CRISPR ribonucleoproteins (RNPs) can generate programmable gene edits, however imprecise editing and efficient delivery to human stem cells are key challenges. Here we describe novel biochemical techniques to assemble various biomolecules and coatings with nanoscale precision around a RNP. First, by modifying the sgRNA with a short S1m RNA aptamer, we developed a modular strategy, termed an “S1mplex,” to assemble Cas9 RNPs with biotinylated moieties. Using S1mplexes with biotinylated short oligonucleotides improves the ratio of precise to imprecise editing up to 18-fold over conventional methods approaching a ratio of 4 precise edits to every imprecise mutation, while assembly with fluorescent molecules allows selection and enrichment for cells with multiplexed gene edits. Second, we developed synthetic coatings for nonviral delivery of RNPs to human pluripotent stem cells. Combined, these strategies, which utilize chemically-defined, off-the-shelf reagents, have significant promise for gene editing applications in vitro (e.g., drug discovery, disease modeling) with human stem cells.
Cell Line Development
Stem Cell Technologies
Clinical Laboratory Scientist3%
Life Science Company4%
Contract Research Organization (Cro)2%