Chapter 18, Problem 29P

F. Port and S. Bullock at the University of Cambridge (UK) designed the elegant plasmid vector pCFD3 for the expression of sgRNAs in Drosophila. The following figure shows a part of this vector. The orange sequences are part of a strong promoter (transcription from this promoter starts at the G in bold—which must be present—and goes from left to right). The purple sequences are a portion of the tracrRNA component of the sgRNA. After cutting the pCFD3 plasmid with the restriction enzyme BbsI (whose recognition site is also shown in the following figure), you will replace the blue sequences in the figure with sequences that will allow the expression of an sgRNA that targets a Drosophila gene called NiPp1.

The last part of the jigsaw puzzle you will need is the following sequence, which shows part of the NiPp1 gene including the triplet corresponding to the start codon. Capital letters are in the gene’s first exon with the coding region in blue; lowercase letters are in the first intron. The NiPp1 protein is 383 amino acids long. Your assignment is to generate a knockout allele of this gene by inducing Cas9 to produce a double-strand break into the gene that will be repaired imprecisely by nonhomologous end-joining (NHEJ).

a.	Identify the two PAM sites in this sequence. Which of these PAM sites would you want to use in order to produce a null allele of the NiPp1 gene? Why would you prefer this site?
b.	If you targeted Cas9 to the proper location in the NiPp1 gene, and the resultant double-strand break was repaired imprecisely by NHEJ (so that a few—usually ≤6 bp are deleted or added at that location), about what percentage of the imprecisely repaired genes could you say with confidence would be null alleles? Explain.
c.	Diagram the pCFD3 vector after it has been cut with the BbsI enzyme. Don’t worry about the small blue fragment that will be removed; the emphasis here is to show the 5′-overhangs that will be made.
d.	Design two 24-nt DNA oligonucleotides that you could anneal together and clone into BbsI-cut pCFD3 vector so that the recombinant plasmid could express an sgRNA useful for making null mutations in the NiPp1 gene.
e.	Show exactly where Cas9 would cut the NiPp1 gene.
f.	Briefly outline what you would do with your recombinant plasmid to make a null mutation in the fly NiPp1 gene.
g.	Briefly outline how you would modify this technique to generate a knockin allele in which the first amino acid in the NiPp1 protein after the initiating Met (that is, Thr) would be changed to Ala.