Question 1

How does Cas9 identify and cut DNA at the target site?

Accepted Answer

Cas9, an endonuclease from Streptococcus pyogenes, uses a synthetic guide RNA (sgRNA) to locate specific DNA sequences. The sgRNA directs Cas9 to the Protospacer Adjacent Motif (PAM) sequence, typically 5'-NGG-3', allowing Cas9 to bind and introduce a double-stranded break at the precise target location.

Question 2

What is the difference between gene insertion and gene deletion using CRISPR-Cas9?

Accepted Answer

Gene insertion uses one sgRNA to guide Cas9 to cleave a site, then homologous recombination inserts new genetic material. Gene deletion employs two sgRNAs directing Cas9 to cut both ends of a target region, excising the DNA fragment, which the cell's repair system then rejoins through non-homologous end joining.

Question 3

How can CRISPR-Cas9 be used to treat HIV infections?

Accepted Answer

CRISPR-Cas9 has been designed to inactivate HIV by excising integrated viral DNA from infected cells. Additionally, engineered systems like Csy4 target free HIV RNA to degrade it, though these antiviral strategies remain experimental and represent emerging therapeutic approaches for combating viral infections.

Question 4

What is the original function of the CRISPR-Cas system in bacteria?

Accepted Answer

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids. Originally discovered in prokaryotes, the antiviral system of bacteria and archaea has been repurposed as a powerful genome-editing tool for laboratory applications across diverse organisms.

Question 5

Can CRISPR-Cas9 edit multiple genes simultaneously?

Accepted Answer

Yes, CRISPR-Cas9 enables multiplexed genome editing, allowing simultaneous modification of multiple genes in a single organism. This capability is particularly advantageous in complex genetic studies and therapeutic applications, such as removing multiple retroviral copies from infected cells in one editing event.

Question 6

What organisms can be edited using CRISPR-Cas9 technology?

Accepted Answer

CRISPR-Cas9 has been reprogrammed to modify the genomes of diverse organisms, including plants, animals, and humans. This versatility makes it a revolutionary tool for genetic engineering across a wide range of biological systems and research applications in both basic and applied science.

Question 7

How does the synthetic guide RNA direct Cas9 to the correct DNA location?

Accepted Answer

The synthetic single guide RNA (sgRNA) contains a complementary sequence that guides Cas9 to a specific genomic locus adjacent to the Protospacer Adjacent Motif (PAM). This precise targeting ensures Cas9 binds and cuts only at the intended DNA location, enabling accurate and efficient genome editing with minimal off-target effects.

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