What is a single guide RNA, and what role does it play in CRISPR-Cas genome editing in eukaryotic cells?

Verified step by step guidance

Understand that a single guide RNA (sgRNA) is a synthetic RNA molecule designed to guide the Cas9 protein to a specific DNA sequence in the genome. It combines two natural RNA components: the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA).

Recognize that the sgRNA contains a 20-nucleotide sequence complementary to the target DNA sequence, which allows it to bind specifically to that region in the genome through base pairing.

Know that the sgRNA forms a complex with the Cas9 nuclease, directing Cas9 to the target DNA site by base pairing between the sgRNA and the DNA sequence adjacent to a protospacer adjacent motif (PAM).

Realize that once the sgRNA-Cas9 complex binds to the target DNA, Cas9 induces a double-strand break at the specific location, which is the critical step for genome editing.

Understand that the cell's natural DNA repair mechanisms then repair this break, allowing for gene disruption, insertion, or correction, depending on the experimental design.

Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Single Guide RNA (sgRNA)

A single guide RNA is a synthetic RNA molecule that combines the functions of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). It directs the Cas9 protein to a specific DNA sequence by complementary base pairing, enabling targeted genome editing.

CRISPR-Cas9 Mechanism

CRISPR-Cas9 is a genome editing tool where the Cas9 enzyme acts as molecular scissors to cut DNA at a location specified by the sgRNA. This targeted DNA cleavage allows for gene disruption, correction, or insertion in eukaryotic cells.

Genome Editing in Eukaryotic Cells

In eukaryotic cells, CRISPR-Cas9 facilitates precise genetic modifications by inducing double-strand breaks repaired via cellular mechanisms like non-homologous end joining or homology-directed repair, enabling gene knockout or insertion.

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Eukaryotic Transcription

Related Practice

Textbook Question

When disrupting a mouse gene by knockout, why is it desirable to breed mice until offspring homozygous (−/−) for the knockout target gene are obtained?

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Textbook Question

Gene targeting and gene editing are both techniques for removing or modifying a particular gene, each of which can produce the same ultimate goal. What is the main technical difference in how DNA is modified that differs between these approaches?

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Textbook Question

The CRISPR-Cas system has great potential but also raises many ethical issues about its potential applications because, theoretically, it can be used to edit any gene in the genome. What do you think are some of the concerns about the use of CRISPR-Cas on humans? Should CRISPR-Cas applications be limited for use on only certain human genes but not others? Explain your answers.

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