6) In the lab, you have been tasked with designing guide RNAs (gRNAs) to direct

Question

6) In the lab, you have been tasked with designing guide RNAs (gRNAs) to direct Cas9 cutting to specific disease alleles. Below is the "Watson" strand of each disease allele, shown in the 5'-3 orientation; remember that the reverse compliment ("Crick" strand) is not indicated here, but is present in the genomic DNA you will be cutting. Also, it does not matter whether you target the "Watson" or "Crick" strand, as long as you direct Cas9 to cut somewhere within the sequence provided for each disease allele The CRISPR target Protospacer (20 bases) PAM: Protospacer-adjacent motif 5 Guide RNA db The Guide RNA always begins with an RNA version of the protospacer sequence Doug Mortlock 2014 Genomic DNA is indicated in blue, and the guide RNA is indicated in red/brown Using the diagram above as a guide, determine the following: N stands for anything For each allele shown below, determine whether it is possible to target Cas9 to this location and, if so, write the 20bp nucleotide sequence(s) that you could use at the 5' end of your guide RNA to target each locus. Be sure to label the 5' and 3' ends of this portion of your gRNA. Note that the "protospacer" sequence must be followed by an NGG "PAM" sequence in order for Cas9 to recognize and cut the target sequence Allele 1: CTACTGATCTTCTACTTAAAATCGAGACTTGGAACATACTACGCTT

Explanation / Answer

For allele 1 & 4, gRNA taregting is possible as they have NGG sequence followed by protospacer

Allele 1: CTACTGATCTTCTACTTAAAATCGAGACTTGGAACATACTACGCTT

gRNA: 5' UUCUACUUAAAAUCGAGACU 3'

Allele 2: CTACTGATCCTCTACTTAAAATCGAGACTTGGAACATACTACGCTT

gRNA: 5' CUCUACUUAAAAUCGAGACU 3'

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