Minimizing the off-target frequency of the CRISPR/Cas9 system via zwitterionic polymer conjugation and peptide fusion

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. The zwitterlated or EKylated CRISPR/Cas9 ribonucleoproteins (RNPs) show reduced off-target DNA editing while maintaining a similar level of on-target gene editing activity. Results show that the off-target efficiency of zwitterlated CRISPR/Cas9 is reduced on average by 70% and can be as high as 90% when compared with naive CRISPR/Cas9 editing. These approaches provide a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biological and therapeutic applications based on CRISPR/Cas9 technology.