Question

Imagine you are a genetic counselor and all CRISPR technologies have become safe and reliable for therapeutic use in humans. One of your patients presents a wasting disease where they fail to produce enough collagen in their bones. You find no mutations in the coding region of the collagen gene and the protein is at healthy levels in all other tissues. Fully explain the steps that you would suggest to treat the disease, including what molecules are necessary for each step?

Answer 1

• Muscle wasting is related with Duchene Muscular Dystrophy (DMD).
• It is associated with DMD gene mutation and expression of protein called Dystrophin.
• This in turn causes, degeneration of myofibrils or muscle wasting.
• CRISPR/ Cas9 system of gene editing may be applied for the correction of such defects.

• CRISPR is Clustered Regulatory Interspaced Short Palindromic Repeats.
• CRISPR associated –Protein 9 nuclease Caspase (Cas-9) is used as a molecular tool for editing of genomes.
• It acts in a similar manner as genetic engineering or construction of GMO (genetically modified organism).

CRISPR/ Cas9 system has components that help in their gene editing:

1. crRNA- guide RNA (g RNA) and sequence recognizing trac rRNA
2. trac rRNA- in stem-loop structure, bind (to be activated) to crRNA
3. Cas9-active form have nuclease, nick formation, single strand/double strand breaks, editing.
4. sgRNA-single guide RNA-with combination of other components

Process:

• The components with the corrected gene may be introduced within the organism through plasmid.
• The plasmid may be introduced by transfection or by direct injections into the target cells.
• Also, some virus mediated vectors may be used or liposomes.
• The recognition point in the target DNA is designated as the PAM (Proto-spacer adjacent motif). The sequence is at the 3’ end of the target sequence(about 20 nucleotides)
• The CAS-9 component recognizes the PAM sequence. CAS- 9 then mediates unwinding or DNA and nick formation (nick is formed at about 3 bp upstream of the PAM).
• The CAS-9 recognition is aided with crRNA.
• The gRNA thus, associated with the crRNA act as recognition sequence, changing of which alters the recognition capabilities of the CAS-9.