Optogenetics + CRISPR, Using Light to Control Genome Editing

Posted by Caroline LaManna on Mar 8, 2016 10:30:00 AM


Scientists around the world have been making major improvements to CRISPR technology since it’s initial applications for genome engineering in 2012. (Check out our CRISPR 101 eBook for everything you need to know about CRISPR.) Like CRISPR, optogenetics has also been making headlines over the past decade. Optogenetics uses genetically encoded tools, such as microbial opsins, to control cellular activities using light. In 2015, scientists combined CRISPR and optogenetics techniques to develop a variety of photoactivatable CRISPR tools. These tools allow scientists to use light to externally control the location, timing, and reversibility of the genome editing process. Read on to learn about the various light-controlled CRISPR tools available to researchers - some readily found at Addgene.

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Shining Light on Transcriptional Activation Using dCas9

Initial photoactivatable CRISPR systems published in early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato’s lab at the University of Tokyo (Nihongaki Y, et al., Chemistry & Biology, 2015 Feb 19; 22(2):169-74) and Charles Gersbach’s lab at Duke University (Polstein LR, et al., Nature Chemical Biology, 2015 Mar; 11(3): 198-200) developed similar systems based on the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create a system that would use light to turn on and off gene expression while imparting spatiotemporal control, reversibility, and repeatability.

The system developed by Nihongaki et al. is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65). Upon expression in the cell, the dCas9-CIB1 fusion binds to the target DNA sequence as directed by the guide RNA (gRNA), while the CRY2PHR-activator fusion floats freely, depicted in the figure below (A). Once triggered by blue light, the CRY2 and CIB1 proteins heterodimerize and move the activator into position to activate gene transcription. The researchers tested a variety of combinations to optimize both fusion proteins, including making alterations to the CIB1 domain, testing various activator probes, and adding various genomic anchors to the N-terminus of both fusion constructs. The best performing combination was NLS-dCas9-trCIB1 and NLSx3-CRYPHR-p65 - it had the lowest background activity in the dark state and highest fold induction at 31X. By using a slit pattern during blue light exposure (470nm), the researchers showed that expression of the human ASCL1 gene could be spatially controlled. The authors also cycled blue light on and off and showed that ASCL1 expression followed suit - control was indeed reversible and repeatable.

With their light-activated CRISPR/Cas9 effector (LACE) system Polstein et al., utilized a similar strategy to develop an optimized photoactivatable CRISPR gene activation system, but settled on a different optimal fusion protein combination. Shown in the figure (B), the optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment of CIB1 and it was fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in HEK293T cells to induce expression of human IL1RN, the researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system was also shown to be reversible and repeatable when blue light (450nm) was cycled on-off-on. Using a slit photomask, the researchers also demonstrated the ability to spatially control gene expression.

Photoactivatable Genome Modifications by NHEJ and HDR

Later in 2015, the Sato lab unveiled a photoactivatable system to cleave a target DNA sequence (Nihongaki Y, et al., Nature Biotechnology, 2015 Jul; 33(7):755-60) resulting in a double strand break (DSB) that can be repaired by either non-homologous end joining (NHEJ) or homology directed repair (HDR). This system is unique in that it utilizes a “split” nuclease - the authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714) halves, rendering the Cas9 non-functional when split but regaining functionality when the halves are reassociated. By fusing a photoinducible, heterodimerizing domain to each of the Cas9 fragments, the authors created a photoactive Cas9 tool, as shown in the figure (C). Although the authors tried a few different photoactivatable designs (some similar to those used in the previous Nihongaki et al. system) their most successful design utilized Magnet photoswitchable proteins derived from the fungal photoreceptor, Vivid (VVD, N. crassa) (Kawano F, et al., Nature Communications, 2015 Feb 24; 6:6256). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and nMagHigh1-C714, this new system had both low background and high fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same PAM and had similar targeting specificity as full-length Cas9 (flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%) and induced modifications by HDR (frequency of 7.2%).

The authors additionally showed that they could lower the background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system’s efficiency at generating mutations when activated with light and lowered background DSBs (non detectable). Like their prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly activated by turning blue light on and off.

As one might expect from the modular nature of Cas9, Nihongaki et al. showed that it was possible to swap out the Cas9 domains in their split fusions and generate a photoactivatable nickase and a photoactivatable repressor (dCas9). The activity of all variants was reversible and repeatable.

Using Chemistry to Photocage CRISPRs

The aforementioned techniques each employed a photoactive strategy which had been engineered from naturally occurring photoactive proteins (i.e. CRY2 and Vivid) - Alexander Deiters’ lab, on the other hand, took a different approach. These researchers used a genetically encoded photocaging technique to insert a light-removable protecting group, specifically a nitrobenzyl photocaged lysine (PCK), on the Cas9 protein (Hemphill J, et al., JACS, 2015 May 6; 137(17):5642-5). In order to insert the PCK into a specific site on the Cas9, the group used an engineered pyrrolysyl tRNA/tRNA synthetase pair which would add the PCK upon reaching the amber stop codon, TAG. (To learn more about site-specific incorporation of amino acids using pyrrolysl tRNA synthetase, read this article).

The group first tested photocaging various lysines in Cas9 to determine which best deactivated the protein’s ability to cleave targeted DNA, settling on photocaging the K866 lysine, as seen in the figure below (D). Next, by using a dual reporter fluorescence assay, Hemphill et al. demonstrated that the Cas9 K866PCK mutant was indeed inactive prior to irradiation with UV light (365nm) and that post-UV exposure it showed cleavage activity similar to the wild-type Cas9. This photocaging technique was also shown to impart spatial control of Cas9 cleavage when using a photomasking technique. Last, Hemphill et al. presented data showing that this genetically encoded, photocaged Cas9 system could silence endogenous gene expression - demonstrating light-induced silencing of transferrin receptor CD71 in HeLa cells.  

photoactive-crispr-optogenetics-schematic-final.png

Comparison of Photoactivatable CRISPR Strategies

CRISPR-optogenetics_table-075566-edited.png

Whether you are looking to activate, repress, or modify a gene, you now have the tools at your disposal to control your genome editing using light. We look forward to more tools as CRISPR and optogenetics continue to evolve and can’t wait to see what cool applications you use these for in the future! 


References

1. CRISPR-Cas9-based Photoactivatable Transcription System. Yuta Nihongaki, Shun Yamamoto, Fuun Kawano, Hideyuki Suzuki, Moritoshi Sato. Chemistry & Biology. 19 February 2015; 22(2):169–174. PubMed PMID: 25619936.

2. A light-inducible CRISPR-​Cas9 system for control of endogenous gene activation. Lauren R Polstein & Charles A Gersbach, Nature Chemical Biology. 2015; 11:198–200. PubMed PMID: 25619936.

3. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Yuta Nihongaki, Fuun Kawano, Takahiro Nakajima, Moritoshi Sato. Nature Biotechnology. 2015; 33:755–760. PubMed PMID: 26076431.

4. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Fuun Kawano, Hideyuki Suzuki, Akihiro Furuya, Moritoshi Sato. Nature Communications. 2015 Feb 24; 6:6256. PubMed PMID: 25708714.

5. Optical Control of CRISPR/Cas9 Gene Editing. Hemphill J, Borchardt EK, Brown K, Asokan A, Deiters A. Journal of the American Chemical Society. 2015 May 6; 137(17):5642-5. PubMed PMID: 25905628.

6. Genetically encoded photocontrol of protein localization in mammalian cells. Gautier A, Nguyen DP, Lusic H, An W, Deiters A, Chin JW. Journal of the American Chemical Society. 2010 Mar 31;132(12):4086-8. PubMed PMID: 20218600.

Additional Resources

  • “Optogenetics Meets CRISPR” by Jef Akst on The Scientist
  • “Photoactivatable #CRISPR-Cas9 systems!” by Doug Mortlock on A CRISPR Blog

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Topics: Optogenetics, CRISPR

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