Many questions were inspired by this CRISPR review.
- PAM stands for “protospacer adjacent motif”. What does this mean?
- Compare CRISPRi and CRISPRa. Which Cas9 variant would be useful for these approaches? What Cas9 fusion protein would you create for CRISPRi? CRISPRa?
- How would you set up your CRISPR-based experiment to preferentially use NHEJ DNA repair pathway? What type of mutation would you expect?
- How would you set up your CRISPR-based experiment to preferentially use HR DNA repair pathway? What kind of mutation could you expect?
- Why would you likely use two gRNAs in Cas9n- and RFN-based experiments? What does it mean that this would reduce your chances of causing off-target effects?
- The ribozyme-gRNA-ribozyme method of creating a sgRNA sounds complex. Why wouldn’t you just clone sgRNA sequence alone in a normal PolII-based promoter? How can this RGR system produce sgRNAs in a tissue-specific manner?
- Discuss the similarity between processing the CRISPR array in bacteria and processing polycistronic tRNA-gRNA (PTG) sequences.
- CRISPR systems are adaptive immune systems in bacteria and archaea that provide resistance to bacteriophage. How has this been adapted to plants? How has this been adapted to animals?
- Compare GMOs and GEs. Why might the latter be more acceptable?
- How do gene drives work?