Problem
In order to understand why these selection and design steps are important for successful CRISPR, consider the following:
1. How could a short insertion or deletion in an ORF result in mutations that disrupt protein function? Provide at least three example scenarios?
2. Why is it important to avoid the 5' and 3' ends of the ORF when choosing a genomic target sequence?
3. What was the purpose of adding additional nucleotides to the 5' ends of the two oligos?
4. The software (identifies not only sequences with high on-target scores, but also lists off-target scores based on sequence similarity to other sequences in the genome of the targeted organism. What could happen if (a) an off-target sequence is identical to the on-target sequence when the plasmid is introduced into target cells? (b) if this off-target event occurs but you do not know, how would that affect your interpretation of the phenotype observed after successful on-target insertion/deletion.