CRISPR/cas 9 systems, Cong and Zheng 2015, Chapter 10

There are 3 known CRISPR systems, types I, II, and III.  Type II CRISPR systems usually only require a single protein, Cas9, to perform the target cleavage. 

Cas9 is a RNA-guided nuclease that is capable of binding to a target DNA and introduce a double strand break in a sequence-specific manner. 

The guide sequence within sgRNA has a length of 20 bp and is the exact complementary sequence of the target site within the genome, sometimes referred to as “protospacer” following the

convention of microbial CRISPR field.

In choosing the target site, it is important to note the requirement of having the “NGG” trinucleotide motif, called protospacer adjacent motif (PAM), right next to the protospacer target on the 3′ end.

The PAM sequence depends on the Cas9 protein employed in the CRISPR-Cas9 system, and hence, the “NGG” PAM sequence applies specifi cally to the Streptococcus pyogenes Cas9 (SpCas9).