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CRISPR technology has greatly facilitated genome engineering with its simplicity, high efficiency and accuracy compared to traditional ZFN and TALEN approaches. Meanwhile, CRISPR has also expanded its potential applications beyond gene editing. With specific fusion proteins, CRISPR system can be employed for gene regulation and genome loci reposition, as well as live cell imaging.
In order to support such broad application with high accuracy and enough flexibility, multiple approaches have been developed to conditionally activate or regulate Cas9 function. Many of them utilize inducers like chemicals or light to work on full length of Cas9, others focus on split Cas9 recombination. Although they can improve CRISPR regulation, these methods lack either universality or reversibility that limits its wider application.
More recently, natural anti-CRISPR (Acr) proteins have been identified as a new type of CRISPR mediator by directly inhibiting CRISPR enzyme activity. Since Acr directly works on CRISPR enzyme, any effect on Acr protein that impacts Acr-Cas9 interaction might be employed for regulating Cas9 activity with much more flexibility. Following this idea, an international research group with bioinformatics and bioengineering background developed a new CRISPR control system based on Acr inhibition by light mediation and published their results in Nature methods.
Compared to previous methods regulating CRISPR function, this new technique named ‘CASANOVA’ demonstrates promising potential for increased accuracy of CRISPR regulation, the majority of which is the engineered Acr with site-specific insertion of a photosensor of LOV2 domain. As LOV2 is a good light-switchable protein that has been widely used as an optical control switch through insertion into specific loops of target proteins, the authors selected this small photosensor to precisely control Acr. Meanwhile, they chose AcrIIA4, one of spCas9 inhibitor, for LOV2 insertion.
As mentioned above, LOV2 needs to insert into specific site of AcrIIA4 for photo-switching. After bioinformatic analysis of AcrIIA4 structure, the L5 loop of AcrIIA4 shows potential for LOV2 insertion. It is relatively easy to destroy AcrIIA4 function by LOV2 insertion leading to conformational alteration, but is harder to keep AcrIIA4 inhibition close to WT after LOV2 insertion without light induction. Therefore, the following work focused on that which went through a series of steps before CASANOVA coming out.
First, the L5 loop region was screened and the site between AcrIIA4 residues E66 and Y67 was identified for LOV2 insertion that exhibited noticeable inhibitory function. However, direct insertion of LOV2 might occupy extra space of AcrIIA4 structure that impairs its inhibitor function resulting from conformational change. So, further stepwise deletion of Acr residues preceding the insertion site was performed to restore its structural integrity, and a mutant with three-amino-acid deletion exhibited the highest Cas9 inhibition without light induction, which is comparable to WT Acr inhibitor. This improvement is probably due to 3 residues deletion that leaves enough space for LOV2 loading into AcrIIA4. More importantly, this variant also showed almost full recovery of Cas9 function after potoactivation of LOV2 for inhibitor derepression. This specific Acr-LOV hybrid variant was subsequently named CASANOVA.
To further promote Acr-LOV hybrid inhibition and make it closer to WT Acr, LOV2 mutation improving docking followed by AcrIIA4 mutation enhancing Cas9 binding affinity were carried out, which generated two variants of CASANOVAT16F and CASANOVAS46D that demonstrated improved Cas9 inhibition with comparable light activation.
CASANOVA and the following two variants were then tested for different applications. They worked well for genome editing, transcription regulation and living cell imaging. More importantly, CASANOVA is a reversible system that enables turn on and off Cas9 function at ease. Additionally, this reversibility is also achievable for working Cas9 that cannot be achieved by some of the previous methods. Therefore, CASANOVA is an advanced CRISPR regulation system expanding CRISPR application.