Epigenetic CRISPR-Cas9 is a modified form of the genome editing platform CRISPR-Cas9 that does not cut DNA but affects gene expression levels. Gene expression levels depend on DNA methylation levels and whether chromatin conformation is in an active or repressed state. Epigenetic CRISPR-Cas9 uses the nuclease dead Cas9 (dCas9) to target the genome and instead of altering the genetic sequence, the system alters the epigenome by bringing transcriptional activators, repressors and chromatin modifiers to the desired genomic region. This technique allows researchers to study how epigenetic marks like DNA methylation, histone acetylation and chromatin modifier proteins affect gene expression in cultured mammalian cells and live mice . Programmable gene regulation via Epigenetic CRISPR-Cas9 has potential clinical applications .
Turning up gene expression
Juan Carlos Izpisua Belmonte of the Salk Institute for Biological Studies, California and his team designed guide RNAs to bring transcriptional activators with dCas9 to activate genes of interest . Instead of fixing the mutated gene in a disease, their approach is to upregulate other genes in the disease pathway that can compensate for the malfunctioning gene. In mouse models for kidney disease and muscular dystrophy their approach improved kidney and muscle function.
Acetylation modifications to histones, the proteins, which package DNA into chromatin change the conformation of the chromatin. Charles Gersbach and Timothy Reddy at Duke University, fused human acetyltransferase, p300 to dCas9. The fusion protein causes histone H3 lysine 27 to become acetylated at target sites, which activated target genes .
DNA methylation is a modification to DNA which regulates the expression of genes. Ronggui Hu’s lab at the Shanghai Institutes for Biological Sciences developed an epigenetic CRISPR-Cas9 system that includes the CpG demethylase enzyme, Tet1 to demethylate the promoter regions of target genes .
Hypermethylation and inactivity of the FMR1 gene is responsible for Fragile X Syndrome, the most common form of intellectual disability in males. Rudolph Jaenisch’s group at the Whitehead Institute for Biomedical Research, Cambridge, developed a version of dCas9-Tet1 which they used to activate FMR1 in induced pluripotent stem cells (iPSCs) from patients with Fragile X Syndrome . When the iPSCs with reactivated FMR1 were differentiated into neurons, neural function was rescued and remained rescued after transplant into mouse brain. Jaenisch’s group also showed that they could also use their system to activate FMR1 in post-mitotic neurons.
Turning down gene expression
Facioscapulohumeral (FSH) Muscular Dystrophy is an epigenetic disease. Abnormal chromatin structure leads to aberrant activation of DUX4-FL, which is normally repressed, leads to disease pathology. Peter L. Jones and his team, now at University of Nevada used dCas9 fused to the KRAB repressor to silence DUX4-FL in muscle cell lines . A method of turning down gene expression by fusing dCas9 with DNA methyltransferase Dnmt3a, which adds methyl groups to target regions of DNA was described by Rudolph Jaenisch’s group .
In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation
Hsin-Kai Liao7, Fumiyuki Hatanaka7, Toshikazu Araoka7, Pradeep Reddy, Min-Zu Wu, Yinghui Sui, Takayoshi Yamauchi, Masahiro Sakurai, David D. O’Keefe, Estrella Núñez-Delicado, Pedro Guillen, Josep M. Campistol, Cheng-Jang Wu, Li-Fan Lu, Concepcion Rodriguez Esteban, Juan Carlos Izpisua Belmonte
Scalpel or Straitjacket: CRISPR/Cas9 Approaches for Muscular Dystrophies
Charis L. Himeda, Takako I. Jones, and Peter L. Jones
Documentaries, videos and podcasts
- CRISPRClustered regularly interspaced short palindromic repeats (CRISPR) is a prokaryotic adaptive immune response that provides immunity against foreign nucleic acids, such as viral DNA and bacterial plasmids, through the use of crRNAs (CRISPR RNAs) and associated Cas genes.
- Cas9Microbial protein found in streptococcus pyogenes m1 gas
- Salk Institute for Biological StudiesLife sciences research institute
- CRISPR-Cas9CRISPR-Cas9 is a genome editing system. CRISPR systems provides immunity to bacteria and archaea from viruses and has been adapted for use as a genome editing tool capable of knocking out genes and rewriting genetic sequences in animal, plant and fungi. CRISPR-Cas9 is being adapted to other applications outside genome editing.