In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation

Abstract

Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner.

Long-lasting Analgesia via Targetedin vivoEpigenetic Repression of Nav1.7

“…To establish robust Na V 1.7 repression, we first compared in vitro repression efficacy of Na V 1.7 using KRAB-dCas9 and ZFP-KRAB constructs. Towards this, we cloned ten guide-RNAs (gRNAs; Supplementary Table 1) -designed by an in silico tool 73 that predicts highly effective gRNAs based on chromatin position and sequence features-into our previously developed split-dCas9 platform 50 . We also cloned the two gRNAs that were predicted to have the highest efficiency (SCN9A-1 and SCN9A-2) into a single construct, since we have previously shown that higher efficacy can be achieved by using multiple gRNAs 50 .…”

“…For these reasons, we have employed a catalytically inactivated "dead" Cas9 (dCas9, also known as CRISPRi), which does not cleave DNA but maintains its ability to bind to the genome via a guide-RNA (gRNA), and fused it to a repressor domain (Krüppel-associated box, KRAB) to enable nonpermanent gene repression of Na V 1.7. Previously, we and others have shown that through addition of a KRAB epigenetic repressor motif to dCas9, gene repression can be enhanced with a high level of specificity both in vitro [45][46][47][48][49] and in vivo 50,51 . This transcriptional modulation system takes advantage of the high specificity of CRISPR-Cas9 while simultaneously increasing the safety profile, as no permanent modification of the genome is performed.…”

Disruptive Technology: CRISPR/Cas-Based Tools and Approaches

“…For clarity, Cpf1, with its staggered DSB, RNA BEs and a plethora of additional tools, such as many additional transcriptional regulators and epigenome regulators [45] and different flavors of paired nickases (two-component RGNs), are not shown. Exclamation mark activation of expression;  Red cross deactivation of expression; STOP sign translation termination (nonsense) codon, dCas9 deactivated Cas9 without endonuclease activity, dsDNA double-stranded DNA, catalytic domains for functional expansion of the RGN complex: CyD cytidine deaminase domain for C > U conversion in the ssDNA loopout, currently with precision of ≤ 2 bp, UGI uracil DNA glycosylase inhibitor domain to prevent base excision repair and removal of base edit, DNMT3a catalytic domain of DNA methyltransferase 3 alpha for DNA methylation and potentially persistent repression of gene expression for affected promoters [82], TET1 catalytic domain of Ten-Eleven Translocation dioxygenase 1 (TET) for DNA demethylation and potentially persistent transcriptional activation of affected promoters [84], VPR VP64 (four tandem repeats of herpes simplex virus VP16) linked to p65 (the transactivation domain of nuclear factor [NF]-κB) and Rta (the Epstein-Barr virus transcriptional activation domain) for broad and potent transcriptional activation of affected promoters [174], KRAB catalytic domain of Krüppel-associated box epigenetic repressor [175], ADAR2 adenosine deaminase acting on RNA 2 [101]…”

In vivo epigenome editing and transcriptional modulation using CRISPR technology

“…A similar split-Cas9 system can be designed to enable AAV delivery of dCas9 fusion proteins and full-length 20nt sgRNAs. For example, in vivo gene repression in mice has been achieved using the AAV-split-KRAB-dCas9 system (Moreno et al 2018). However, the use of two separate AAV vectors to co-express split Cas9 or dCas9 fragments may reduce the delivery and gene modulation efficiency.…”

“…Currently, adeno-associated virus (AAV) is the most commonly used viral vectors for packaging and delivery of CRISPR components in vivo (Moreno et al 2018; Senis et al 2014; Swiech et al 2015; Thakore et al 2018; Yang et al 2016). The development of dual-vector AAV system and recent discoveries of small Cas9 orthologues (e.g.…”