Question

This month, there was fanfare about the first case of using CRISPR invivo to treat Leber’s congenital amaurosis 10 (LCA10) which is a leading cause of blindness in childhood. This is due to mutations in a large gene, CEP290.

The article comments that:

“For the latest trial, the components of the gene-editing system – encoded in the genome of a virus — are injected directly into the eye, near photoreceptor cells.”

A. Specifically, what components are the authors referring to when they say “the components of the gene-editing system – encoded in the genome of a virus”?

B. How is the zinc finger technology similar to the CRISPR approach?

C. What is a potential risk of using the CRISPR technology invivo?

D. If there was a suitable safe harbor, or the possibility to use a AAV virus to replace the mutant CEP290 gene in the photoreceptor cells (Similar to replacing the CNG3B channel using AAV5). What would be the potential difficulties compared to fixing the gene with CRISPR?

E. If the mutation in the CEP290 gene was known to be a premature stop codon, would the gene therapy be the safest approach to treat this disease?

Answer 1

Solution :

A) CRISPR/Cas 9 system is a recently developed technique that allows precise alteration to DNA through a process called as genome editing. This technique is a copy cat mechanism derived from the natural technic exibited by bacterial cells against foreign bacteriophage DNA( bacteriophages are viruses which infect bacteria).

To adapt CRISPR to function in virtually any eukaryotic cell type, minimal system consisting of a nuclease(here Cas 9) and an engineered guide RNA( gRNA or SgRNA ) have been devoloped. The gRNA contains a target specific guide sequence that directs the nuclease Cas9 to cleave target genomic DNA. This will introduce site-specific double-stranded breaks (DSBs). DSBs are overruled through either non-homologous end joining (NHEJ) or homology-directed repair (HDR).

Presently there are no approved therapies for patients having Leber congenital amaurosis 10( LCA 10). LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. Recently researchers showed that guide RNA pairs coupled with Cas9 were used for removing the intronic splice mutation and restoring the expression of wild-type CEP290 gene. Alongside they have demonstrated that a dual adeno-associated virus  (AAV) system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina.

B) Both Zinc-finger technology and CRISPR/Cas 9 technology are used to target specific DNA sequences. Both Cas 9 nuclease and zinc finger nuclease targets and induce double stranded breaks in DNA.