hspCas9 linearized Smart Nuclease vectors

Designer zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases

(TALENs), and homing meganucleases have made site-specific genome modifications a reality in many different
model organisms ranging from zebrafish to mammalian cells.

Genome editing tools

Type II prokaryotic CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
system, originally discovered in the bacterium Streptococcus pyogenes as a mechanism to defend against viruses
and foreign DNA

sRNA or small RNAs as guides to cleave DNA in a sequence-specific manner.

Guide sequences to target specific sequences

(unlike ZFNs and TALENs where construct assembly can be laborious
and time-consuming), as well as its targeting efficiency, this system has the potential to be a disruptive technology in
the field of genome-engineering.

The CRISPR/CRISPR-associated (Cas) system involves

• 1) retention of foreign genetic material, called “spacers”, in clustered arrays in the host genome
• 2) expression of short guiding RNAs (crRNAs) from the spacers
• 3) binding of the crRNAs to specific portions of the foreign DNA called protospacers
• 4) degradation of protospacers by CRISPR-associated nucleases (Cas).

A well-characterized Type II CRISPR system like inbacterium Streptococcus pyogenes, where four genes (Cas9, Cas1, Cas2, Csn1) and two non-coding small RNAs (pre-crRNA and tracrRNA) act in concert to target and degrade foreign DNA in a sequence-specific manner.