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Gene therapy is the application of genetic techniques to alter specific genes that cause disease in humans. Today’s gene therapy involves gene editing, which is targeted genome editing using nucleases to provide a method for inducing deletions or insertions.

The most recent technique involves using CRISPR – associated protein 9 (Cas9) to provide better specificity, simplicity, speed and pricing.

Students are asked to use CRISPR/Cas9 to edit a gene in Saccharomyces, a commonly used genus of yeast. Cas9 recognizes a defined site in the genome, as directed by appropriate guide RNAs. The students use the system to delete a gene used in galactose fermentation.

The CRISPR target sequence is 20 bases long as part of each CRISPR locus. The galactose gene in Saccharomyces contains the nucleotide sequence 5’-AGGTCACTGGCAAATGTACT-3’.

Cas9 proteins select the correct location on the host's genome by utilizing the sequence for base pair bonding with the host DNA.

Which of the following sequences would be the complementary sequence on Cas9 for this gene, and why?

A

3’-AGGTCACTGGCAAATGTACT-5’ because the matching sequence should be identical to the original sequence, with a reverse orientation.

B

5’- TCCAGTGACCGTTTACATGT-3’ because the matching sequence should be complementary to the original sequence and in the same orientation.

C

3’-TCCAGTGACCGTTTACATGT-5’ because the matching sequence should be complementary to the original sequence, with a reverse orientation.

D

3’-UCCAGUGACCGUUUACAUGA-5’ because the matching sequence should be made of RNA and with a reverse orientation.

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