What is CRISPR and why is it a revolutionary tool?

We are witnessing one of the most fascinating discoveries in science as it unfolds.  In 2012, a landmark paper was published by Doudna and colleagues,1 launching a then little-known terminology called CRISPR into the scientific space and propelling a revolution that could very well still be in its infancy. As scientists understand its enormous potential and attempt to overcome its limitations, CRISPR has since become a "lab-hold" name. Since the demonstration of how CRISPR can be used as a gene-editing tool in 2012, there has been a 15-fold increase in the number of publications involving this tool to date (PubMed). While the potential of CRISPR in gene editing has been shown in a variety of applications, the technique has also stirred up controversy. Nevertheless, the progress attained so far using CRISPR and the promise of it being used as a standard genetic engineering tool for gene therapy is simply undeniable.  In this article, we’ll take you through the exciting CRISPR journey to see what this genetic tool is capable of achieving, the variety of applications it has had, its reach and penetrance, and review the benefits and ramifications of CRISPR research. CRISPR as a gene-editing tool is used in several applications and Roche products have been successfully integrated into some of them, such as human embryonic stem cell research and single-cell molecular screening. Roche products have also been useful in the identification of genome-wide off target cleavage sites of CRISPR/Cas9 using CIRCLE-Seq. We’ll also discuss some of these applications.

What is CRISPR and what makes it unique?

The acronym CRISPR expands into a mouthful: clustered regularly interspaced short palindromic repeats. It was first identified as an interesting mix of repeat and non-repeat elements of unknown significance in Escherichia coli 2 and didn’t garner much interest. However, years later, the presence of these genetic elements was  recognized as a defense mechanism against invading viruses in Streptococcus thermophilus strains.3 Just like how humans elicit an immune defense against antigens using immunological memory, bacteria hoist a defense response against invading phages by storing portions of the genome of the virus (called spacers) that attacked them earlier. The spacers are interspersed among a small cluster of palindromic repeats (CRISPR). As new viruses attack the bacteria, their genomes also get incorporated among the clustered repeats forming variations of spacers. This integration marks the first phase, the adaptation phase, in bacterial defense against viruses. 

While a few variations of CRISPR defense mechanism have been identified for the subsequent phases, the simplistic and the most commonly used one is the Type II system. In simple terms, in the next phase, the RNA biogenesis phase, the CRISPR RNA (crRNA) transcribed from spacers pair with small trans-activating RNA (called TracrRNA) that is complementary to the repeat sequence and form a dual RNA complex. This pairing recruits specific CRISPR-associated proteins (Cas proteins), which are endonucleases, and triggers the formation of effector complexes comprising the dual RNA complex and Cas proteins.

During subsequent viral infections, the effector complex recognizes short motifs near the target sequence (called protospacer adjacent motifs, PAMs) in the viral (target) genetic material and binds with sequences complementary to crRNA, initiating the Cas protein then to cleave the viral genome by causing a double-stranded break (DSB). The DBS caused by the exonuclease in this final interference phase can then be repaired by the cell with its natural repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways.4

How did CRISPR democratize gene editing?

From simple molecular scissors such as restriction endonucleases to sophisticated programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), several genetic engineering tools have existed before CRISPR came into the molecular biologist’s portmanteau. Through the integration of DNA-binding domains and a non-specific endonuclease, ZFNs and TALENs are used to manipulate specific target sites. While they have been successful in advancing gene therapy efforts, they are both complicated and require adequate protein engineering expertise , especially in terms of designing these nucleases for recognizing new sequences. Because of its dependence on RNA instead of proteins (with their innate complexity), CRISPR seemed promising and attainable. 

But, does this gene-editing mechanism work only in bacteria or can it be manipulated to edit any genome? The scientific curiosity behind this question was exactly what caused the CRISPR revolution.  The simple RNA-guided DNA base pairing that induces cleavage suggested that any crRNA binding with its complementary DNA should be able to induce a DBS by Cas protein in any given genome. Jinek et al. in 2012 in their seminal paper1 showed that crRNA and TracrRNA could be fused into a chimeric single guide RNA, which could be engineered to cleave GFP DNA. This ability demonstrated that in principle, a guide RNA (gRNA) could be designed to cleave any genome and manipulate it, thus opening up the field of genetic engineering and raising it to its new glory. Now genes could be deleted, inserted or otherwise modified with ease. 

Unlike ZFNs and TALENs, CRISPR system was simple, easy to engineer specific binding and also cost effective. From bacteria to yeast to plants to rodents to humans, gene editing using the CRISPR/Cas system has been successfully demonstrated.5 Proof-of-concept experiments have been carried out for preventing conditions such as Duchenne Muscular Dystrophy in mice,6 suggesting the prospects for using CRISPR/Cas for therapeutic purposes.  Genome-wide CRISPR libraries have been constructed for drug screening, the use of CRISPR in cancer therapy is being explored widely 7 and clinical trials have been initiated for using CRISPR/Cas genome editing in humans.8 CRISPR/Cas9 has demonstrated high-fidelity gene editing in human embryonic stem cells9 and  CRISPR-based single-cell molecular screens are being developed.10 On a larger scale, CRISPR/Cas9 dropout screening for systematically investigating genes associated with lethal phenotypes has been explored.11 These, along with the numerous next-generation research and clinical applications of CRISPR technology that applies or have the potential to apply CRISPR,12 indicate the strong likelihood of CRISPR being used as a standard procedure for correcting genetic diseases and improving cell therapies. 

Limitations and ramifications of CRISPR research

The specificity of CRISPR/Cas9 system is afforded by the ability of the effector complex to accurately recognize the cleavage sites in the target genome and inducing the break specifically at those sites. However, mismatches encountered before the PAM sequence have triggered breaks at unintended target sites.13 Repair by NHEJ is also error prone and could introduce insertions, deletions and frame-shift mutations. The off-target effects of such unintended gene editing have caused wide-spread concern and efforts to identify and rectify them are underway.13 Novel techniques for identifying genome-wide off-target cleavage sites of CRISPR/Cas9 such as CIRCLE-Seq are being explored.14 Ethics and standards for the use of this technique are still being considered and established in scientific community around the world, especially for using CRISPR in germline editing.15

The simplicity and robustness of CRISPR has transformed our ability to manipulate genomes and stretched its application from basic science to translational research and medicine within a historically short time. With solutions being worked out with respect to correcting off-target effects and safety measures being put in place, the limits to which CRISPR technology can be used in rectifying genetic diseases and enhancing existing therapies is beyond imagination. 

Given the power of gene editing using the CRISPR/Cas9 system and its potential to alter humankind in ways not feasible until now, it is imperative to ensure that every step of the process is carried out with utmost care. 

  1. Jinek M, Chylinski K, et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012.

  2.  Ishino Y, Shinagawa H, Makino K. et al. Nucleotide sequence of the iap gene responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 1987. 169: 5429–5433.

  3. Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007. 80; 315: 1709–1712.

  4. Loureiro A and da Silva GJ. CRISPR-Cas: converting a bacterial defense mechanism into a state-of-the-art genetic manipulation tool. Antibiotics. 2019. 8,18: doi:10.3390/antibiotics8010018

  5. Lin S, Staahl B, Alla R, et al. Enhanced homology-directed human genome engineered by controlled timing of CRISPR/Cas9 delivery. Elife,2014. 

  6. Long C, McAnally JR, Shelton JM et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014. 345:1184-1188.

  7. Martinez-Lage M, Puig-Serra P, Menendez P et al. CRISR/Cs9 for cancer therapy: hopes and challeges. Biomedicines. 2018. 6;4:105.
  8. Sheridan C. Go-ahead for first in-body CRISPR medicine testing. Nature Biotechnol. December 2018. News article.
  9. Yang L, Mali P, Kim-Kiselak C et al. CRISPR-Cas mediated targeted genome editing in human cells. In: Storici F. (eds) Gene Correction. Methods in Molecular Biology (Methods and Protocols), vol 1114. Humana Press, Totowa, NJ.
  10. Hill AJ, McFaline-Figuero JL, Starita LM et al. On the design of CRISPR-based single-cell molecular screens. Nature Methods. 2018. 15;4:271-274.
  11. Wu K and Malek S. CRISPR/Cas9-based gene dropout screens. Chronic lymphocytic leukemia. Methods and protocols. Malek SN (ed). Methods in Molecular Biology 1881; Springer Protocols. Chapter 15.
  12. Pickar-Oliver A and Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nature Reviews. Mol Cell Biol. 2019. s41580-019-0131-5
  13. Stenberg S and Doudna JA. Expanding the biologist’s toolkit with CRISPR/Cas9. Molecular Cell. 2015.58:568-574.
  14. Tsai SQ, Nguyen NT, Malagon-Lopez J et al. CIRCLE-Seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off targets. Nature Methods.2017.14:607-614.
  15. Lander R, Baylis F, Zhang F et al. Adopt a moratorium on heritable genome editing. Nature. March 2019. Comment.

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