CRISPR - What's All The Excitement About

9/25/2016
CRISPR:What'sAlltheExcitementAbout?
Biotech and Pharmaceutical Industries
What are Transcription Factors?

The role these factors have in gene expression
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Credit: Martin McCarthy/E+/Getty Images
By Theresa Phillips
Updated June 07, 2016
What are transcription factors? With this review, improve your understanding of the
important role they play in gene expression.
Understanding Transcription Factors

https://www.thebalance.com/crisprwhatsalltheexcitementabout375716
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In order for our bodies to have different types of cells, there has to be some mechanism
for controlling the expression of our genes. In some cells, certain genes are turned off,
while in other cells they are transcribed and translated into proteins.
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Transcription factors are one of the most common tools that our cells use to control gene
expression.
A Brief Definition
Transcription factors (TFs) are molecules involved in regulating gene expression. They are
usually proteins, although they can also consist of short, non-coding RNA. TFs are also
usually found working in groups or complexes, forming multiple interactions that allow
for varying degrees of control over rates of transcription.
Turning Genes Off and On

In people (and other eukaryotes), genes are usually in a default "off" state, so TFs serve
mainly to turn gene expression "on." In bacteria, the reverse is often true, and genes are
expressed "constitutively" until a TF turns it "off." TFs work by recognizing certain
nucleotide sequences (motifs) before or after the gene on the chromosome (up- and
downstream).
Genes and Eukaryotes

Eukaryotes often have a promoter region upstream from the gene, or enhancer regions
up or downstream from the gene, with certain specific motifs that are recognized by the
various types of TF.
The TFs bind, attract other TFs and create a complex that eventually facilitates binding by
RNA polymerase, thus beginning the process of transcription.
Why Transcription Factors Are Significant

Transcription factors are only one of the means by which our cells express different
combinations of genes, allowing for differentiation into the various types of cells, tissues
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and organs that make up our bodies.
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However, this mechanism of control is extremely important, especially in light of the

findings of the Human Genome Project that we actually have fewer genes in our genome,
or on our chromosomes, than originally thought.
What this means is different cells have not arisen from differential expression of
completely different sets of genes, but are more likely to have varying levels of selective
expression of the same groups of genes.
The Cascade Effect

TFs can also control gene expression by creating a "cascade" effect, wherein the presence
of small amounts of one protein trigger the production of larger amounts of a second,
which triggers production of even larger amounts of a third, and so on. The mechanisms
through which significant effects are induced by very small amounts of the initial material
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or stimulus are the basic models of today's biotechnological advances in Smart Polymer
research.
Gene Expression and Life Expectancy

Manipulating TFs to reverse the cell differentiation process is the basis of methods for
deriving stem cells from adult tissues.
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The ability to control gene expression, along with knowledge obtained from studying the
human genome and genomics in other organisms, has led to the theory that we can
prolong our lives,
if we just control the genes that regulate the aging process in our cells.
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CRISPR: What's All the Excitement About?

A New Tool for Gene Manipulation
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Rafe Swan/Cultura/Getty Images
By Paul Diehl
Updated August 11, 2016
Recently scientists have found an exciting new tool with which to engineer DNA. The
CRISPR system has nothing to do with keeping your vegetables fresh in the refrigerator. It
is the acronym for the newest system to manipulate genomic DNA in almost any animal.
Researchers have been able to knockout or eliminate genes, repress gene expression, and
up-regulate genes to increase expression with the CRISPR technology.
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It is a very flexible techniquethat researchers can use toeasily alterthe expression

ofgenesto better understand theirfunction.
What Exactly Is CRISPR?

CRISPR stands for Clustered Regularly-Interspaced Short Palindromic Repeatsan
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incredibly boring name for an exciting technology. Why the tedious name? It is because,
when they were first discovered in the late 1980s in bacteria, no one knew what the short
stretches of repeatedDNA separated by random DNA sequences were for. They were just
some strange feature in the genomic DNA of some bacteria.
It took almost 20 years until Jennifer Doudna at the University of California figured out
that these sequences matched parts of certain viral DNA that infected the bacteria. As it
turned out, the CRISPR sequences were asort ofimmune system for the bacteria.
How Does CRISPR Work?

Doudna and her collaborator, Emmanuelle
Chapentier, eventually worked out that,
when infected by a virus, bacteria that had these short repeating DNA piecesthatmatched
the viral DNA would use them to make RNA that bound to theDNA of the invading virus.
Then, a second piece of RNA made from the random DNA that separated the CRISPR
repeatsinteracted with a protein called Cas9. This protein would cleave the virus DNA
andinactivatethe virus.
Researchers quickly realized they could exploit this capability of CRISPRto cut apart
specific DNA sequences to knock out genes.
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While there are other techniques, such as zinc finger nucleases and TALENS that can be
used to target and cut specific locations in genomic DNA, these approaches rely on bulky
proteins to target the alternations to specific regions in the DNA. It is difficult to design
and carry out modification on a large scale with lots of genes using these earlier
approaches.
What Makes CRISPR so Useful?

The CRISPR systemonly relies on twoshort pieces of RNA: one that matches the targeted
DNA region,and a second that binds to a protein called Cas9. In fact, though, it turns out
that both these short RNA pieces can be combined into a dual-function single-guide RNA
molecule that bothtargets a specific DNA sequenceand recruits theCas9 cleaving protein.
This means that the Cas9 protein and one short piece of RNA that is 85 bases long is all
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that's needed to cut a DNA at almost anywhere in the genome.It is relatively simple to
introduce DNA to producea single-guide RNA and the Cas9 proteinalmost any cells
making CRISPR generally applicable.
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However, convenient targeting is not the only advantage of the CRISPR technology over
other TALENS and zinc fingers. The CRISPR system is also much more efficient than these
alternative approaches. For example, a group at Harvard found that CRISPR deleted a
targeted gene in 51%79% of the cases, whereas TALENS efficiency was less than 34%. Due
to this high efficiency, another group was able to use CRISPR technology to directly
knockout genes in embryonic mice to produce transgenic mice in a single generation.
The standard approach requires a couple generations of breeding to get the mutation in
both copies of a targeted gene.
What Else CanCRISPR Do?

In addition to deleting a gene, some groupshave also realized that, with a
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fewalternations, the system can be used for other sorts of genetic manipulation. For
example, in the beginning of 2013, a group from MIT showed that CRISPR can be used to
insert new genes into genomic DNA. Shortly thereafter a group at UCSF used a modified
version of the system dubbed CRISPRi to repress expression of target genes in bacteria.
More recently, a group at Duke University also set up a variation of the system to activate
sets of genes. Several groups are also now working with variations of these approachesto
screen large numbers of genes at onceto figure out which one are involved in different
biological responses.
The Shiny New Toy of Genetic Engineering

Certainly there is tremendous excitement about this new tool for genetic engineering and
the rush to apply it for a variety of applications. However, there are still some challenges
that need to be overcome and, as is often the case with new technology, it takes a while to
work out where the limitations are. Researchers at Harvard, for example, have found that
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CRISPR targeting
may not be as precise as initially thought. Off-target effects of the
CRISPR complex can led to unintended changes when altering DNA.

Despite the challenges, though, CRISPR clearly has shown enormous potential to facilitate
alteration of genomic DNA that will help researchers more quickly understand how the
tens of thousands of genes in the human genome function. This alone has important
implications for improvements disease treatment and diagnosis. Further, with additional
development, the technology itself may be useful for a novel type of therapeutics. It may
provide anew approachforgene therapy. However, these advances are a ways off. For
now, it is just exciting to watch the rapid development of this new research tool and think
about the types of experiments it may allow.
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(Posted: Sept 30, 2013)
What Are GMOs and How Are They Made?

The Basics of Genetic Modification
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Andrew Brookes/Cultura/Getty Images
By Paul Diehl
Updated August 11, 2016
What Is a GMO?
GMO is short for "genetically modified organism." Genetic modification has been around
for decades, andis the most effective and rapid way to create a plant or animal with a
specific trait or characteristic. It enables precise specific changes to the DNA sequence.
Because DNAessentially comprisesthe blueprint for the whole organism, changes to the
DNA change the functions the organism is capable of.
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There is really no other way to do this except by using the techniques developed over the
last 40 years to directly manipulate the DNA.
How do you genetically modify an organism? Actually, this is a pretty broad question. An
organism can be a plant, animal, fungus, or bacteria and all of these can be, and have
been, genetically engineered for almost 40 years. The first genetically engineered
organisms were bacteria in the early 1970s. Since then, genetically modified bacteria
have become the work horse of hundreds of thousands of labs doing genetic
modifications on both plants and animals. Most of the basic gene shuffling and
modifications are designed and prepared using bacteria, mainly some variation of E. coli,
then transferred to target organisms.
The general approach to genetically alter plants, animals, or microbes is conceptually
pretty similar. However, there are some differences in the specific techniques due to
general differences between plant and animal cells.
For example, plant cells have cell walls and animal cells do not.
Reasons for Genetic Modifications of Plants and Animals

GM animals are primarily made for research purposes, often as model biological systems
used for drug development. There have been some GM animals developed for other
commercial purposes, such as fluorescent fish as pets, and GM mosquitoes to help
control disease carrying mosquitoes.
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However, these are relatively limited application outside of basic biological research. So
far, no GM animals have been approved as a food source. Soon, though, that may change
with the AquaAdvantage Salmon that is making its way through the approval process.
With plants, however, the situation is different. While a lot of plants are modified for
research, the objective of most crop genetic modification is to make a plant strains that
are commercially or socially beneficial. For example, yields can be increased if plants are
engineered with improved resistance to a disease-causing pest like the Rainbow Papaya,
or the ability to grow in an inhospitable, perhaps colder region. Fruit that stays ripe
longer, such as Endless Summer Tomatoes, provides more time for shelf time after
harvest for use. Also, traits that enhance the nutritional value, such as Golden Rice
designed to be rich in vitamin A, or utility of the fruit, such as non-browning Arctic
Apples have also been made.

Essentially, any trait that can be made manifest with the addition or inhibition of a specific
gene, can be introduced.
Traits that require multiple genes could also be managed, but this requires a more
complicated process that has not yet been achieved with commercial crops.
What Is a Gene?
Before explaining how new genes are put into organisms, it is important to understand
what a gene is. As many probably know, genes are made of DNA, which is partly
composed for four bases commonly noted as simply A, T, C, G. The linear order of these
bases in a row down a DNA strand of a gene can be thought as a code for a specific
protein, just as letters in a line of text code for a sentence.
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Proteins are large biological molecules made of amino-acids linked together in various
combinations. When the right combination of amino acids is linked together, the amino
acid chain folds together into a protein with a specific shape and the right chemical
features together to enable it to perform a particular function or reaction. Living things
are made up largely of proteins. Some proteins are enzymes that catalyze chemical
reactions; others transport material into the cells, and some act as switches activating or
deactivating other proteins or protein cascades. So, when a new gene is introduced, it
gives the cell the code sequence to enable it to make a new the protein.
How Do Cells Organize their Genes?

In plants and animal cells, almost all of the DNA is ordered in several long strands wound
up into chromosomes. The genes are actually just small sections of the long sequence of
DNA making up a chromosome. Every time a cell replicates, all the chromosomes are
replicated first. This is the central set of instructions for the cell, and each progeny cell
gets a copy. So, to introduce a new gene that enables the cell to make a new protein that
confers a particular trait, one simply needs to insert a bit of DNA into one of the long
chromosome strands. Once inserted, the DNA will be passed to any daughter cells when
they cell replicate, just like all the other genes.
In fact, certain types of DNA can be maintained in cells separate from the chromosomes
and genes can be introduced using these structures so they do not integrate into the
chromosomal DNA. However, with this approach, since the cells chromosomal DNA is
altered is usually not maintained in all cells after several replications. For permanent and
inheritable genetic modification, such as those processes used for crop engineering,
chromosomal modifications are used.
How Is a New Gene Inserted?

Genetic engineering simply refers to inserting a new DNA base sequence (usually
corresponding to a whole gene) into the chromosomal DNA of the organism. This may
seem conceptually straightforward, but technically, it gets a little more complicated.
There are many technical details involved in getting the right DNA sequence with the right
signals into the chromosome in the right context that enables the cells to recognize it is a
gene and use it to make a new protein.
There are four key elements that are common to almost all genetic engineering
procedures:
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1. First, you need a gene. This means you need the physical DNA molecule with the
particular base sequences. Traditionally, these sequences were obtained directly from
an organism using any of several laborious techniques. Nowadays, rather than
extracting DNA from an organism, scientists typically just synthesize from the basic A,
T, C, G chemicals. Once obtained, the sequence can be inserted into a piece of bacterial
DNA that is like a small chromosome (a plasmid) and, since bacterial replicate rapidly,
as much of the gene as needed can be made.
2. Once you have the gene, you need to place it in a DNA strand surrounded with the right
surrounding DNA sequence to enable the cell to recognize it and express it. Principally,
this means you need a small DNA sequence called a promoter that signals the cell to
express the gene.
3. In addition to the main gene that is to be inserted, often a second gene is needed to
provide a marker or selection. This second gene is essentially a tool used to identify the
cells that contain the gene.
4. Finally, it is necessary to have a method of delivering the new DNA (i.e., promoter, new
gene, and selection marker) into the organism's cells. There are a number of ways to do
this. For plants, my favorite is the gene gun approach that uses a modified 22 rifle to
shoot DNA-coated tungsten or gold particles into cells.
With animal cells, there are a number of tranfection reagents that coat or complex the
DNA and enable it to pass through the cell membranes. It is also common for the DNA to
be spliced together with modified viral DNA that that can be used as a gene vector to
carry the gene into the cells. The modified viral DNA can be encapsulated with normal
viral proteins to make a pseudovirus which can infect cells and insert the DNA carrying
the gene, but not replicate to make new virus.
For many dicot plants, the gene can be placed in a modified variant of the T-DNA carrier
of the Agrobacterium tumefaciens bacteria. There are a few other approaches as well.
However, with most, only a small number of cells pick up the gene making selection of the
engineered cells a critical part of this process. This is why a selection or marker gene is
typically necessary.
But, How Do You Make a Genetically Engineered Mouse or Tomato?

A GMO is an organism with millions of cells and the technique above only really describes
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how to genetically engineer single cells. However, the process to generate a whole
organism essentially involves using these genetic engineering techniques on germ cells
(i.e., sperm and egg cells). Once the key gene is inserted, the rest of the process basically
uses genetic breeding techniques to produce plants or animals that contain the new gene
in all the cells in their body. Genetic engineering is really just done to cells. Biology does
the rest.
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CRISPR - What's All The Excitement About

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9/25/2016

Biotech and Pharmaceutical Industries

What are Transcription Factors?

Credit: Martin McCarthy/E+/Getty Images

Understanding Transcription Factors

Turning Genes Off and On

Genes and Eukaryotes

Why Transcription Factors Are Significant

and organs that make up our bodies.

However, this mechanism of control is extremely important, especially in light of the

The Cascade Effect

Gene Expression and Life Expectancy

Biotech and Pharmaceutical Industries

CRISPR: What's All the Excitement About?

Rafe Swan/Cultura/Getty Images

It is a very flexible techniquethat researchers can use toeasily alterthe expression

What Exactly Is CRISPR?

How Does CRISPR Work?

What Makes CRISPR so Useful?

What Else CanCRISPR Do?

The Shiny New Toy of Genetic Engineering

CRISPR complex can led to unintended changes when altering DNA.

(Posted: Sept 30, 2013)

Biotech and Pharmaceutical Industries

What Are GMOs and How Are They Made?

Andrew Brookes/Cultura/Getty Images

Reasons for Genetic Modifications of Plants and Animals

Apples have also been made.

How Do Cells Organize their Genes?

How Is a New Gene Inserted?

But, How Do You Make a Genetically Engineered Mouse or Tomato?

7 Essential Tools for Protein

What Is siRNA and How Is It Used

4 Common Societal Concerns with Germ Line Gene Therapy

Common Biotech Terms You

How the Polymerase Chain

What Are Restriction Enzymes?

What Are GMOs, and Why Do

Techniques for Sequencing

PCR: The Technology Behind DNA

How Has Food Biotechnology

Biotechnology and Biotech

Get Daily Money Tips to Your Inbox

2016 About, Inc. All rights reserved.

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