CRISPRCas9 Flashcards
What role does dystrophin play in muscle function, and what disease arises from mutations in the dystrophin gene?
Dystrophin is a protein that links the cytoskeleton of muscle cells to the surrounding membrane, providing structural integrity. Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease.
What is the significance of “exon skipping” in the context of DMD treatment strategies?
Exon skipping aims to bypass mutations in the dystrophin gene by inducing the cellular machinery to “skip over” affected exons during protein synthesis. This can produce a partially functional dystrophin protein, potentially mitigating the severity of DMD.
How does CRISPR-Cas9 technology differ from traditional gene therapy approaches?
Traditional gene therapy often involves delivering a functional copy of a gene to replace a faulty one. CRISPR-Cas9, however, acts as a programmable “molecular scissor” to precisely edit the existing gene within the cell’s DNA.
Describe the concept of homology-directed repair (HDR) and its importance in CRISPR-Cas9-mediated gene editing.
HDR is a cellular DNA repair mechanism that uses a homologous DNA template to repair double-strand breaks. In CRISPR gene editing, an ssODN can serve as a template to introduce specific changes or corrections into the target gene sequence.
What are off-target effects in CRISPR-Cas9 gene editing, and why are they a concern?
Off-target effects occur when the CRISPR-Cas9 complex cuts DNA at unintended locations, potentially leading to unwanted mutations and genomic instability. This is a major safety concern for therapeutic applications.
Explain the experimental approach used in the study on correcting a MYBPC3 gene mutation in human embryos
Ma et al., (2017) injected a CRISPR-Cas9 complex targeting a mutation in the MYBPC3 gene (associated with hypertrophic cardiomyopathy) into human zygotes. They assessed editing efficiency, mosaicism, and potential off-target effects by analyzing individual blastomeres.
What is the role of single-stranded oligonucleotide donor DNA (ssODN) in CRISPR-Cas9-mediated gene editing?
ssODN is a short, single-stranded DNA molecule that carries the desired gene sequence. When co-injected with the CRISPR-Cas9 complex, it acts as a repair template, guiding HDR to introduce the specific genetic change.
Describe the safety assessments conducted in the preclinical study on CRISPR-Cas9-based therapy for sickle cell disease
The study by Dever et al. (2021) evaluated the long-term toxicity and tumorigenicity potential of CRISPR-Cas9-edited hematopoietic stem and progenitor cells (HSPCs) in mice. They monitored animals for adverse events, assessed engraftment of edited cells, and performed histopathological analysis of various tissues.
How can the size of the Cas9 protein be a limitation in gene therapy applications, and what strategies are being explored to address this?
The large size of the Cas9 protein can hinder its delivery using viral vectors, which have limited packaging capacity. Researchers are exploring smaller Cas proteins (like CasMINI) and alternative delivery methods to overcome this challenge.
What are the potential advantages of using Fanzor, a recently discovered CRISPR-like system, for gene editing compared to Cas9?
Fanzor is a smaller and more compact system than Cas9, potentially offering advantages in packaging and delivery. Additionally, it exhibits distinct PAM sequence requirements, potentially expanding the range of genomic targets amenable to editing.
What is CRISPR and how does it work?
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a naturally occurring bacterial defense system that has been adapted for genome editing. It works by using a guide RNA (gRNA) molecule to direct the Cas9 enzyme to a specific location in the genome. The Cas9 enzyme then cuts the DNA at that location. This cut can then be repaired by the cell’s natural DNA repair mechanisms, which can be harnessed to introduce precise changes into the genome.
What are the potential applications of CRISPR technology?
Treating genetic diseases: CRISPR can be used to correct genetic mutations that cause diseases like Duchenne muscular dystrophy (DMD), hypertrophic cardiomyopathy, and sickle cell disease.
Developing new disease models: CRISPR can be used to create animal models of human diseases, which can be used to study the disease process and test new therapies.
Improving crop yields and agricultural practices: CRISPR can be used to engineer crops that are more resistant to pests, diseases, and environmental stresses.
Developing new diagnostic tools: CRISPR can be used to develop new diagnostic tools for a variety of diseases.
What are the potential risks associated with CRISPR gene editing?
Off-target effects: The Cas9 enzyme can sometimes cut DNA at unintended locations in the genome, which could lead to unintended consequences.
Mosaicism: CRISPR editing may not be successful in all cells of an organism, leading to a mixture of edited and unedited cells, known as mosaicism.
Ethical concerns: There are ethical concerns about the use of CRISPR to edit the human germline, as this could have unpredictable consequences for future generations.
What are the different types of CRISPR systems?
- CRISPR-Cas9 system (main system)
- Cas12a (also known as Cpf1) - recognizes a different PAM sequence than Cas9, which expands the range of genomic targets that can be edited
- Cas13- targets RNA rather than DNA, which makes it useful for applications like RNA knockdown and RNA editing.
How is CRISPR being used to treat genetic diseases in humans?
CRISPR is being investigated as a potential treatment for a variety of genetic diseases. One approach is to use CRISPR to edit the genes of patients’ cells in vitro and then transplant the edited cells back into the patient. Another approach is to deliver CRISPR components directly into the patient’s body to edit the genes of cells in vivo. Clinical trials are currently underway to evaluate the safety and efficacy of CRISPR-based therapies for diseases like sickle cell disease and cancer.
