NEXT GENERATION GENOME EDITING TECHNOLOGIES Next-Generation-Genome-Editing-Technologies | Page 11
ALTERNATIVE SOLUTIONS:
With the ongoing strife for rights to CRISPR acquisition, other researchers who have keen
interest in gene editing tools or who need developing a much better tool than CAS9 which
could overcome the problems related to it have laid their eyes on two new emerging tools:
1. Cpf1
The history of gene editing became interesting when a new nuclease came into picture. Zhang’s
lab recently published a paper describing two genes from Cpf1 family that have cleaving ability
in mammalian cells.
1. It is a class II nuclease (CRISPR from Prevotella and Francisella) and is classified as type V
CRISPR system.
2. It has RuvC-like endonuclease which is similar to CAS9’s but lacks in HNH domain.
3. It requires only one RNA whereas CAS9 required two (trRNA and crRNA).
4. It cleaves DNA in a staggered manner and gives 5’overhang 18-20 bp away from PAM sites.
CAS9 produces blunt ended double strands (3nt upstream of PAM sites).
5. The Cpf1 crRNA has a much simpler structure (short stem loop in direct repeat).
Benefits:
Due to staggered cleavage, directional gene transfer is possible
Sticky end mediated gene transfer can be used to target nondividing cells, which are difficult
to modify through HDR.
AT- rich regions that did not have enough 3’-NGG PAM sites can also be treated as targets.