Transient expression of the CRISPR-Cas9 machinery is desirable to reduce the risks of off-targets and immune responses. Electroporation of Cas9 ribonucleoproteins (RNPs) is the most common delivery method to achieve transient Cas9 expression. Recently, retroviral capsids have been used for delivering Cas9 RNPs, in which Cas9 was fused to the viral proteins. The fusion strategy may cause relative low capsid assembly efficiency. We recently developed virus-like particles (VLPs) consisting of lentiviral capsid and Cas9 RNPs using the specific interactions between aptamer and aptamer-binding protein (ABP), and obtained near-normal capsid assembly efficiency. Here we test whether highly active Cas9 (SpCas9) RNP V... More
Transient expression of the CRISPR-Cas9 machinery is desirable to reduce the risks of off-targets and immune responses. Electroporation of Cas9 ribonucleoproteins (RNPs) is the most common delivery method to achieve transient Cas9 expression. Recently, retroviral capsids have been used for delivering Cas9 RNPs, in which Cas9 was fused to the viral proteins. The fusion strategy may cause relative low capsid assembly efficiency. We recently developed virus-like particles (VLPs) consisting of lentiviral capsid and Cas9 RNPs using the specific interactions between aptamer and aptamer-binding protein (ABP), and obtained near-normal capsid assembly efficiency. Here we test whether highly active Cas9 (SpCas9) RNP VLPs can be generated with high efficiency by aptamer/ABP interaction. We found that by optimizing the locations and types of aptamer used for single guide RNA modification, highly active SpCas9 RNP VLPs can be generated efficiently. VLP-delivered SpCas9 generated lower off-target insertions and deletions than SpCas9 RNPs delivered by electroporation. VLPs containing Cas9 from different species and targeting multiple genes can be efficiently prepared in single-particle preparation. Multiple-target VLPs were more efficient than the combination of single-target VLPs for simultaneous targeting of multiple genes. Thus, in addition to better safety features, the Cas9 VLPs are especially suited for multiplex genome editing. In summary, our VLPs offer safe, efficient, and flexible multiplex genome editing.
