Question

1. In CRISPR, why does it make sense to design our crRNA first before making any other changes to the genomic DNA?

2. In CRISPR, when searching for PAMs, GGNGG makes a much better PAM than NGG - why is this?

Answer 1

CRISPER-cas9 is a method of gene editing which uses cas9 enzyme which used as scissors to cut at a specific region of double bond genomic DNA , whereas, a small predesigned RNA sequence called guide-RNA or gRNA-Now the gRNA or crRNA is made in such a way that they will have the sequence which is complementary with the target DNA region. Next a complementary sequence will first find and attached to the target sequence of DNA to be edited and cas9 will next cut the region at specific location. Thus before starting any procesure related to genomic editing it is important to design gRNA or crRNA.

2. PAM or Protospacer adjacent motif is a DNA sequence containing 2-6 base pairs which is followed by cas9 enzyme. In absence of PAM, cas9 is failed to bind and cleave efficiently the target DNA site. In general PAM sequence is consists of NGG, where N can be any nucleotidre and G is guanine. But it has been found that a GGNGG motif which containes double guanine is able to bind more effectively at target site than single gunanine motif.