Question

CRISPS is used to treat a mutation that causes congenital cardiac hypertrophy. (12pts total)

a. What are the fundamental components of the technology that usually need to be injected into

cells to replace the disease allele with a healthy one (i.e., via homology-dependent repair)?

b. Which components did they find might be unnecessary in early stage embryos and why?

c. Why were the scientists concerned about detecting mosaicism and off-target mutations?

Answer 1

CRISPR technology is a simple yet powerful tool for editing genomes. It allows researchers to easily alter DNA sequences and modify gene function. ... The protein cas 9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA.

a.

CRISPR-Cas9 Mechanism

CRISPR relies on just two components: the molecular scissors, a CRISPR-associated (Cas) nuclease, and the GPS guiding it to the appropriate site, the guide RNA (gRNA). In natural, the gRNA consists of two distinct segments of RNA: CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA).

b.

The most popular way to edit genes relies on a system called CRISPR–Cas9. Co-opted from a mechanism that some microbes use to defend themselves against viruses, it uses an enzyme called Cas9 to make cuts to DNA. A scientist can supply a snippet of RNA to guide Cas9 to a specific site in the genome. But Cas9 and enzymes like it have been known to cut DNA at other sites, too, particularly when there are DNA sequences in the genome similar to the target (see ‘Off-target effects’). Such ‘off target’ cuts could result in health problems: a change to a gene that suppresses tumour growth, for example, might lead to cancer.

C.

CRISPR-Cas9-induced DNA recombination can lead to mosaicism or different types of mutations in disparate cells. ... It is widely accepted that off-target effects (off-targets) and mosaicism are the two major hurdles to the application of CRISPR-Cas9 to repair genetic defects in human embryos.

Off-target genome editing. ... Specifically, off target mutation consist of unintended point mutations, deletions, insertions inversions, and translocations. Designer nuclease systems such as CRISPR-cas9 are becoming increasingly popular research tools as a result of their simplicity, scalability and affordability.

Genetic mosaicism is the presence of more than one genotype in one individual.Mosaicism can result from numerous mechanisms. These include natural mechanisms such as chromosome non-disjunction, anaphase lag, endoreplication, and mutations arising during development.