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

Abstract

Akin to Zinc Finger and Transcription Activator Like Effector based transcriptional modulators, nuclease--null CRISPR--Cas9 provides a groundbreaking programmable DNA binding platform, begetting an arsenal of targetable regulators for transcriptional and epigenetic perturbation, by either directly tethering, or recruiting, transcription enhancing effectors to either component of the Cas9/guide RNA complex. Application of these programmable regulators is now gaining traction for the modulation of disease--causing genes or activation of therapeutic genes, in vivo. Adeno--Associated Virus (AAV) is an optimal delivery vehicle for in vivo delivery of such regulators to adult somatic tissue, due to the efficacy of viral delivery with minimal concerns about immunogenicity or integration. However, present Cas9 activator systems are notably beyond the packaging capacity of a single AAV delivery vector capsid. Here, we engineer a compact CRISPR--Cas9 activator for convenient AAV--mediated delivery. We validate efficacy of the CRISPR--Cas9 transcriptional activation using AAV delivery in several cell lines.

Enhanced Cas12a multi-gene regulation using a CRISPR array separator

“…To study the consequences of varying the nonsense spacer, we transfected the HEK293T cells with constructs encoding the CRISPR array and nuclease-deactivated Cas12a fused to the miniaturized VP64-p65-Rta activator (Vora et al, 2018) and mCherry (subsequently denoted dCas12a-miniVPR). If the nonsense spacer did not influence the performance of the downstream crRNA, we should see equal GFP activation in all cells.…”

“…Cells were transfected with constructs carrying 1) the nuclease-deactivated (D832A) dCas12a (from Lachnospiraceae bacterium, human codon-optimized) (Zetsche et al, 2015) fused either to the VP64-p65-Rta (VPR) activator (Chavez et al, 2015) and mCherry, or to mini-VPR (Vora et al, 2018) and mCherry; 2) a CRISPR array-expressing plasmid. For Figures 1 and 3, a CRISPR array construct consisting of firefly luciferase immediately followed by a CRISPR array and an SV40 pA terminator, expressed under the CAG promoter element, was used ( Supplementary File 1).…”

Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays

“…When the sequence was used twice (2× sNRP-1 polyA), then the potency was as efficient as a single SV40polyA sequence [ 165 , 166 ]. Yet, the two sNRP-1 polyA were less suitable for specific transcripts compared to bGHpolyA or spA [ 167 ]. The effects of polyadenylation sequences for specific transcripts are still less well understood.…”

“…Recently, a single rAAV expression vector has been developed driving the gRNA by a U6 promoter (360 bp) and a shortened but 3× less active VPR (500 bp) and dSaCas9 (3200 bp) from an SCP1 promoter (80 bp) attached to 2×-sNRP-1 polyA signal (34 bp). A modified rAAV-vector version with a full-length CMV promoter and the bGHpolyA efficiently upregulated a gene ( Actc1 , Neurog2, or Hbb ) upon infection of N2A neuron derived cells by 50–150× in vitro [ 167 ]. A single rAAV vector expressing dCas9 fusion protein, as well as a sgRNA, shows excellent potential for positive or negative regulation of transcription in many genetic pathways involved in retinal diseases.…”

In vivo epigenome editing and transcriptional modulation using CRISPR technology

“…Instead of truncating the dSaCas9, a recent study has truncated the VPR activation effector to reduce the size of the dSaCas9-VPR fusion protein from 5.0kb to 4.3kb (Vora et al 2018) (Figure 4C). VPR consists of a fusion of VP64, p65 and Rta domains.…”