Top 10 Crispiest CRISPR Applications*

*that are NOT Gene Editing

Watch my full CRISPR address at The Oxford Union here. https://youtu.be/QW2TU3p7uyA

There is a heady and hysterical goldrush to CRISPR ALL THE THINGS. And with good reason. These are not your grandpa’s GMOs.

“Second-generation” genome-editing tools can now precisely convert a single base into another without the need for double strand break or incorporating a gene from another organism. At the drop of a “nickase,” C can be converted to T, and A to G, generating a STOP codon and abolishing the need for complex knockout — strategies. (Review CRISPR fundamentals here.)

Like the immeasurable heaven of the Laniakea supercluster, the applications of CRISPR seem to know no bounds. But, the most exciting applications for CRISPR have little to do with gene editing. At the rate of CRISPR publications (1000s per year), you may forgive yourself for not being able to stay up on the literature.

I have compiled some of my favorite (for about a minute) CRISPR applications. The breathless future of CRISPR means these will likely be overturned faster than an ubiquitinated protein.

1. Gene “Scaring” for Massively Parallel Whole Organism or Single Cell Lineage Tracing- In the absence of a template for homologous repair, Cas9 produces short insertions or deletions (indels) at its target sites, which are variable in their length and position. This generates hundreds of scars- essentially “barcoding” each cell (either in a transgenic reporter line or whole organism) in order to simultaneously trace the lineage of and profile the transcriptome of thousands of single cells. Massively parallel profiling strategies are being adopted across the board for large-scale functional screenings.

2. Fluorescent Gene Tagging — Fluorescent markers suitable for live-cell imaging are incorporated into endogenous genes. This new method is easier, faster and cheaper than traditional FP tagging.

3. RNA Editing for Programmable A to I Replacement- New ‘REPAIR’ system edits RNA, rather than DNA; and has potential to treat diseases without permanently affecting the genome. REPAIR has the ability to target individual RNA letters, or nucleosides, switching adenosines to inosines (read as guanosines by the cell). These letters are involved in single-base changes known to regularly cause disease in humans. In human disease, a mutation from G to A is extremely common; these alterations have been implicated in, for example, cases of focal epilepsy, Duchenne muscular dystrophy, and Parkinson’s disease.

4. RNA Scissors to Regulate Translation- The discovery of another component of the CRISPR system, a special pair of RNA scissors- allows for us to now regulate protein translation too. Just announced in February of 2018- I am speculating that this will quickly become a multi-functional tool.

5. Light-Inducible ON/Off Toggle Switch — 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.

6. CRISPR interference (CRISPRi) and CRISPR Activation (CRISPRa)- In CRISPRi, a steric block halts transcript elongation by RNA polymerase, resulting in the knockdown or repression of the target gene. CRISPRa technology allows for overexpression of genes through recruitment of transcription activators for targeted gene activation in their endogenous context and is applicable to both coding and noncoding genes.

7. Changing the Epigenetic Signature — Epigentics is itself a new science. There are multiple layers of epigenetic regulatory mechanisms operating in the genome. Among the well-described ones are DNA methylation, histone posttranslational modifications, and non-coding RNAs (short and long). Guiding Cas9 to a target sequence with a –methylase or –acetylase can increase or reduce DNA and histone methylation, and acetylation on lysine tails.

8. Live–Cell Chromatin Imaging- The organization of chromatin in 3D space plays a critical role in regulating gene expression. Multi-colored tracking of native (ie. unfixed live cell) chromatin loci has started to illuminate the positioning of transcriptionally active and inactive regions of chromatin in the 3D nuclear space throughout the cell cycle.

9. Synthetic Chromatin Biology: Do you remember the scene in the movie Contact when S.R. Hadden describes how the registration marks line the images up in three-dimensional space reveal the primer? That is like a chromatin loop. Chromatin loop-structures bring distant pieces of DNA (enhancers and promoters) close to regulate gene expression. These loops can be directly engineered with CRISPR to either promote or inhibit the enhancer — promoter activity. Synthetic DNA regulation systems of unprecedented sophistication can be created with by combining spatiotemporal layers of chromatin regulation.

10. Gene Drives- Simply put, a gene drive gives a phenotype an advantage, a “drive” that allows the gene to be passed on to more than 50% of its offspring, as in conventional reproduction. In several examples with mosquitos researchers were able to push a gene that inhibits the transmission of malaria to more than 95% of the progeny.

These are my current top ten but, new and intriguing developments are already on the horizon. For example, the “docking” sites for CRISPR are genome segments that have at one end a specific three-base trio: N, where N is any of DNA’s four bases, followed by two guanines (Gs). Only about one-sixteenth of the 3.2-billion-base human genome has the right sequence. In February of 2018, a newly engineered xCas9 promises to release the PAM restrictions and allow access to 4X more genomic docking sites with greater precision and less off target effects.

References

Introduction

· Kuscu, C. et al. CRISPR-STOP: Gene silencing through base editing-induced nonsense mutations. Nat. Methods14, 710–712 (2017).

1. Genome “Scar”

· Massively parallel whole-organism lineage tracing using CRISPR/Cas9 induced genetic scars. Jan Philipp Junker, Bastiaan Spanjaard, Josi Peterson-Maduro, Anna Alemany, Bo Hu, Maria Florescu, Alexander van Oudenaarden doi: https://doi.org/10.1101/056499

· Massively parallel single cell lineage tracing using CRISPR/Cas9 induced genetic scars. Bastiaan Spanjaard, Bo Hu, Nina Mitic, View ORCID ProfileJan Philipp Junker doi: https://doi.org/10.1101/205971 Nature Biotechnology doi: 10.1038/nbt.4124

2. Fluorescent protein tagging

· A generic strategy for CRISPR-Cas9-mediated gene tagging Daniel H. Lackner, Alexia Carré, Paloma M. Guzzardo, Carina Banning, Ramu Mangena, Tom Henley, Sarah Oberndorfer, Bianca V. Gapp, Sebastian M.B. Nijman, Thijn R. Brummelkamp & Tilmann Bürckstümmer Nature Communications volume 6, Article number: 10237 (2015) doi:10.1038/ncomms10237

3. RNA Nuceloside Editing

· Cox DBT, Gootenberg JS, Abudayyeh OO, et al. RNA editing with CRISPR-Cas13. Science. Online October 25, 2017. DOI: 10.1126/science.aaq0180

4. RNA Scissors

· Juliane Behler et al. The host-encoded RNase E endonuclease as the crRNA maturation enzyme in a CRISPR–Cas subtype III-Bv system, Nature Microbiology (2018). DOI: 10.1038/s41564–017–0103–5

5. Optogenic Toggle

· CRISPR-Cas9-based photoactivatable transcription system. Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato MChem Biol. 2015 Feb 19; 22(2):169–74.

· A light-inducible CRISPR/Cas9 system for control of endogenous gene activation. Lauren R. Polstein and Charles A. Gersbach

6. CRISPRi and CRISPRa

· CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS Cell. 2013 Jul 18; 154(2):442–51.

· L. A. Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 159, 647–661 (2014).

· S. Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 517, 583–588 (2015).

7. Epigenetic Regulation

· Editing DNA Methylation in the Mammalian Genome. Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, Young RA, Jaenisch R Cell. 2016 Sep 22; 167(1):233–247.e17.

· Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Hilton IB, D’Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA Nat Biotechnol. 2015 May; 33(5):510–7.

· Rapid and reversible epigenome editing by endogenous chromatin regulators. Braun SMG, Kirkland JG, Chory EJ, Husmann D, Calarco JP, Crabtree GR Nat Commun. 2017 Sep 15; 8(1):560.

8. Live Cell Chromatin Imaging

· Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B Cell. 2013 Dec 19; 155(7):1479–91.

· Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9. Qin P, Parlak M, Kuscu C, Bandaria J, Mir M, Szlachta K, Singh R, Darzacq X, Yildiz A, Adli M Nat Commun. 2017 Mar 14; 8():14725.

9. Chromatin looping

· Programmable DNA looping using engineered bivalent dCas9 complexes. Hao N, Shearwin KE, Dodd IB Nat Commun. 2017 Nov 20; 8(1):1628.

· Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Stefanie L. Morgan, Natasha C. Mariano, Abel Bermudez, Nicole L. Arruda, Fangting Wu, Yunhai Luo, Gautam Shankar, Lin Jia, Huiling Chen, Ji-Fan Hu, Andrew R. Hoffman, Chiao-Chain Huang, Sharon J. Pitteri & Kevin C. Wang. Nature Communications volume 8, Article number: 15993 (2017) doi:10.1038/ncomms15993

10. Gene Drives

· Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Gantz VM, Jasinskiene N, Tatarenkova O, Fazekas A, Macias VM, Bier E, James AA

· A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C, Katsanos D, Gribble M, Baker D, Marois E, Russell S, Burt A, Windbichler N, Crisanti A, Nolan T Nat Biotechnol. 2016 Jan; 34(1):78–83.

Conclusion

· Evolved Cas9 variants with broad PAM compatibility and high DNA specificity Johnny H. Hu, Shannon M. Miller, Maarten H. Geurts, Weixin Tang, Liwei Chen, Ning Sun, Christina M. Zeina, Xue Gao, Holly A. Rees, Zhi Lin & David R. Liu Nature volume 556, pages 57–63 (05 April 2018) doi:10.1038/nature26155