CRISPRCas9- Lucky dip Flashcards
What is Duchenne Muscular Dystrophy (DMD)?
How can CRISPR be used to treat DMD?
DMD is a genetic disorder primarily affecting boys, characterized by progressive muscle degeneration and atrophy. It arises from mutations in the dystrophin gene, which plays a crucial role in maintaining the structural integrity of muscle cells.
CRISPR can be used to correct the mutations in the dystrophin gene responsible for DMD. This can be achieved by using CRISPR-Cas9 to cut the DNA at the mutation site and then providing a template for the cell’s repair mechanisms to use, resulting in a corrected gene sequence.
How does CRISPR correct the MYBPC3 gene mutation in human embryos?
CRISPR-Cas9 is injected into human embryos carrying a mutation in the MYBPC3 gene, which is often implicated in HCM. The Cas9 enzyme cuts the DNA at the mutation site, and the embryo’s own repair mechanisms use a provided template to correct the gene sequence, leading to the potential for healthy heart development.
What are the potential off-target effects of CRISPR?
CRISPR-Cas9 can sometimes cut DNA at unintended sites, known as off-target effects. These effects can potentially lead to unintended genetic modifications. Researchers are actively developing strategies to minimize off-target effects and improve the precision of CRISPR gene editing.
What is Fanzor, and how does it work?
Fanzor is a newly discovered RNA-guided DNA endonuclease, similar to CRISPR-Cas12. It utilizes a guide RNA called ωRNA to target specific DNA sequences and cleaves the DNA at the target site. Fanzor has a unique structure and cleaves DNA with a different pattern than other known RNA-guided enzymes
What are the advantages of using SMRT-seq with UMI for analyzing gene editing outcomes?
SMRT-seq with UMI (Single Molecule, Real-Time sequencing with Unique Molecular Identifiers) allows for long-read sequencing and accurate quantification of gene editing outcomes, including unintended large deletions and insertions that may be missed by short-read sequencing methods. This provides a comprehensive understanding of the genetic modifications induced by CRISPR.
What is the role of dystrophin in muscle function, and what are the consequences of mutations in the dystrophin gene?
Dystrophin is a protein that links the cytoskeleton to the muscle cell membrane, providing structural support and maintaining membrane integrity. Mutations in the dystrophin gene lead to Duchenne muscular dystrophy (DMD), characterized by progressive muscle degeneration and weakness.
Describe the experimental design used in the study “Correction of a pathogenic gene mutation in human embryos” to assess the efficiency of CRISPR-Cas9 in correcting the MYBPC3 mutation.
The study involved injecting a CRISPR-Cas9 system using the homologous maternal WT strand, targeting the MYBPC3 mutation into human embryos at different cell cycle stages. The embryos were then analyzed for the presence of the corrected gene using whole-genome sequencing, allowing the researchers to compare the efficiency of gene correction at different injection timings.
How does the study on HBB gene correction in CD34+ HSPCs for sickle cell disease demonstrate the safety and feasibility of this approach for clinical applications?
The study showed high-frequency HBB gene correction in HSPCs from both healthy donors and patients with SCD, with efficient engraftment in mice models. The absence of genotoxicity and tumorigenicity in long-term studies supports the safety and feasibility of this approach.
What are the potential unintended consequences of CRISPR-Cas9 gene editing, and what strategies can be employed to minimize these risks?
Unintended consequences include off-target editing, large deletions, and mosaicism. Strategies to minimize these risks include using high-fidelity Cas9 enzymes, optimizing guide RNA design, and employing sensitive detection methods for off-target events.
Explain how CasMINI, a miniaturized Cas9 protein, overcomes the size limitations associated with traditional Cas9 for gene editing applications.
CasMINI is a significantly smaller version of Cas9, enabling its packaging into viral vectors with limited carrying capacity. This allows for more efficient delivery of the gene editing machinery to target cells, expanding the potential applications of CRISPR-Cas9.
Describe the structure and function of Fanzor, a recently discovered transposon-associated RNA-guided DNA endonuclease.
Fanzor is a prokaryotic RNA-guided DNA endonuclease that utilizes a unique omegaRNA (ωRNA) molecule for target recognition and cleavage. It exhibits a distinct domain architecture and a preference for a specific target-adjacent motif (TAM) for DNA cleavage.
What are the ethical concerns surrounding the use of CRISPR-Cas9 for germline editing, as highlighted in the scientific critique of the gene-edited babies study?
The gene-edited babies study raised ethical concerns due to the potential for unintended consequences and long-term health effects of germline editing, the lack of informed consent from the edited individuals, and the potential for exacerbating societal inequalities.
Explain the limitations of short-range PCR in detecting unintended large deletions caused by CRISPR-Cas9, and how long-range PCR and droplet digital PCR can address this issue.
Short-range PCR can miss large deletions as the primers may anneal outside the deleted region, leading to false-negative results. Long-range PCR and droplet digital PCR can amplify larger DNA fragments, increasing the sensitivity for detecting large deletions.
Describe the concept of “NICER” gene editing and how it leverages nickase activity to enhance the precision and efficiency of gene correction.
NICER gene editing uses Cas9 nickases to create single-strand breaks at the target site, promoting gene correction through homology-directed repair. This approach minimizes the formation of unintended double-strand breaks, reducing the likelihood of undesired insertions and deletions.
What are the potential advantages of using nickases over traditional Cas9 nucleases for therapeutic gene editing?
Nickases induce less DNA damage compared to Cas9 nucleases, reducing the risk of off-target effects and large deletions. This increased specificity makes nickases a promising tool for therapeutic gene editing, particularly for applications requiring high precision and safety.
