Targeted Transcriptional Activation in Human Cells and in the Mouse Brain Tissue by Capsid-modified AAV and Evolved CRISPRa


Student thesis: Doctoral Thesis

View graph of relations


Related Research Unit(s)


Awarding Institution
Award date31 May 2021


Transcriptional dysregulation has been implicated in a number of progressive neurodegenerative diseases. To correct transcriptional misregulation, emerging CRISPR tools such as CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) could be used. In addition, efficient and precise transcriptional activation in broad brain areas and multiple neuroanatomical structures can be achieved by systemic delivery of an all-in-one adeno-associated virus (AAV) vector bearing CRISPRa and its guide sequence (sgRNA) to the brain tissue via a single AAV vector platform. However, AAV packaging and inefficient blood-brain barrier (BBB) crossing are two major challenges need to be resolved to realize such application. Furthermore, co-injection of multiple AAV vectors often is required to simultaneously express different sgRNAs for synergistic transcription activation. To enable the use of a single AAV vector platform with only one sgRNA for effective transcription activation in the brain tissue, a potent all-in-one CRISPRa is warranted.

Firstly, to enable AAV packaging of an all-in-one CRISPR vector, we constructed minimal CRISPRa and CRISPRi transgenes by fusing catalytically inactive Staphylococcus aureus Cas9 (dSaCas9) to the transcriptional activator (VP64 and VP160) and repressor (KRAB and SID4X) domains. These CRISPRa and CRISPRi were also linked to truncated regulatory elements (promoter and polyadenylation signal) and a guide sequence (sgRNA). We then evaluated the performance of these constructs in two episomal reporter assays (bioluminescent and fluorescent), five endogenous genes (Camk2a, Mycn, Nrf2, Keap1, and PDGFRA), and two cell lines (N2a and U87) by targeting the promoter and/or enhancer regions. Despite these all-in-one vectors being minimal in size, we showed that their transgene expression and functionality were not compromised.

Next, to enable systemic delivery of AAVs to the mouse brain tissue in vivo, we have generated an AAV1-PHP.B by inserting a 7-mer PHP.B peptide on AAV1 capsid. We showed that AAV1-PHP.B can efficiently cross the blood-brain barrier (BBB) and be taken up by the brain tissue upon lateral tail vein injection in mice. Importantly, a single-dose intravenous administration of AAV1-PHP.B expressing CRISPRa was shown to achieve targeted transgene activation in the mouse brain.

Finally, to enable precise transcription activation using an all-in-one CRISPRa with only one sgRNA, we attempted to improve the CRISPRa potency by deriving a VP64 with gain-of-function mutations through introduction of a large spectrum of substitution mutations to the VP64 transgene in the living cells using base editors. We firstly generated diverse VP64 variants using CRISPRa-GFP expressing HEK293FT cells. Because the sgRNA of CRISPRa was designed to target its own promoter, VP64 with enhanced function would lead to improved self-activation of CRISPRa, thereby higher GFP fluorescence intensity than the wild-type VP64. This self-activation strategy allows positive selection of VP64 mutants with enhanced potency for sorting, expanding, and characterization. These newly identified VP64 mutants were then validated using episomal reporter assays and by targeting endogenous genes. Finally, the most promising VP64 mutant was selected to reconstruct all-in-one CRISPRa for targeted gene activation using only one sgRNA. Their performance in vivo was evaluated by packaging evolved CRISPRa into the AAV1-PHP.B vectors for systemic delivery to the brain of adult mice. In this case, the CMV-driven CRISPRa was used to target the hSyn1 promoter of fluorescent transgene that was co-delivered as a separate AAV vector. Collectively, we have successfully reengineered CRISPRa bearing a VP64 variant with gain-of-function mutation for precise transgene activation in vitro and in vivo via a single AAV vector platform.

An integration use of this enhanced all-in-one CRISPRa construct and AAV-PHP.B delivery vehicle can realize the applications of a non-invasive, specific and potent AAV-CRISPRa system for correcting transcriptional misregulation in broad brain areas and multiple neuroanatomical structures. The use of a single AAV vector platform also can simplify AAV production, improve CRISPRa delivery efficiency and gene activation potency by ensuring the uptake and co-localize of both CRISPRa and its sgRNA in an individual cell. Ultimately, this enhanced AAV-CRISPRa system can be potentially used for therapeutics treatment of human neurodegenerative diseases such as Alzheimer’s disease and Parkinson's disease.

    Research areas

  • AAV Capsid, Adeno-associated Virus, Base Editor, Cis-regulatory Elements, CRISPR Activation, CRISPR Interference