Download THR_Paper2_CRISPR

A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
Tatiana H. Riordan
Math 89s Duke University
November 1st, 2016
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
2
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 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 unrelated from one another, known as
spacers (Marraffini, Luciano A., and Erik J. Sontheimer). As repeated sequences in
bacterial genomes, especially with interrupted spacers, are 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 in
countless 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
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
3
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 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 have the
technology to sort through large DNA sequence databases, they began to line up
stretches of DNA with thousands of different species. Through this process they
discovered that the CRISPR 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 simpler CRISPR 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.
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
4
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 placed into 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 transcribe spacers 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 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).
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
5
.
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 its base-pairing potential, 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 protospacer-adjacent motif (PAM) sequence, is fundamental for 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
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
6
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. 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 offense system. Because the CRISPR-Cas9
system is extremely 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.
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
7
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. Essentially, 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 for years, 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 CRISPRCas9 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 should 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
endogenous β-globin gene, but the efficiency of the directed repair was low and the
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
8
embryos were mosaic. 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 mutation was caused by the editing attempt.
They concluded that there is a “pressing 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 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
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
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.
9
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
10
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
A Brief Overview of the CRISPR-Cas System: It’s History, Mechanism, and Applications
11
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 Genetics 11.3 (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." Nature 526.7571 (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." Cell 163.4 (2015): 840-53. Web.
Zetsche, Bernd, Jonathan S. Gootenberg, Omar O. Abudayyeh, Ian M. Slaymaker, Kira S.
Makarova, Patrick Essletzbichler, Sara E. Volz, Julia Joung, John Van Der Oost, Aviv
Regev, Eugene V. Koonin, and Feng Zhang. "Cpf1 Is a Single RNA-Guided Endonuclease of a
Class 2 CRISPR-Cas System." Cell 163.3 (2015): 759-71. Web.