A3
Paper #6203
Disclaimer — This paper partially fulfills a writing requirement for first year (freshman) engineering students at the
University of Pittsburgh Swanson School of Engineering. This paper is a student, not a professional, paper. This paper
is based on publicly available information and may not be provide complete analyses of all relevant data. If this paper is used
for any purpose other than these authors’ partial fulfillment of a writing requirement for first year (freshman)
engineering students at the University of Pittsburgh Swanson School of Engineering, the user does so at his or
her own risk.
CRISPR/CAS9: A REVOLUATIONARY TOOL FOR GENOME EDITING
Jacob Meadows, jmm367@pitt.edu, Vidic 2:00, Hannah Kendrick, hmk25@pitt.edu, Mahboobin 4:00
Revised Proposal — Since the complete mapping of the
human genome in 2003, advances in gene sequencing
technology have made it significantly easier, quicker, and
less expensive to gather genetic data. Now, with relatively
cheap and simple methods to obtain genetic data, the focus of
researchers shifts to converting this data into functionally
and clinically relevant knowledge. To accomplish this,
researchers must identify dependable and efficient methods to
determine how genotype influences phenotype [1]. Previous
methods
for
determining
this
genotype-phenotype
relationship, such as homologous recombination and
knockdown with RNAi, are powerful tools for evaluating
specific gene functions. Despite this, each has downsides
including low efficiency, laborious selection and screening
requirements, and off-target or only temporary effects [1].
In recent years, a novel genome editing technology, the
CRISPR/Cas9 system, has been developed that surpasses its
predecessors in terms of customizability, efficiency, and
specificity for genome editing [2].
These other genome
editing technologies include zinc-finger nucleases (ZFNs)
and transcription activator-like effectors nucleases (TALENs)
[1].
The CRISPR/Cas9 system, a naturally-occurring
prokaryotic
immune
defense
mechanism
against
bacteriophages, has been engineered to cleave DNA at sitespecific locations based on a guide-RNA. This genome
editing system can be used to insert new DNA sequences into
an organism’s genome and silence specific genes through
inducing double-stranded DNA breaks and initiating errorprone DNA repair mechanisms [2]. CRISPR genome editing
is already a useful tool for basic and translational research,
but recent improvements in the specificity of the Cas9
nuclease by investigators at Harvard Medical School show a
decrease in off-target effects below detectable levels, thereby
opening up greater possibilities for the technology [3].
Current and future applications of CRISPR/Cas9 genome
editing are extensive, but this paper will specifically discuss
general basic research applications and translational
research applications to cancer biology, eradication of
malaria, and gene therapy for muscular dystrophy [4]. With
the recent developments of new high fidelity Cas9 variants,
these applications and many others have great potential to
succeed, as one of the main limitations to CRISPR/Cas9
University of Pittsburgh Swanson School of Engineering 1
Submission Date
genome editing was previously the potential dangers of offtarget effects [3].
This technology is significant to bioengineers because it
makes available a relatively easy, swift, and cost-effective
method for modifying the DNA sequence of human and model
organism cells. In addition, CRISPR was recognized as
Science Magazine’s “Breakthrough of the Year” for 2015
because of its increasing importance as a tool in biology [5].
CRISPR/Cas9 genome editing also directly applies to
medicine, but ethical and technical aspects limit current use
in humans.
This paper will examine the history and development of
CRISPR/Cas9 technology, assess its function and biological
mechanisms, explore its current and future applications, and
consider its ethical and social implications.
REFERENCES
[1] T. Gaj, et al. (2013) “ZFN, TALEN, and CRISPR/Casbased methods for genome engineering.” Trends in
Biotechnology
(online
article).
http://www.sciencedirect.com/science/article/pii/S016777991
3000875
[2] J. Sander, K. Joung, et al. (2014) “CRISPR-Cas systems
for editing, regulating, and targeting genomes.” Nature
Biotechnology
(online
article).
http://www.nature.com/nbt/journal/v32/n4/full/nbt.2842.html
[3] B. Kleinstiver, et al. (2016) “High-fidelity CRISPR–Cas9
nucleases with no detectable genome-wide off-target effects.”
Nature
(online
article).
http://www.nature.com/nature/journal/vaop/ncurrent/full/natu
re16526.html
[4] S. Novella. (2015) “CRISPR and the Ethics of Gene
Editing.” Science Based Medicine (online article).
https://www.sciencebasedmedicine.org/crispr-and-the-ethicsof-gene-editing/
[5] R. Sanders. (2015) “Science magazine names CRISPR
‘Breakthrough of the Year’.” Berkeley News (online news).
http://news.berkeley.edu/2015/12/18/science-magazinenames-crispr-breakthrough-of-the-year/
Jacob Meadows
Hannah Kendrick
(online
article).
http://www.sciencedirect.com/science/article/pii/S016777991
3000875
This article, from a professional peer-reviewed journal
focused on multidisciplinary applied biosciences, outlines the
potential of ZFNs, TALENs, and CRISPR/Cas9
endonucleases to redefine the boundaries of biological
research and influence the development of personalized
medicine. The article details the history and differences
between each genome editing technology and delves into the
technical function and therapeutic applications of genome
editing. Information from this article will help us establish
an understanding of the technical aspects of genome editing
and the recent developments in CRISPR/Cas9 technology.
SOURCES CONSULTED
J. Choi. (2013) “Genome Editing.” Stanford Huntington’s
Outreach
Project
(online
article).
http://web.stanford.edu/group/hopes/cgibin/hopes_test/genome-editing/#what-is-genome-editing
L. Cong, F. Ran, D. Cox, et al. (2013) “Multiplex Genome
Engineering Using CRISPR/Cas9 Systems.” Science (online
article).
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795411/
“How to Choose a Topic” (2015) University of Pittsburgh
Library System (video)
http://www.library.pitt.edu/other/files/il/fresheng/index.html
E. Pak. (2014) “CRISPR: A game-changing genetic
engineering technique.” Harvard Medical School SITN
(online article). http://sitn.hms.harvard.edu/flash/2014/crispra-game-changing-genetic-engineering-technique/
S. Young. (2014) “Genome Surgery.” MIT Technology
Review
(online
article).
http://www.technologyreview.com/review/524451/genomesurgery/
B. Kleinstiver, et al. (2016) “High-fidelity CRISPR–Cas9
nucleases with no detectable genome-wide off-target effects.”
Nature
(online
article).
http://www.nature.com/nature/journal/vaop/ncurrent/full/natu
re16526.html
This article, from an international and interdisciplinary
peer-reviewed journal, unveils the development of a novel
high-fidelity CRISPR-Cas9 nuclease, SpCas9-HF1, which
exhibits no detectable levels of off-target effects. The article
explains why off-target effects from conventional Cas9 are a
major impediment and how they reduced off-target effects
such that it retained robust on-target activity but had
diminished ability to cut incorrect off-target sites.
Information from this article will be used to illustrate the
increasing specificity of CRISPR-Cas technology and explain
why the creation of SpCas9-HF1 is especially promising for
genome editing applications.
ANNOTATED BIBLIOGRAPHY
J. Choi. (2013) “Genome Editing.” Stanford Huntington’s
Outreach
Project
(online
article).
http://web.stanford.edu/group/hopes/cgibin/hopes_test/genome-editing/#what-is-genome-editing
This article, from the Stanford Huntington’s Outreach
Project, defines genome editing, lays out the underlying
technical principals and explains some technologies such as
zinc finger nucleases (ZFNs), transcription activator-like
effector nucleases (TALENs), meganucleases, and
CRISPR/Cas9. Information from this article will be used in
our description of why CRISPR is a more efficient and less
expensive technique of genome editing as well as explaining
how CRISPR is proposed to be implemented therapeutically.
S. Novella. (2015) “CRISPR and the Ethics of Gene Editing.”
Science Based Medicine (online article).
https://www.sciencebasedmedicine.org/crispr-and-theethics-of-gene-editing/
This article, from a website that explores issues and
controversies in science and medicine, discusses the ethical
implications of CRISPR and gene editing as a whole. The
article summarizes the ethical considerations of
CRISPR/Cas9 technology from multiple points of view and
suggests medical applications of CRISPR that may be
ethically controversial. Information from this article will
help initiate a discussion on the ethics of CRISPR/Cas9
technology and how prominent scientists, engineers, and
regulatory organizations advise proceeding with applying this
technology to medicine.
L. Cong, F. Ran, D. Cox, et al. (2013) “Multiplex Genome
Engineering Using CRISPR/Cas9 Systems.” Science (online
article).
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795411/
This article, published in a high-impact peer-reviewed
scientific journal, details how the Zhang group at MIT used
CRISPR/Cas systems with RNA guides to induce precise
cleavage at endogenous genomic loci in human and mouse
cells. This source shows the development of CRISPR/Cas
prokaryotic immunity systems into genome editing tools and
will be used to explain the history of origin of CRISPR/Cas9
technology.
E. Pak. (2014) “CRISPR: A game-changing genetic
engineering technique.” Harvard Medical School SITN
(online article). http://sitn.hms.harvard.edu/flash/2014/crispra-game-changing-genetic-engineering-technique/
This article, from a Harvard Medical School scientific
communication website, summarizes the steps of actually
applying CRISPR/Cas9 technology and explains the role of
T. Gaj, et al. (2013) “ZFN, TALEN, and CRISPR/Cas-based
methods for genome engineering.” Trends in Biotechnology
2
Jacob Meadows
Hannah Kendrick
CRISPR in industry and academia. This article includes
useful visual aids for understanding the function of
CRISPR/Cas9 systems and will aid in our technical
description of the process and the Cas9 enzyme itself, as well
as applications in industry.
J. Sander, K. Joung, et al. (2014) “CRISPR-Cas systems for
editing, regulating, and targeting genomes.” Nature
Biotechnology
(online
article).
http://www.nature.com/nbt/journal/v32/n4/full/nbt.2842.html
This article, from a professional and high-impact peerreviewed journal focused on biological technologies and
methodologies, examines the function and use of the
CRISPR/Cas9 system as a genome editing technology in
detail and explains its significance to biological researchers.
The article includes figures and explanations that detail the
conceptual basis of CRISPR-Cas9 systems and the practical
considerations for implementing CRISPR-Cas technology.
Information from this article will be used to formulate
comprehensive view of CRISPR/Cas9 function in biological
systems and to point out potential pitfalls of the technology.
R. Sanders. (2015) “Science magazine names CRISPR
‘Breakthrough of the Year’.” Berkeley News (online news).
http://news.berkeley.edu/2015/12/18/science-magazinenames-crispr-breakthrough-of-the-year/
This article, from the University of California Berkeley
News website, reports that Science magazine has named
CRISPR the ‘Breakthrough of the Year’ for 2015. The article
explains why CRISPR was so influential in 2015 and how
this it has affected every area of genetics. Information in this
article will be used to reveal the significance of the
CRISPR/Cas9 technology to the general public and to
emphasize how applications of CRISPR are already
pervading into everyday life.
S. Young. (2014) “Genome Surgery.” MIT Technology
Review
(online
article).
http://www.technologyreview.com/review/524451/genomesurgery/
This article, from MIT Technology Review, presents the
pros and cons of CRISPR/Cas9 technology, TALENs, and
ZFNs and explains how these pros and cons arise from their
function and composition. The review also delineates
approaches to cure sickle-cell anemia, HIV, and cystic
fibrosis with these technologies and the current status of these
endeavors. This information will be used in comparing
CRISPR/Cas9 to other genome editing technologies and in
discussing the potential gene therapy applications.
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