Question 1

How do genome editing techniques create targeted mutations in DNA?

Accepted Answer

Genome editing methods use engineered nuclease enzymes to create small cuts or double-strand breaks in specific DNA sequences. Cells attempt to repair this damage through nonhomologous end joining or homology-directed repair. During repair, mutations are introduced into the targeted region with much higher efficiency than classical gene targeting methods.

Question 2

What is the difference between NHEJ and HDR in DNA repair?

Accepted Answer

Nonhomologous end joining (NHEJ) directly reseals broken DNA ends but often imperfectly, deleting or adding bases and mutating the target sequence. Homology-directed repair (HDR) fixes damage by copying from a homologous template, allowing researchers to add specific sequences to direct precise changes at the target site.

Question 3

How do zinc finger nucleases and TALENs differ in their targeting approach?

Accepted Answer

Zinc finger nucleases (ZFNs) fuse DNA-binding zinc finger domains to the FokI endonuclease, with each domain recognizing a specific nucleotide triplet. TALENs join FokI to bacterial transcription activator-like effector domains, where each domain recognizes a single unique DNA base, providing different levels of sequence specificity.

Question 4

Why is the CRISPR-Cas9 system advantageous compared to protein-based genome editing methods?

Accepted Answer

The CRISPR-Cas9 system uses customizable RNA guide sequences called single guide RNAs (sgRNAs) to target Cas9 to desired genetic sequences. This RNA-based approach provides distinct advantages over protein-based methods like ZFNs and TALENs because the guide sequence can be easily modified to target almost any genomic location in mammalian cells or other systems.

Question 5

What role does the protospacer adjacent motif play in CRISPR-Cas9 targeting?

Accepted Answer

The protospacer adjacent motif (PAM) is a 3-base pair sequence located at the 3' end of the target DNA region. Cas9 requires the PAM to recognize and cleave the target DNA, and the exact PAM sequence is specific to the organism from which the Cas9 enzyme originated, making it essential for successful genome editing.

Question 6

How are CRISPR guide sequences synthesized and incorporated into cells?

Accepted Answer

Two complementary oligonucleotides matching the target sequence are synthesized, denatured, and annealed. The resulting guide sequence is ligated into a plasmid containing an sgRNA scaffold using restriction enzymes and ligase. The CRISPR construct is then co-transfected with a Cas9-encoding construct into target cells, which are cultured and screened by PCR or sequencing for desired mutations.

Question 7

What are some current applications of genome editing in research?

Accepted Answer

Researchers use genome editing to insert reporter sequences like green fluorescent protein into specific genes, generate knockout organisms where genes are rendered non-functional, and create large genetic deletions by inducing two double-strand breaks in the same chromosomal region. These applications enable study of gene function and disease mechanisms in model organisms and cultured cells.

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