02 - Code Breakers: Using CRISPR to Rewrite Genetics
Designing gRNA to Target LacZ
1. IDENTIFY and HIGHLIGHT five PAM sites in the target sequence. For this experiment, as - sume that you are using a Cas9 enzyme which uses an 5’-NGG-3’ PAM site. In this notation, the “N” can be any nucleotide. This means that the Cas9 will only bind to sequences imme- diately upstream (in the 5’ direction) of an AGG, TGG, CGG, or GGG sequence. NOTE: Since Cas9 can bind to either of the complementary DNA strands, remember to examine both for PAM sequences. 2. IDENTIFY and UNDERLINE the binding regions upstream of your highlighted PAM sites. In CRISPR, the guide RNA recognizes and binds to 20 nucleotides on the DNA strand opposite from the NGG PAM site. Binding always occurs upstream (in the 5’ direction) of the PAM sites. 3. Using base pairing rules, CREATE a guide sequence for the gRNA. This sequence should bind to the underlined region identified in step 2. For complimentary binding, Adenine (A) pairs with Thymine (T) and Cytosine (C) pairs with Guanine (G). Remember that for the bottom strand, a 5’ to 3’ direction means recording the sequence from right to left. 4. USING steps 1-3, DESIGN five candidate gRNAs for the LacZ Gene Sequence (0-200 bp). RECORD your answers in Table 1.
EXAMPLE SEQUENCE: 5’-TTCACTGCGTTCAGCAAAAAGTGAATTCTTGGTTACTGC 3’-AAGTGACGCAAGTCGTTTTTCACTTAAGAACCAATGACG
LacZ Gene Sequence (0-200 bp) 5’- ATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTG 3’- TACTGGTACTAATGCCTAAGTGACCGGCAGCAAAATGTTGCAGCACTGAC GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT CCTTTTGGGACCGCAATGGGTTGAATTAGCGGAACGTCGTGTAGGGGGAA TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA AGCGGTCGACCGCATTATCGCTTCTCCGGGCGTGGCTAGCGGGAAGGGAA CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACC - 3’ GTCAACGCGTCGGACTTACCGCTTACCGCGAAACGGACCAAAGGCCGTGG - 5’
Guide Sequence TABLE 1
PAM Sequence
gRNA Name Example gRNA 1 gRNA 2 gRNA 3 gRNA 4
TTCAGCAAAAAGTGAATTCT
TGG
20
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