Improving CRISPR-Cas Performance with Chemical Modifications in Guide RNAs and Single-Cell DNA Analysis

As gene editing systems move rapidly from research to therapeutic applications, further improving their performance while better understanding their editing characteristics at the single-cell level becomes fundamentally important. Leveraging our capability to robustly chemically synthesize RNAs of more than 160 nucleotides in length we have developed novel approaches for improving the activity and specificity of CRISPR systems using chemical modifications in guide RNAs. In addition, to mapping positions where chemical modifications can enhance the stability of guide RNAs while maintaining function, we developed a novel approach for enhancing specificity by employing site-specific modifications in the 20-nucleotide DNA recognition sequence in single-guide RNAs (sgRNAs). Our deep sequencing results from human cells transfected with pre-formed Cas9-sgRNA complexes show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or “MP”) incorporated at select sites in the ribose-phosphate backbone of sgRNAs can markedly reduce off-target cleavage activities, while maintaining high on-target performance. Subsequent structural studies showed these positions map onto contacts between Cas9 protein sidechains involved in monitoring the complementarity of the gRNA–target DNA duplex for sufficient helix formation and specificity to allow cleavage.

A more complete understanding of the exact nature of induced mutations in each cell in a population can enable further advances in optimizing the frequencies of on- and off-target gene edits especially when multiple events in each cell are desired. To demonstrate the potential value of single-cell DNA analysis we will present results using the Tapestri® platform, a high-throughput, single-cell DNA analysis system which enables the sensitive detection of multiple types of editing events including single nucleotide edits, deletions, insertions, and translocations, and can identify the co-occurrence and zygosity of these changes in different cells in a population of CRISPR edited cells.