Question

In the CRISPR-Cas9 system, two key mechanisms of DNA repair, non-homologous end joining (NHEJ) and homologous recombination (HR), play crucial roles in the modification of target DNA. Can these repair mechanisms be intentionally controlled or manipulated, and what effects do they have on the target DNA?

A) Both repair mechanisms can be precisely controlled, allowing researchers to choose the desired outcome for target DNA modification.
B) Only NHEJ can be intentionally manipulated, leading to unpredictable changes in the target DNA.
C) Repair mechanisms cannot be controlled, but the effects on target DNA depend on the specific genetic context.
D) The control of repair mechanisms depends on the specific CRISPR-Cas9 technology used, and their effects on target DNA vary.

If the repair mechanisms cannot be controlled, how can the determination of which mechanism occurred in the target DNA be made, and is it possible to distinguish between the desired and undesired repair mechanisms?

A) Distinguishing between repair mechanisms is challenging, and it is not possible to determine which one has occurred accurately.
B) Techniques such as prime editing allow precise control, making it easy to distinguish between desired and undesired repair mechanisms.
C) Distinguishing between repair mechanisms is possible through various methods, and careful analysis can differentiate between the desired and undesired outcomes.
D) The determination of repair mechanisms is solely dependent on the cell type, and distinguishing between desired and undesired outcomes is not feasible.

Answer 1

While CRISPR-Cas9 DNA editing can be guided, control over DNA repair mechanisms NHEJ and HR is imperfect, and outcomes can vary. Distinguishing between repair outcomes is possible through detailed analysis, and improved precision can be achieved with techniques like prime editing.

Step-by-step explanation:

Control and Distinguishing of DNA Repair Mechanisms

In the CRISPR-Cas9 system, it is challenging to precisely control whether non-homologous end joining (NHEJ) or homologous recombination (HR) will be the mechanism used to repair double-stranded breaks in the DNA. While both mechanisms play crucial roles in the process, HR requires the presence of a homologous sequence to serve as a template for repair, whereas NHEJ joins the broken DNA ends directly, often introducing mutations.

Distinguishing between these outcomes is possible by analyzing the edited sequences post-repair. Sequencing techniques can identify the hallmark imprecisions of NHEJ like insertions or deletions, known as indels, while HR outcomes will be precise if a template was provided. For predictable editing outcomes, newer techniques such as prime editing have been developed, improving the precision of repairs.

While the control over DNA repair mechanisms can be somewhat directed by manipulating the cellular conditions or by providing a DNA template to influence HR, the inherent nature of these processes means there is always a degree of unpredictability. Therefore, researchers must verify the outcome of CRISPR-Cas9 editing by examining the genetic sequence of the edited cells and comparing it to the intended design.