Question

PART 3: Designing a Guide RNA You will now design your own guide RNA to inactivate a butterfly gene like Robert Reed did. Your goal is to knock out the optix gene in a species of butterfly called the painted lady (Vanessa cardu). Activity Stander Biointeractive Using CRISP other Your guide RNA must match a target DNA sequence in the gene that you want to knock out. A partial sequence of an exon from the butterfly's aptix gene is shown below. 5'-CGACACCGGTTCCAGCGCTCGAGCCACGCGAAGCTTCAGGCGCTGTGGCTGGAAG 3-GCTGTGGCCAAGGTCGCGAGCTCGGTGCGCTTCGAAGTCCGCGACACCGACCTTC CGCACTACCAGGAAGCGGAGCGCCTCCGCGGTCGCCCGCTCGGGCCCGTCGACAA GCGTGATGGTCCTTCGCCTCGCGGAGGCGCCAGCGGGCGAGCCCGGGCAGCTGTT GTACOGGGTGCGGAAGAAGTTOCCTCTGCCGAGGACTATTTGGGACGGCGAAGAG-3' CATGGCCCACGCCTTCTTCAAGGGAGACGGCTCCTGATAAACCCTGCCGCTTGTC-5' 1. Underline a 20-nucleotide target DNA sequence in the top (5 to 3) strand of the exon above. Remember that this sequence should be directly upstream on the end) of a PAM sequence (5'NGG-3'). 2. Highlight the PAM sequence that is next to your target DNA sequence. This is where Case wil bind. 3. Rewrite the target DNA sequence and its complement below, indicating where Cas9 would cut both strands of DNA with a large space or vertical line (1) 4. Record the 20-nucleotide guide RNA sequence that matches your target DNA sequence. This sequence should not include the PAM. 5. Robert Reed's tab at Cornell University designed guide RNAs to knock out the optik ene in three species of Focus

Answer 1

Answer:

According to data-

• Crispr-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system is a recent technique of genetic engineering utilising the bacterial endonuclease Cas9 (Crispr associated endonuclease).
• In this technique, a guideRNA is used to find out the target DNA sequence which needs to be modified (knock-out/ repression/ activation). Guide RNA contains 2 moieties, the first one is the anchor which contains the target sequence and the second one is the scaffold which binds Cas9 protein.
• Cas9 binds to the guide RNA scaffold by surface expressed positively charged groups forming a riboprotein complex. The target sequence is approximately 10-20 base pairs in length, should be unique and must contain a PAM sequence just downstream where Cas9 endonuclease can act upon.
• PAM sequence or protospacer adjacent motif is the region which is recognized by Cas9 (for example Cas9 endonuclease from Streptococcus pyogenes recognizes the sequence 5’-NGG-3’).
• Cas9 can bind to any PAM sequence available but its endonuclease activity will depend upon the binding intensity with which the guide RNA binds the target sequence.

Answer 1:

Answer 2:

5'-CGACACCGGTTCCAGCGCTCGAGCCACGCGAAGCTTCAGGCGCTGTGGCTGGAAG 3'-GCTGTGGCCAAGGTCGCGAGCTCGGTGCGCTTCGAAGTCCGCGACACCGACCTTC The probable PAM sequences 5'- NGG-3' PAM sequences are underlined in red CGCACTACCAGGAAGCGGAGCGCCTCCGCGGTCGCCCGCTCGGGCCCGTCGACAA GCGTGATGGTCCTTCGCCTCGCGGAGGCGCCAGCGGGCGAGCCCGGGCAGCTGTT GTACCGGGTGCGGAAGAAGTTCCCTCTGCCGAGGACTATTTGGGACGGCGAACAG-3' CATGGCCCACGCCTTCTTCAAGGGAGACGGCTCCTGATAAACCCTGCCGCTTGTC-5' The target sequence upstream of the PAM is underlined in blue

5’-...CTCGAGCCACGCGAAGCTTCAGG...-3’

Target PAM

Answer 3:

Cas9 cleaves 3 nucleotides upstream of the PAM sequence.

Cas9 cleavage site 5'-...CTCGAGCCACGCGAAGC TTCAGG...-3' 3'-...GAGCTCGGTGCGCTTCGAAGTCC...-5'

Answer 4:

Guide RNA sequence that is complementary to the target sequence

3’-GAGCUCGGTGCGCUUCGAAG-5’

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