The clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated protein 9 (Cas9) system is a powerful genome-editing tool that is widely used in many different applications. However, the high-frequency mutations induced by RNA-guided Cas9 at sites other than the intended on-target sites are a major concern that impedes therapeutic and clinical applications. A deeper analysis shows that most off-target events result from the non-specific mismatch between single guide RNA (sgRNA) and target DNA. Therefore, minimizing the non-specific RNA–DNA interaction can be an effective solution to this issue. Here we provide two novel methods at the protein and mRNA levels to minimize this mismatch issue by chemically conjugating Cas9 with zwitterionic pCB polymers or genetically fusing Cas9 with zwitterionic (EK)n peptides.
