Exam 4: CRISPR-Cas9

Question 1

Compare and contrast the CRISPR-Cas9 system to restriction enzyme digesting.

Answer 1

CRISPR-Cas9: It is a targeted and programmable system that uses a guide RNA molecule to direct the Cas9 enzyme to a specific DNA PAM sequence, where it induces a double-strand break. The cell’s natural repair machinery then introduces changes at the cut site. Highly specific and can be programmed to target virtually any DNA sequence by designing a specific guide RNA.

Restriction Enzyme Digestion: Restriction enzymes recognize specific DNA sequences and cleave the DNA at or near these recognition sites, producing blunt or sticky ends. This process is not as programmable or targeted as CRISPR-Cas9. If similar sequences appear elsewhere in the genome, non-specific cutting may occur.

Question 2

Describe how the CRISPR-Cas9 system can be used to edit a genome.

Answer 2

-Scientists design a small piece of RNA called guide RNA (gRNA), which is complementary to the specific DNA sequence they want to modify.
The gRNA serves as a guide to locate the target DNA sequence within the genome.

-The gRNA is combined with the Cas9 enzyme to create the CRISPR-Cas9 complex.

-The Cas9 enzyme acts as a pair of molecular scissors that can cut the DNA at the precise location identified by the gRNA.

-Once inside the cell, the CRISPR-Cas9 complex moves toward the cell nucleus, where the genomic DNA resides. The gRNA guides the Cas9 enzyme to the specific target sequence in the genome.

-The Cas9 enzyme cuts the DNA at the targeted location. This creates a break in the DNA strand.

-Cells have mechanisms to repair DNA breaks. There are two main repair pathways: Non-Homologous End Joining (NHEJ) and Homology-Directed Repair (HDR).

NHEJ often introduces small insertions or deletions (indels) at the site of the cut, leading to gene disruptions.

HDR, if provided with a DNA template, can be used to introduce specific changes or insertions at the target site.

Question 3

Describe the types of alterations that CRISPR can generate and relate them to the treatment of a genetic disorder like Duchene Muscular Dystrophy (DMD).

Answer 3

If a patient has a specific point mutation in the DMD gene, CRISPR could be used to correct that mutation, restoring the normal reading frame and enabling the production of a functional dystrophin protein. In cases where the DMD gene is severely mutated or missing, CRISPR could be used to insert a functional copy of the DMD gene, allowing the cells to produce dystrophin and potentially ameliorating the symptoms of DMD.

Question 4

Describe the PAM site and it’s role in CRISPR.

Answer 4

Cas9, the CRISPR-associated protein, recognizes and binds to the PAM sequence in the target DNA. The presence of a PAM sequence is essential for Cas9 to initiate the process of DNA recognition and cleavage.

Question 5

CRISPR

Answer 5

Clustered
Regularly
Interspaced
Palindromic
Repeats

Question 6

What is the difference between non-homologous end joining (NHEJ) and homology directed repair (HDR)?

Answer 6

At its core, NHEJ break-ends can be ligated without a template strand, whereas HDR breaks require a template strand to repair. NHEJ occurs more frequently because while it is a crude repair, it is efficient.

Non-Homologous End Joining (NHEJ):
-Has an error-prone mechanism that directly rejoins the broken ends of the DNA without the need for a homologous template.
-Directly ligates the broken ends, often resulting in loss or addition of nucleotides at the junction.

Homology-Directed Repair (HDR):
-High-fidelity repair mechanism that uses a homologous DNA template to guide the repair of the broken DNA strands.
-HDR is more accurate compared to NHEJ, as it uses a template with the correct genetic information to restore the sequence at the break site.