A Brief Overview of the CRISPR-Cas System: It S History, Mechanism, and Applications

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A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications

A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications

Tatiana H. Riordan

Math 89s Duke University

November 1st, 2016

Introduction

The CRISPR-Cas system is a well-known groundbreaking form of gene editing technology, but the history, mechanism, and application of it is rarely well understood. In this paper I provide a general explanation of the CRISPR-Cas system and its importance.

The CRISPR-Cas system is a prokaryotic immune system that provides a form of acquired immunity through recognizing and cutting exogenous[JF1][TR2][TR3] genetic elements and inserting them into its own genetic code for future resistance. It is famously known for being the most powerful gene editing technology of our time.

History

The earliest discovery of the CRISPR system was in 1897, when Yoshizumi Ishino, a researcher at the University of Osaka, cloned and sequenced the iap gene in E coli. While reading the strand of DNA, Ishino found a set of 29-nucleotide repeats separated by short sequences, all different and unrelatedfrom one another, known as spacers (Marraffini, Luciano A., and Erik J. Sontheimer). [JF4]As repeated sequences in bacterial genomes, especially with interrupted spacers, are is very unusual, Ishino realized that there must be a purpose for this. In 2002, Jansen et al. named this system ‘Clustered Regularly Interspaced Short Palindromic Repeats’, more commonly referred to as ‘CRISPR’ (Mojica, Francisco J. et.al). Various CRISPR’s were documented incountless genomes of bacteria, demonstrating that CIRSPR is a universal feature of prokaryotes (Mojica, Francisco J,.et.al)), . Janson et al. also observed that CRISPR was accompanied in the prokaryotic genomes by a set of homologous genes, cas genes, or CRISPR associated genes. Four cas genes (cas 1-4) were recognized and were consistently located adjacent to a CRISPR locus, and showed patterns representative for helicases[JF5] and exonucleases, implying that these genes are involved in DNA metabolism or gene expression. These findings suggest a functional relationship between the cas genes and CRISPR loci (Mojica, Francisco J. et.al).

However, the CRISPR-Cas function remained unknown until 2004 when a key supplement to the understanding of CRISPR was found. Now that scientists haved the technology to sort earch (sift/sort?) through large DNA sequence databases, they began tothey canbegan to line up stretches of DNA with thousands of different species. Through this process they discovered that theCRISPR loci in the bacteria matched virus DNA. Essentially, they found out that, somehow, the spacers are pieces of DNA gathered from viruses that previously tried to attack the bacteria cell (“Antibodies Part 1: Crispr”). The discovery of the spacers’ source revealed that the CRISPR-Cas system could play a role in adaptive immunity in bacteria.

Within the next half decade, multiple experiments provided experimental evidence that CRISPR is an adaptive immune system and that CRISPR and the cas system cuts both phages and plasmid DNA (Hsu, Patrick D., Eric S. Lander, and Feng Zhang, Garneau, Josiane E.,et.al).

In 2012, a simplerCRISPR system that relies on the protein Cas 9 was discovered. The Cas9 endonuclease consists of a four-component system than includes two RNA molecules (Barrangou, Rodolphe);. (Zetsche, Bernd et.al). The importance of the CRISPR-Cas9 will be discussed in the application section of the paper.

Mechanism of CRISPR/Cas systems

Prokaryotic viruses (bacteriophages or phages) are one of the most plentiful organisms on earth, and a major threat to bacteria. CRISPR-Cas immunity is a process of bacteria and archaea to prevent bacteriophage infection by providing quick and forceful adaptation to the rapidly evolving viruses of bacteria and archaea. The acquisition and utilization of spacer sequences constitute the two main stages of CRISPR immunity (Marraffini, Luciano A). Without the CRISPR system, when a bacteriophage injects its DNA into a microbe, the bacteriophage would capture the cell, producing countless more bacteriophages until it eventually kills the cell.

Adaption/Space Acquisition Stage. When a microorganism is invaded by a virus, the first stage, known as ‘adaptation’ or ‘space acquisition’ beings. The viral genome is taken and placedinto a CRISPR locus in the form of a spacer, as seen in figure 1. Although the actual mechanism to produce crRNAs vary among CRISPR-Cas system, all Cas proteins [JF6]transcribe spacers through some [JF7]process to produce CRISPR RNA (crRNA) (Brouns, S.J., M. M. Lundgren, et.al). The crRNA directs the cell division of the virus by Cas nucleases, an enzyme that cleaves nucleic acids (Brouns, S. J., et.al).This allows the cell to adapt rapidly to the invaders in close proximity of the environment, which is why this phase of the CRISPR complex is referred to as the ‘adaption’ phase. The information collected in the spacers is used to resist invaders in the next phase of CRISPR interference, the ‘defense’ stage (Marraffini, Luciano A., and Erik J. Sontheimer). Immunity through spacer acquisition allows for a population to acquire rapid resistance to its predators and pass this resistance mechanism off to its offspring (Nuñez, James K.et.al).

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Figure 1. Image of CRISPR-Cas Spacer

Defense/ crRNA Biogenesis Stage. In the defense stage, also known as the ‘crRNA biogenesis’ sage, the CRISPR complex must process the information transcribed in the adaption phase and use it to direct the interference machinery to invasive targets. Once transcribed, the crRNA serves as a guide for the recognition of the invasive target through tits base-pairing potential[JF8], as seen in figure 2.

Interference Stage. The third stage, ‘interference’, is the phase in which the invading virus is destroyed. Although there are varying systems of interference depending on the life form, a PAM sequence, protospacer-adjacent motif[JF9], f (PAM) sequence, is fundamental forto avoiding auto-immunity because it is a component of the invading virus or plasmid and not of the bacterial CRISPR locus. Evidence shows that even a single spacer/target mismatch compromises CRISPR interference (Marraffini, Luciano A). The correct base pairing between the crRNA and the PAM indicates a conformational change in Cascade that employs a protein, Cas3 or Cas9, depending on the system,for DNA decomposition (Wang, Jiuyu., et.al). Detailing the mechanism of CRISPR immunity is difficult because of the exceptional diversity of CRISPRs and its associated genes; different Cas proteins participate in crRNA biogenesis and target recognition in different system. The process described above is a general mechanism that can be applied to CRISPR systems.

Figure 2. Process of CRISPR-Cas gene editing technology

Application

The CRISPR-Cas9 system allows for cheap, precise, and possibly universal technology for gene editing technology. Jennifer Doudna, a biochemist at the University of California Berkeley, saw the CRISPR-Cas9 system and thought of the idea of turning this gene editing defense system into an and offense [JF10]system. Because the CRISPR-Cas9 system is extremelyso precise at cutting genes, Doudna had the idea of replacing the target gene, usually a bacteriophage or a phage, with a different gene such as genes that have Huntington’s disease of hemophilia. In order to replace the removed gene, Doudna states that placing a replacement gene in the proximity of the cut area is enough for repair enzymes to recognize the break, see the good gene, and insert it into the break.

Research by many scientists have shown that when changing the target gene and inserting the new CRISPR-Cas9 system into a mouse, it will find that specific gene and cut it out("Genome Editing: Efficient CRISPR Experiments in Mouse Cells”).

This experiment has not only proved to be successful in mice, but in all species tested; as of now, scientists have not found a single life form that this exact system doesn’t work on[JF11]; n. Eessentially, as of what current research shows,we can take these CRISPR-Cas9 systems out of bacteria and place them into anything, from corn that is vulnerable to a certain pesticide to a human with cancer cells.

Although we have been experimenting with genetic engineering foryears, we have never had technology as powerful as this. Previous gene editing technology costed around $5,000, would take six months to do, and was not precise at all. The CRISPR-Cas9 system costs around $75, takes a couple of weeks, and is extremely accurate (“Antibodies Part 1: CRISPR”). Currently, the CRISPR-Cas9 system has been used to control transcription, modify epigenomes, conduct genome-wide screens and imaging chromosomes. Two clinical trials for targeting cancer therapies using CRIPSR-Cas9 have been approved in the United States and China (Barrangou, Rodolphe, and Jennifer A. Doudna).

The main controversy surrounding the CRISPR-Cas9 gene editing technology is how far this technology shouldcan be taken. Testing on human embryos was the feared yet widely expected result of the CRISPR-Cas9 findings. In 2015, Chinese researchers used defective human embryos, tripronuclear zygotes, to investigate CRISPR-Cas9 mediated gene editing in human cells. They found that the CRISPR-cas9 system could split the an endogenousβ-globin [JF12]gene, but the efficiency of the directed repair was low and the embryos were mosaic[JF13]. Essentially, none of the 85 human embryos they injected satisfied their goals. In almost every case, the embryo either died or the DNA was not altered. Collateral damage was also evident – DNA mutationwas caused by the editing attempt. They concluded that there is a “pressing [JF14]need to further improve the fidelity and specificity of the CRISPR-Cas9 platform, a prerequisite for any clinical applications of CRISPR-Cas9-mediated editing” (Liang, Puping, Yanwen Xy, et.al).

Some researchers worry that research done by the Chinese scientists ithis [JF15]paper is just the beginning and that more attempts will be made with complete alternation of babies as the end goal. The two main issue with this potential outcome is the lack of information about the consequences of editing human genes, as well as the general ethical issue of altering a humans being. Rudolf Jaenisch, biology professor at M.I.T., asks why anyone would want to edit the genes of human embryos to prevent disease. Because of the way genes are distributed in embryos, when one parent has a gene with a genetic disorder, only half of the parent’s embryos will inherit it. If gene editing is performed, half of the embryos would be altered for no reason.

Conclusion

CRISPR-Cas gene editing is an incredible mechanism with lots of potential application. Due to its inexpensive, quick, and precise qualities its use could be incredibly beneficial in altering humans, animals, and food crops. However, due to the lack of understanding surrounding its consequences and bioethical concerns, further research must be done before this technology can be used for clinical use. Through my research for this paper, I found the CRISPR-Cas technology fascinating and potentially a major breakthrough in both the medical and the agricultural industry. I hope to see the CRISPR-Cas system used to improve crops and save lives in the future.

Works Cited

Barrangou, Rodolphe, and Jennifer A. Doudna. "Applications of CRISPR Technologies in Research and beyond."Nature Biotechnology(2016): 933-41. Web.

Barrangou, Rodolphe. "Diversity of CRISPR-Cas Immune Systems and Molecular Machines."Genome Biology. BioMed Central, 2015. Web. 01 Nov. 2016.

Brouns, S. J., M. M. Jore, M. Lundgren, E. R. Westra, R. J. Slijkhuis, A. P. Snijders, M. J. Dickman, K. S. Makarova, E. V. Koonin, and J. Can Der Oost. "Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes."PubMed(2008): n. pag.NCBI. Web.

By Giving Anyone an Advantage Prior to Entering That Struggle, You Are Reducing Their Need to Persevere Which Will Affect the Outcome of Their DNA as They Age. Our Genetic Composition Is Simply a Starting Point, It's Experience That Determines Who We Beco. "Antibodies Part 1: CRISPR."Radiolab Podcast Articles. Radio Lab, n.d. Web. 01 Nov. 2016.

Garneau, Josiane E., Marie-Ève Dupius, Manuela Villon, Dennis A. Romero, Rodolphe Barrangou, Patrick Boyaval, Chritophe Fremaux, Philippe Horvath, Alfonso H. Magadán, and Sylvian Moineau. "The CRISPR/Cas Bacterial Immune System Cleaves Bacteriophage and Plasmid DNA."Nature. 2010 Macmillan Pubishers, 19 Apr. 2010. Web.

"Genome Editing: Efficient CRISPR Experiments in Mouse Cells."Genome Editing: Efficient CRISPR Experiments in Mouse Cells. PHYSORG, 25 Oct. 2016. Web. 01 Nov. 2016.

Hsu, Patrick D., Eric S. Lander, and Feng Zhang. "Development and Applications of CRISPR-Cas9 for Genome Engineering."Cell. U.S. National Library of Medicine, 05 June 2014. Web. 01 Nov. 2016.

Liang, Puping, Yanwen Xy, Xiya Zhang, Chenhui Ding, Rui Huang, Zhen Zhang, Jie Lv, Xiaowei Xie, Yuxi Chen, Yujing Li, Ying Sun, Yaofu Bai, Zhou Songyang, Wenbin Ma, Conquan Zhou, and Junjiu Huang. "CRISPR/Cas9-mediated Gene Editing in Human Tripronuclear Zygotes."SpringerLink. 2016 Springer International Publishing, 18 Apr. 2015. Web. 01 Nov. 2016.

Marraffini, Luciano A., and Erik J. Sontheimer. "CRISPR Interference: RNA-directed Adaptive Immunity in Bacteria and Archaea."Nature Reviews Genetics11.3 (2010): 181-90. Web.

Marraffini, Luciano A., and Erik J. Sontheimer. "CRISPR Interference: RNA-directed Adaptive Immunity in Bacteria and Archaea."Nature Reviews. Genetics. U.S. National Library of Medicine, Mar. 2010. Web. 01 Nov. 2016.

Marraffini, Luciano A. "CRISPR-Cas Immunity in Prokaryotes."Nature526.7571 (2015): 55-61. Web.

Mojica, Francisco J., Cesar Díez-Villaseñor, Elena Soria, and Guadalupe Juez. "Biological Significance of a Family of Regularly Spaced Repeats in the Genomes of Archaea, Bacteria and Mitochondria."Wiley Online Library. John Wiley & Sons, Inc., n.d. Web.

Nuñez, James K., Philip J. Kranzusch, Jonas Noeske, Addison V. Wright, Christopher W. Davies, and Jennifer A. Doudna. "Cas1–Cas2 Complex Formation Mediates Spacer Acquisition during CRISPR–Cas Adaptive Immunity."Nature Structural & Molecular Biology. U.S. National Library of Medicine, June 2014. Web. 01 åNov. 2016.

Wang, Jiuyu, Jiazhi Li, Hongtu Zhao, Gang Sheng, Min Wang, Maolu Yin, and Yanli Wang. "Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems."Cell163.4 (2015): 840-53. Web.

Zetsche, Bernd, JonathanÂS. Gootenberg, OmarÂO. Abudayyeh, IanÂM. Slaymaker, KiraÂS. Makarova, Patrick Essletzbichler, SaraÂE. Volz, Julia Joung, John VanÂDerÂOost, Aviv Regev, EugeneÂV. Koonin, and Feng Zhang. "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System."Cell163.3 (2015): 759-71. Web.

[JF1]Consider foot note?

[TR2]

[TR3]

[JF4]Consider re reading this sentence. Run on? Maybe say like and he named it a spacer or something

[JF5]Consider footnote

[JF6]You say although but ur not comparing it to anything. Ilek anough they are usually difference the norm is.. or something u know

[JF7]This kinda makes it sound like u don’t understand ir

[JF8]No clue what that is haha

[JF9]I think u should write protospacer-adjacent motif (PAM)

[JF10]Maybe delete the and. Not sure what ur trying to say heeh

[JF11]This experiment only is successful in mice, but also in every single life form that his experiment has been tested on..?

[JF12]Foot note?

[JF13]Possible footnote

[JF14]U never ended the quoation

[JF15]What paper