Q7 - Designing a CRISPR Knockout of Gene X

Theoretical A Real exam question - full text reproduced under IBO's CC BY-NC-SA 4.0 license

Recombinant DNA & Biotechnology Techniques

Michael Smith (1932-2000) invented site-directed mutagenesis. Recently scientists invented CRISPR- Cas9 technology to carry out mutagenesis more easily. Cas9 protein from Streptococcus pyogenes is guided by a 20bp guideRNA which base-pairs with target DNA. This Cas9 enzyme can only make double strand cuts in DNA three bases upstream from a 5’-NGG-3’ site (1). Double strand cuts are attacked by DNA degrading enzymes, and then re-joined. Gene X, which you want to stop from functioning with this enzyme, is shown (2). An alternative way to knockout genes is to use a pair of modified Cas9’s which can only make single strand cuts, and guideRNA’s which target adjacent sites.

Figure for Q7: Designing a CRISPR Knockout of Gene X

This question mixes multiple-choice/numeric-estimate parts with True/False statements - answer each part using the information and data above.

A. Which of the five guideRNA sequences should be used to guide Cas9 to cut Gene X as early as possible in its coding sequence?

B. Which labelled NGG site (I-V) should Cas9 cut at to disrupt Gene X most effectively?

C. Using the pair of modified Cas9s and guideRNAs increases off-target damage to other genes.

D. The Streptococcus pyogenes genome has more NGG sites than expected by chance.

E. If the gene has no GG sequences, Cas9s from other species should be investigated for alternatives.

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Question reproduced from IBO 2017, Theoretical Exam A, licensed under CC BY-NC-SA 4.0 - attributed to the International Biology Olympiad. Open the full exam PDF · Official answer key & worked solutions