long insertions, in human cultured cells and reduce our reliance on exogenous delivery of DNA template, a barrier in mitochondrial gene editing. Finally, we develop a useful methodological pipeline that can quickly quantify the colocalization of different engineered proteins in mitochondria using immunocytochemistry, high-throughput fluorescent imaging, and automated analysis written in Python. We hope this resource may help researchers empirically engineer new DNA writers that are efficiently imported into human mitochondria. Overall, the new genetic technologies and pipelines described here may assist future scientists in engineering new gene editing approaches to cure developmental or mitochondrial disorders