- CRISPR/Cas system (Cas9/gRNA) Off-Targeter
Note: The address of CasOT was changed to http://casot.cbi.pku.edu.cn/


CasOT
CasOT is a genome-wide potential Cas9/gRNA off-target searching tool.



Cas9/gRNA
Cas9/gRNA system, one type of the CRISPR/Cas systems, is a kind of engineered endonuclease (EEN). It consists of two components: Cas9/gRNA recognizes and cleaves the target DNA and causes a DSB (double-strand break), which provides the opportunity of gene mutagensis and other types of genome manipulation.


Reference:
22745249 (in vitro) 23287718 23287722 (human cell)


On- & off-target effects of Cas9/gRNA
The sequence in one strand of target DNA identical to the gRNA is called the protospacer.

A particular motif is required for Cas9/gRNA recognizing & cleavage in the 3'-end out of the protospacer, which is usually -NGG, named as PAM (protospacer adjacent motif).

Some reports suggest the PAM type, and the mismatch numbers in different regions (seed or non-seed region) of protospacer, show different effects on the efficiency of Cas9/gRNA. In brief, the off-target effects of Cas9/gRNA is under discovery.


Reference:
23360965 (in vitro) 24022156 (a review of human cell)


Paired-gRNA strategy of Cas9
The paried-gRNA strategy is reported to show higher specificity than the above single-gRNA strategy, it consists of three components: The two close nicks induced by the gRNA pair can cause a DSB. However, single nick in a potential off-target induced by only one gRNA would be difficult to cause DSB.


Reference:
23907171 23992846




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School of Life Sciences, Peking University, Beijing, China
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