Question

In the CRISPR-Cas technology for editing genes, what is (are) the function(s) of sgRNA? a. to bind to the target gene b. to bind to Cas9 c. to cause a double-strand break in the target gene d. all of the above e. both a and b

Short answer

The functions of sgRNA in the CRISPR-Cas technology for gene editing are to bind to the target gene and to bind to Cas9. Thus, the correct answer is e. both a and b.

Step-by-step solution

Understand the CRISPR-Cas technology

CRISPR-Cas is a gene-editing tool. It consists of the Cas9 enzyme and sgRNA or guide RNA. It is important to understand that the Cas9 enzyme and sgRNA form a complex which locates and alters target DNA sequences.

Understand the role of sgRNA

sgRNA, or single-guide RNA, is a pivotal component in the CRISPR-Cas system. The primary functions of sgRNA are: 1. To bind to the target gene: The sgRNA forms a complex with the Cas9 enzyme and guides it to the target gene location. 2. To bind to Cas9: The sgRNA is essential to form the sgRNA-Cas9 complex. It bridges the interaction between Cas9 and the target DNA.

Analyze the options

Option c is incorrect because the actual double-strand break in the target gene is caused by the Cas9 enzyme, not the sgRNA. Now looking at options d and e, option d states all of the above which is incorrect as we just determined that option c is not a function of the sgRNA. Therefore, by elimination and the functions analyzed, it can be concluded that both option a and option b are functions of the the sgRNA, meaning option e is the correct answer.

Key concepts

Gene Editing

Gene editing, sometimes referred to as genome editing, is a type of genetic engineering in which DNA is modified, deleted, added, or replaced in the genome of a living organism. Unlike traditional genetics where entire genes were turned on or off, gene editing allows for much more precise changes at the level of individual base pairs, the 'letters' of DNA. This precision is what makes modern gene editing a powerful tool for scientific research, medicine, and biotechnology.

The CRISPR-Cas system, a prominent gene editing technology, uses a pair of molecular scissors called Cas9 and a piece of RNA called sgRNA to cut DNA at a specific location. Once the DNA is cut, the cell's natural repair mechanisms kick in to fix the break, which can be harnessed to either disable a problematic gene or insert a new segment of DNA, thus giving researchers the ability to rewrite the genetic code.

sgRNA Function

The single-guide RNA (sgRNA) has a fundamental role in the CRISPR-Cas gene editing machinery. It serves multiple functions:

• Target Recognition: sgRNA is designed to match the DNA sequence of a specific target gene. By base pairing with the DNA, it directs the Cas9 enzyme to the exact location where a cut should be made, ensuring the precision of gene editing.
• Complex Formation: sgRNA also binds to the Cas9 protein, forming a stable complex. This structure is essential as it allows the sgRNA to act as a bridge between Cas9 and the target DNA.

The beauty of sgRNA is in its design; by changing the sequence of sgRNA, researchers can target any gene of interest. This versatility is why CRISPR-Cas is an advantageous tool over previous gene-editing techniques.

Cas9 Mechanism

Cas9 is often likened to a pair of molecular scissors. But how do these scissors know where to cut? The answer lies in the elegant mechanism of the Cas9 enzyme.

• Binding: Cas9 begins its work by forming a complex with the sgRNA. Once bound, the sgRNA guides Cas9 to its target sequence in the DNA through base pairing.
• Double-Strand Break: Upon recognition of the target DNA, Cas9 performs a precise double-strand break, cutting both strands of the DNA helix. This cut can trigger one of the cell's repair pathways, which can be exploited to introduce changes to the genome.
• Repair Pathways: After the cut, the cell can repair the DNA break by non-homologous end joining (NHEJ), which can lead to gene disruption, or homology-directed repair (HDR), which can insert or replace sequences with an external template DNA.

The specificity and flexibility of the Cas9 enzyme make CRISPR-Cas a transformative technology for gene editing. Its potential applications range from correcting genetic disorders to enhancing crop resistance to diseases and pests.