Genome Editing with CRISPR

For biotechnology

Many questions were inspired by this CRISPR review.

1. PAM stands for “protospacer adjacent motif”. What does this mean?
2. Compare CRISPRi and CRISPRa. Which Cas9 variant would be useful for these approaches?  What Cas9 fusion protein would you create for CRISPRi?  CRISPRa?
3. How would you set up your CRISPR-based experiment to preferentially use NHEJ DNA repair pathway? What type of mutation would you expect?
4. How would you set up your CRISPR-based experiment to preferentially use HR DNA repair pathway? What kind of mutation could you expect?
5. 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?
6. 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?
7. Discuss the similarity between processing the CRISPR array in bacteria and processing polycistronic tRNA-gRNA (PTG) sequences.
8. 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?
9. Compare GMOs and GEs. Why might the latter be more acceptable?
10. How do gene drives work?