For cell line engineering, can you design gRNAs to exons and introns?
Yes, gRNAs are designed to exons or introns depending on what is more appropraite for the project. For knockout projects we aim to design gRNAs to conserved exons in the first third of the gene. For knock-in projects where a donor is required to introduce a change in the coding sequence, gRNAs are designed near to the required change in the exon. For knock-in donors, if a gRNA is designed to an exon, in order to prevent re-cutting of the targeted allele, we may also introduce silent mutations in the donor to disrupt the gRNA target site/PAM site. However, depending on gene structure, it is also possible to site the gRNA in the intron distal to that in which, for example, an antibiotic resistance marker is located. While this would have an impact on targeting frequency, and would require modification of intronic sequence to prevent re-cutting of the targeted allele, it would mean that exonic silent mutations would not be required.