Rational Design of Mini-Cas9 for Transcriptional Activation

Abstract

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope

“…In addition, we have demonstrated the versatility of SauriCas9, which can be adapted for base editing. Recently, mini-SaCas9 that only retains DNA binding activity has been developed [33,34]. Mini-SaCas9 can be used for a variety of applications by fusing with other effectors.…”

Rational design of a compact CRISPR-Cas9 activator for AAV-mediated delivery

“…As such, while this manuscript was under preparation, a similar work was published demonstrating feasibility of delivering a Cas9 activator in a singular AAV vector. 33 Looking forward, Cas9 activator mediated activation remains an exciting alternative method for the treatment of genetic disorders; yet, an AAV based system alone does not solve all potential related issues of toxicity or delivery. While the AAV system itself has now generally been considered benign and effective for delivery in clinical setting, Cas9 itself has yet to be thoroughly vetted for toxicity clinically.…”

In vivo epigenome editing and transcriptional modulation using CRISPR technology

“…Alternatively, intein-mediated split dSaCas9 reconstitution system enabled the introduction of a large dSaCas9 fusion proteins into the cells through two separate AAV vectors. Using the similar concept as described for the SpCas9-based split system (Chew et al 2016), the dSaCas9 was split at residue 739 into dSaCas9 C and dSaCas9 N (Ma et al 2018). The dSaCas9 C -VPR and dSaCas9 N -gRNA were then packaged into two separate AAV vectors for co-expression.…”

“…The dSaCas9-VPR miniature activator was also significantly more robust than the previously established dCas9-VP64 of the SAM system (Konermann et al 2015). Another example of effective AAV packaging strategy was the adaption of a mini-SaCas9-VTR by fusing a truncated SaCas9 (mini-SaCas9) to a downsized tripartite VPR transactivation domain (VTR) (Ma et al 2018). By deleting conserved functional domains of SaCas9, the resulting mini-SaCas9 was nuclease-defective but retained efficient DNA binding activity.…”

“…In addition, self-assembled arrays of split SpyTag:SpyCatch or MoonTag:MoonCatcher peptides have been fused to the mini-SaCas9-VTR for recruiting multiple copies of VTR activator (Ma et al 2018). SpyTag and MoonTag scaffold systems were smaller than the SunTag system for efficient packaging into the AAV vectors.…”