Development of CRISPR/dCas9-Effector Systems to Address Fibrosis in Muscle Wasting Disorders. / March, James.

2021. 623 p.

Research output: ThesisDoctoral Thesis



  • James T. March PhD Thesis

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    Embargo ends: 2/12/23


Fibrosis, which is characterised by excessive extracellular matrix material deposition, arises as a pathological element in multiple chronic disease contexts in diverse tissues and organs. Through fibrotic remodelling, normal tissue is replaced by extensive and disordered connective tissue that compromises tissue and organ function. Disease contexts affected by fibrosis include muscle wasting disorders, like Duchenne muscular dystrophy (DMD). DMD is an archetypal fibroproliferative muscle wasting disorder, where muscle dysfunction is accompanied by pathological fibrosis, which contributes to the lethal disease outcome. However, such fibrosis in DMD is not specifically addressed by treatment approaches in development. Therefore, anti-fibrotic therapeutics should be developed for use in dystrophic muscle wasting contexts.
A range of cellular and molecular factors contribute to fibrogenesis, including the matricellular protein Periostin and the cytokine Connective Tissue Growth Factor (CTGF), which function as pro-fibrotic mediators of the fibrosis master regulator Transforming Growth Factor Beta-1 (TGFβ1). In contrast, other factors, such as the peptide hormone Relaxin, appear to function as natural suppressors of fibrosis, through antagonism of TGFβ1 signalling, in a range of models.
It is hypothesised that Periostin or CTGF downregulation and Relaxin upregulation may reduce fibrosis in muscle wasting disorders. Further, it is considered that the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated-9 (Cas9) protein RNA-guided endonuclease system has potential for specific modulation of expression of these genes. The CRISPR/Cas9 system can be utilised for gene expression modulation by catalytic inactivation of Cas9 (dCas9) and its coupling to transcriptional repressor and activator domains. For instance, dCas9 has been fused to the Krüppel associated box (KRAB) effector for repression, as well as the tripartite activator VP64-p65-Rta (VPR) and the catalytic core of the histone acetyltransferase p300 for activation. Critically, such dCas9-effectors can be targeted to genomic sites of interest through programming of short guide RNA (sgRNA) sequences.
On this basis, CRISPR/dCas9-effector systems were developed for targeted downregulation of Periostin and CTGF and upregulation of Relaxin genes in human and mouse (POSTN/Postn, CTGF/Ctgf and RLN2/Rln1). Multiple sgRNAs of maximal predicted efficiency and specificity were designed using bioinformatics tools to target regions surrounding the transcriptional start sites of these genes. Following this, designed sgRNA sequences were cloned into an expression plasmid and screened through assessment of genome modification efficiency, by transfection into HEK293T/Cas9 for human and Neuro2A/Cas9 cells for mouse sgRNAs. This evaluation of gene editing efficiency was carried out by Tracking of Indels by Decomposition (TIDE) analysis, whilst POSTN and RLN2 sgRNAs were also analysed by T7EI assay. Of 13 human and 10 mouse sgRNAs targeting the three genes, 10 human and 8 mouse sgRNAs showing high levels of genome modification were identified as high performers.
Moreover, pUbC-dCas9-effector-GFP and dual pAAV-split-dCas9-effector systems, utilising split-intein protein trans-splicing for full length dCas9-effector reconstitution, were developed for screening of sgRNA target gene expression modulation efficiency and as a potential strategy for delivery of optimal sgRNA and dCas9-effectors in vivo respectively. Construct expression was confirmed in the mouse myoblast C2C12 cell line.
Subsequently, high performing designed mouse Postn, Ctgf, and Rln1 sgRNAs identified through TIDE screening were cloned into plasmids bearing dCas9-effector-GFP transgenes: the pUbC-dCas9-VPR-GFP and pUbC-dCas9-p300-GFP constructs for Rln1 sgRNAs and the dCas9-KRAB-GFP construct for Postn and Ctgf sgRNAs. These sgRNAs were screened for target gene modulation efficiency at the RNA level by transfection into C2C12s, followed by qRT-PCR to test the hypotheses that the developed systems could upregulate Rln1 and downregulate Postn and Ctgf gene expression. In the case of Rln1, upregulation was found to be robust and significant and a clear best performer sgRNA was found. Additionally, the VPR effector was found to be substantially better than the p300 effector for Rln1 activation. For Postn and Ctgf downregulation, best performing sgRNAs were identified, with the best performing Postn but not Ctgf sgRNA able to significantly repress target gene expression even when C2C12s were treated with profibrotic TGFβ1. For the Ctgf screen, more experiments are required to see if significant downregulation can be detected.
Following identification of best performing sgRNA-UbC-dCas9-effector-GFP systems, the efficacy of these systems for target gene protein modulation in C2C12s was determined by Western blots and ELISA (for Relaxin-1). It was found that the best performing Rln1 psgRNA-UbC-dCas9-VPR-GFP construct was able to significantly increase expression of Relaxin-1, whilst the best performing Postn psgRNA-UbC-dCas9-KRAB-GFP construct was able to significantly decrease expression of Periostin. No significant decrease in expression of CTGF was detected with the best performing Ctgf psgRNA-UbC-dCas9-KRAB-GFP construct.
Finally, best performing sgRNAs were cloned into pAAV-split-dCas9-effector plasmids. The efficiency of the best Postn and Rln1 sgRNAs in combination with split-dCas9-effector constructs for target gene modulation at the RNA level was determined by transfection of C2C12s followed by qRT-PCR. The best performing Rln1 sgRNA with pAAV-split-dCas9-VPR system was capable of significantly upregulating Rln1 expression, which validated this system for Rln1 upregulation in vitro, and suggests that the system holds potential for the upregulation of other gene targets. However, Postn expression was not found to be significantly downregulated with the best performing Postn sgRNA with pAAV-split-dCas9-KRAB system. More experiments are therefore required to validate the functionality of this system.
Overall, this novel study supplies in vitro proof-of-principle of dCas9-effector potential for targeted RLN2/Rln1 upregulation and POSTN/Postn and CTGF/Ctgf downregulation in skeletal muscle, particularly in the cases of RLN2/Rln1 and POSTN/Postn. The next stage of the project aims to supply an in vivo demonstration of the functionality of best performing Rln1 sgRNA with split-dCas9-VPR constructs delivered by AAV8 viral particles for Rln1 upregulation. The anti-fibrotic efficacy of these constructs in the context of skeletal muscle fibrosis will also be determined.
Original languageEnglish
Awarding Institution
Thesis sponsors
  • Muscular Dystrophy UK
Award date1 Dec 2021
Publication statusUnpublished - 2021

ID: 43842378