Problem $3(16$ pts$)$: Individuals with BRCA$1$ mutations often inherit a mutation in one copy

of their BRCA$1$ gene. The most common mutation is nonsense mutation that changes

arginine $1443$ to a stop codon. You would like to use CRISPR to treat BRCA$1+/-$ patients.

Wild type BRCA$1$:

$5-$TGA TGT GCT TTG TTC TGG ATT TCG CAG GTC GTC AAC GGC AGA AGA$-3$

$3-$ACT ACA CGA AAC AAG ACC TAA AGC GTC CAG GAG TTC CCG TCT TCT$-5$

S$1450-$ T$1449-$S$1448-$Q$1447-$E$1446-$P$1445-$N$1444-$R$1443-$L$1442-$ D$1441-$E$1440-$L$1439-$A$1438-$ S$1437-$ S$1436$

Mutant BRCA$1$: Arg$1443$X

$5-$TGA TGT GCT TTG TTC TGG ATT TCA CAG GTC GTC AAC GGC AGA AGA$-3$

$3-$ACT ACA CGA AAC AAG ACC TAA AGT GTC CAG GAG TTC CCG TCT TCT$-5$

X$1443-$L$1442-$ D$1441-$E$1440-$L$1439-$A$1438-$ S$1437-$ S$1436$

$1.$ Which allele will you target?

$2.$ Is there a $(5-$NGG$-3)$ PAM site present? If so$,$ circle it$/$them in the sequence.

$3.$ Write the $20$bp of complementary RNA sequence you will use to generate your

crRNA.

$4.$ Will you include anything else to $$fix$$ the mutant allele?

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