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← Theoretical B

Q29 — Cas9 Target Sites, Donor Design, and Mutation Severity

Theoretical B Real exam question — full text reproduced under IBO's CC BY-NC-SA 4.0 license
Recombinant DNA & Biotechnology Techniques

Cas9 is an enzyme utilized in genome engineering, where it cleaves the DNA at sites complementary to the gRNA sequence. To cleave the DNA, Cas9 requires an NGG (N is any nucleotide) sequence next to the target site. Repair of Cas9-cleaved DNA results in either random mutations or precise introduction of the desired change. The latter requires a donor template having a homologous sequence to the target site.

Panel A: a schematic of Cas9 bound to gRNA cleaving genomic DNA, branching into mutagenic repair or donor-template-guided recombination. Panel B: the target DNA sequence with gRNA-matching region highlighted green and NGG highlighted red, plus two donor sequences DN-1 and DN-2 with the desired change in blue. Panel C: the wild-type sequence compared to four random mutational outcomes Mut-1 through Mut-4, each with a small deletion, insertion, or substitution highlighted in pink. Figure 1. A. Repair outcomes of Cas9-mediated DNA cleavage. B. The DNA sequence encoding an N-terminal fragment of the target protein, the gRNA sequence (highlighted in green), and two donor sequences (DN-1 and DN-2) having the desired change highlighted in blue (the sequences highlighted in orange are silent mutations introduced to the Donor sequences) are shown. The trinucleotide highlighted in red is NGG. C. The random mutational outcome (in pink) at the Cas9-mediated cleavage site without supplying donor sequence is represented.

On your answer sheet, indicate “T” for true statements and “F” for false ones.

A. There are a total of two Cas9-targetable sites in the given DNA.
B. DN-2 is a better choice than DN-1 for introducing the desired mutation.
C. Mutation in Mut-2 is potentially more deleterious than mutation in Mut-3 (find the start codon).
D. Mutation in Mut-4 is potentially more deleterious than mutation in Mut-1 (find the start codon).
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Question reproduced from IBO 2024, Theoretical Exam Part B, licensed under CC BY-NC-SA 4.0 — attributed to the International Biology Olympiad. Open the full exam PDF