Problem 1: Why does CRISPR not always generate a non-functional protein?
Problem 2: When using 'Strategy 1' to inactivate a gene product, why is it better to find a Protospacer in an earlier exon rather than a latter one?
Problem 3: For CRISPR design, why is it important to remove the PAM sequence from your Protospacer sequence in your sgRNA?
Problem 4: When using strategy 2 to inactivate a gene product, why does it not matter whether the first and last exons encode for amino acids?
Problem 5: Which Primer sets using strategy 2 will generate a PCR product following KO? Will the PCR product size change pre- and post- gene removal? If so, then will the PCR product be larger or smaller when amplifying from the KO locus?