Genome editing using CRISPR/Cas9 Flashcards
lecture 13
What is CRISPR?
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an adaptive immune system found in bacteria. It helps protect against invading viruses by recognizing and cutting their DNA. The CRISPR/Cas system includes three components: Cas9 (protein), crRNA (RNA), and tracrRNA (RNA).
How does CRISPR function in bacteria?
Bacteria use CRISPR as an immune response by integrating fragments of viral DNA (protospacers) into their genome. Upon reinfection, the bacterial cell transcribes the protospacers into RNA, which guides Cas9 to cleave the viral DNA, preventing re-infection.
What are the key components of the CRISPR/Cas system?
Cas9: Protein that cuts DNA.
crRNA: RNA that guides Cas9 to the target DNA.
tracrRNA: RNA that helps stabilize the crRNA and Cas9 complex.
Together, they form the guide RNA (gRNA), which directs Cas9 to specific DNA sequences.
What is the role of PAM in CRISPR-mediated DNA cleavage?
The Protospacer Adjacent Motif (PAM) is a short DNA sequence required for Cas9 to bind and cut DNA. PAM sequences are not present in the CRISPR locus, ensuring the system does not target its own genome.
How is CRISPR used in genome editing?
CRISPR can be used to create targeted DNA breaks at specific loci. The breaks are repaired by the cell’s repair mechanisms (HDR or NHEJ), enabling precise genetic modifications such as insertions, deletions, or mutations.
What is the CRISPR knock-out strategy?
In a knock-out strategy, CRISPR/Cas9 creates a double-strand break in the target gene. The cell repairs the break using NHEJ, leading to insertions or deletions (indels) that disrupt the gene’s function, effectively knocking it out.
What is the CRISPR knock-in strategy?
In a knock-in strategy, a double-strand break is introduced by CRISPR/Cas9, and a donor DNA template is provided. The cell uses homology-directed repair (HDR) to insert new genetic material at the targeted locus.
How is CRISPR used in prostate cancer research?
CRISPR is used to knock out or modify the androgen receptor (AR) gene in prostate cancer cell lines. This allows researchers to study the role of AR in cancer progression and test potential therapies targeting AR.
Describe the two CRISPR-based strategies used in prostate cancer research.
Knock-out strategy: Target AR gene with CRISPR/Cas9 to create a cell line that lacks AR expression.
Knock-in strategy: Create a modified prostate cancer cell line to study aberrant androgen receptor variants (AR-Vs), which are involved in castrate-resistant prostate cancer.
What are some challenges with CRISPR in human treatments?
Challenges include off-target effects, regulatory concerns, immune responses, mosaicism (incomplete editing), and efficacy of delivery.
How has CRISPR been applied in HIV treatment?
CRISPR has been used to edit the CCR5 gene, which codes for a receptor used by HIV to enter cells. Disrupting this gene makes cells resistant to HIV infection, as seen in the “Berlin Patient” case.
What are the methods of CRISPR delivery in vivo and ex vivo?
Ex-vivo: Cells are removed from the patient, edited, and then reintroduced.
In-vivo: CRISPR/Cas9 is delivered directly into the patient’s body, often through viral vectors or nanoparticles.
What are some ethical considerations in CRISPR gene editing?
Considerations include the potential for off-target effects, germline versus somatic editing, regulatory guidelines, and the implications of editing human embryos or germline cells.
How is CRISPR used for gene editing in eukaryotes?
In eukaryotes, CRISPR/Cas9 is used to target specific genomic sequences for modification. The Cas9 protein creates a double-strand break, which is then repaired by cellular repair mechanisms like HDR or NHEJ, enabling precise genetic changes, such as mutations or insertions.
Why is correct gRNA design essential for CRISPR gene editing?
Proper gRNA design ensures that the Cas9 protein is guided to the correct genomic target, minimizing off-target effects. The gRNA must be specific to the target sequence and have the appropriate PAM sequence nearby to allow Cas9 cleavage.
