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University of California
Los Angeles

Sample Paper - Clustered regularly interspaced
short palindromic repeats (CRISPR)

A dissertation submitted in partial satisfaction
of the requirements for the degree
Doctor of Philosophy in Bioscience

by

2016

c Copyright by


2016

The dissertation of is approved.

University of California, Los Angeles
2016

2

Table of Contents

1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

3 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7

3

List of Figures
1.1

Spectrum of gene . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

2

CHAPTER 1
Abstract
A major addition to the understanding of CRISPR came with Jansen’s observation
that the prokaryote repeat cluster was accompanied by a set of homologous genes
that make up CRISPR-associated systems or cas genes. Four cas genes (cas 1 to 4)
were initially recognized. The Cas proteins showed helicase and nuclease motifs,
suggesting a role in the dynamic structure of the CRISPR loci.In this publication
the acronym CRISPR was coined as the universal name of this pattern. However,
the CRISPR function remained enigmatic.[BM02, ?]
Simplified diagram of a CRISPR locus. The three major components of a
CRISPR locus are shown: cas genes, a leader sequence, and a repeat-spacer array.
Repeats are shown as gray boxes and spacers are colored bars. The arrangement
of the three components is not always as shown.In addition, several CRISPRs
with similar sequences can be present in a single genome, only one of which is
associated with cas genes
In 2005, three independent research groups showed that some CRISPR spacers are
derived from phage DNA and extrachromosomal DNA such as plasmids.In effect,
the spacers are fragments of DNA gathered from viruses that previously tried to
attack the cell. The source of the spacers was a sign that the CRISPR/cas system
could have a role in adaptive immunity in bacteria.All three studies proposing
this idea were initially rejected by high-profile journals, but eventually appeared
in other journals.
The first publication[26] proposing a role of CRISPR-Cas in microbial immu-

1

Figure 1.1: Spectrum of gene
nity, by Mojica’s group, predicted a role for the RNA transcript of spacers on
target recognition in a mechanism that could be analogous to the RNA interference system used by eukaryotic cells. Koonin and colleagues extended that
hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins. Others hypothesized
that CRISPR sequences directed Cas enzymes to degrade viral DNA.
Experimental work by several groups revealed the basic mechanisms of CRISPRCas immunity. In 2007 the first experimental evidence that CRISPR was an
adaptive immune system was published. A CRISPR region in Streptococcus thermophilus acquired spacers from the DNA of an infecting bacteriophage. The
researchers manipulated the resistance of S. thermophilus to phage by adding and
deleting spacers whose sequence matched those found in the tested phages.[31][32]
In 2008, Brouns and colleagues identified a complex of Cas protein that in E. coli
cut the CRISPR RNA within the repeats into spacer-containing RNA molecules,
which remained bound to the protein complex. That year Marraffini and Son-

2

theimer showed that a CRISPR sequence of S. epidermidis targeted DNA and not
RNA to prevent conjugation. This finding was at odds with the proposed RNAinterference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas
system that targets foreign RNA was later found in Pyrococcus furiosus.A 2010
study showed that CRISPR-Cas cuts both strands of phage and plasmid DNA in
S. thermophilus.[33]
A major addition to the understanding of CRISPR came with Jansen’s observation that the prokaryote repeat cluster was accompanied by a set of homologous
genes that make up CRISPR-associated systems or cas genes. Four cas genes (cas
1 to 4) were initially recognized. The Cas proteins showed helicase and nuclease
motifs, suggesting a role in the dynamic structure of the CRISPR loci.In this publication the acronym CRISPR was coined as the universal name of this pattern.
However, the CRISPR function remained enigmatic.

3

CHAPTER 2
Introduction
Simplified diagram of a CRISPR locus. The three major components of a CRISPR
locus are shown: cas genes, a leader sequence, and a repeat-spacer array. Repeats
are shown as gray boxes and spacers are colored bars. The arrangement of the
three components is not always as shown. In addition, several CRISPRs with
similar sequences can be present in a single genome, only one of which is associated
with cas genes.[8]
In 2005, three independent research groups showed that some CRISPR spacers are
derived from phage DNA and extrachromosomal DNA such as plasmids.In effect,
the spacers are fragments of DNA gathered from viruses that previously tried to
attack the cell. The source of the spacers was a sign that the CRISPR/cas system
could have a role in adaptive immunity in bacteria.All three studies proposing
this idea were initially rejected by high-profile journals, but eventually appeared
in other journals.

4


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