Week 5 CRISPR Notes Sp18

Lecture Notes Week 5: The CRISPR Revolution
SLIDE: Gene Editing
● Gene editing is the process by which a specific, pre-determined modification

is made to DNA in living cells.
● The possible types of modifications include DNA removal, addition or
replacement.
● Gene editing is a powerful tool for biological research, because it can help to
understand gene function and chromosome biology.
● Gene editing also has numerous applications, from creating new crop
varieties to curing genetic disease.
● In order to edit a genome, molecular tools are needed, the most widely used
being CRISPR/Cas.
In the following slides, we will describe how gene editing works, how CRISPR/Cas
reagents enable gene editing, and we will consider some of the applications of the
technology…from agriculture to human health.
SLIDE: Gene Editing is Enabled by Harnessing DNA Repair Pathways
In last week’s lecture, we discussed different forms of DNA damage and how cells
respond to this damage. One type of damage is the formation of a DNA double strand
break. When a break occurs, there are typically two ways in which the broken
chromosome can be repaired:
● Non-homologous end joining (NHEJ) is the direct rejoining of a broken
chromosome. Often, the chromosome is rejoined precisely. However,
sometimes nucleotides are lost (or more rarely gained) at the break site,
causing a mutation (shown in red in the slide). Typically, the gain or loss of
nucleotides is from one to fifteen or so base pairs.
● Breaks also can be repaired by copying information from a homologous

template. Most often, the template is a homologous chromosome or sister
chromatid. DNA sequences in the template that differ from the target (shown
in green) are copied into the locus. In gene editing, we deliver to the cell a
template with DNA sequence changes we want incorporated into the
chromosome, thereby achieving precise gene edits.
SLIDE: Targeting DNA Double Strand Breaks
Gene editing begins with a DNA double strand break. To create a targeted break, we
use one of three classes of nucleases: zinc finger nucleases, TALENs (Transcription
Activator-Like Effector Nucleases, and CRISPR (Clustered Regularly Interspaced
Short Palindromic Repeats). Each class of nuclease can be engineered to recognize a
specific DNA sequence you want to modify.
1
● Zinc finger nucleases and TALENs are chimeric proteins made up of a DNA
binding domain and a nuclease domain. For zinc finger nucleases, DNA
binding is achieved by the zinc finger protein motif. Each zinc finger typically
recognizes three base pairs, and different zinc fingers exist that recognize
different three base combinations. Zinc fingers can be stitched together to
form an array that recognizes an extended DNA sequence.
● TALENs recognize DNA by a TAL motif, which comes from a plant pathogenic
bacterium. Each TAL motif recognizes a single base, and motifs can be
stitched together to form an array with unique DNA sequence specificity.
The zinc finger arrays or TALE arrays are fused to the nuclease FokI. FokI
functions as a dimer, and so zinc finger nucleases and TALENs are engineered
to bind DNA sequences in close proximity in order for FokI to dimerize and
cleave.
● DNA targeting is achieved in CRISPR systems through base-pairing between a

guide RNA (gRNA) and the target DNA. The Cas protein is a nuclease that
introduces a break in the RNA/DNA duplex. This simple mechanism of DNA
targeting – through base pairing – is one of the main reasons CRISPR systems
have emerged as the reagent of choice for gene editing.
SLIDE: In Nature, CRISPR Systems Are Used by Bacteria to Fight DNA Invaders
1. Acquisition. When a virus or plasmid invades a cell, DNA sequences from the
intruder are captured in the CRISPR array. The CRISPR array is really an archive of
DNA sequences from viruses and plasmids that have entered the cell in the past.
2. Expression. The CRISPR array is expressed as a pre-crRNA and then processed

into individual crRNAs that are bound by a Cas protein.
3. Interference. If a plasmid or virus enters the cell with sequence identity to the
crRNA, the RNA pairs with the plasmid or viral DNA. The Cas protein then cleaves
the DNA, thwarting plasmid or viral entry. Diverse CRISPR systems exist in nature;
however, most operate by this mechanism in which sequence information is
archived and used to prevent future infections, hence it is a form of adaptive
immunity.
SLIDE: CRISPR Systems Can Be Designed to Introduce Targeted DNA Breaks in

Any Locus of Interest
● The most widely used CRISPR system comes from Streptococcus pyogenes;
the Cas protein in this system is called Cas9.
● A 20 nt region on the gRNA (the spacer sequence) pairs with the target DNA.
The 20nt region can be designed to target any locus of interest.
2
● One requirement for targeting is the so-called PAM sequence (NGG, shown in
red). Cas9 scans the genome for NGG motifs, and if pairing occurs with the 20
nt spacer sequence, then cleavage occurs.
SLIDE: Targeted Mutagenesis Through Non-Homologous End Joining
This slide and the next reinforce concepts in DNA repair that were described above
and in last week’s lecture.
● Mutations can be introduced into a target by designing a gRNA to recognize

the gene of interest.
● After cleavage, the DNA is most often rejoined precisely. However,
occasionally, small insertions or deletions are introduced at the break site.
● If the mutations occur in coding sequences, then they may cause frameshift
mutations that render the gene non-functional.
SLIDE: Targeted Mutagenesis Through Homologous Recombination
● Precise DNA sequence alterations can be created by introducing a break with

CRISPR/Cas and also providing a DNA repair template.
● The repair template is identical in sequence to the broken chromosome
except for the sequence alterations that are desired to be incorporated (note
that the red base is a little hard to see).
● The process of homologous recombination copies the sequence alterations
into the target locus. The sequence alterations can range from one base pair
(shown here) to several thousand base pairs.
SLIDE: Gene Editing and the Food Supply
● Gene editing is being used to create new crops, including those that produce
healthier foods.
● One of the first products derived from gene editing that will enter the food
supply is a soybean oil that has a fatty acid composition similar to olive oil,
namely low in unsaturated fats and free of trans fatty acids.
● The soybean variety was created using TALENs at a biotechnology company
called Calyxt in New Brighton, Minnesota. TALENs were invented at the UMN.
TALENs were invented before CRISPR/Cas, and many CRISPR-derived food
products will appear in the coming years.
SLIDE: Gene Editing and Human Health
● One of the first therapeutic applications of gene editing will be to combat

cancer.
● For example, white blood cells (T cells) can be harvested from patients and
edited so that they attack cancer cells. The first targets for this type of
therapy are cancers of the blood and lymph system, such as leukemia.
3
● In 2015, a young child – Layla – was on the verge of death. She was
administered T cells designed to target leukemia. The engineered cells
completely cleared the leukemia, and almost three years later, Layla is cancer
free.
SLIDE: Future Prospects
● There are many applications of CRISPR reagents besides gene editing.

● For example, nuclease inactive Cas proteins can be fused to transcriptional
activator or repressor domains and targeted to genes to control their
expression.
● Due to CRISPRs simple, effective DNA targeting mechanism, almost daily new
applications of CRISPR reagents are being described that help advance
biological research and promise to solve real-world problems.

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Lecture Notes Week 5: The CRISPR Revolution

SLIDE: Gene Editing

● Gene editing is the process by which a specific, pre-determined modification

SLIDE: Gene Editing is Enabled by Harnessing DNA Repair Pathways

● Breaks also can be repaired by copying information from a homologous

SLIDE: Targeting DNA Double Strand Breaks

● DNA targeting is achieved in CRISPR systems through base-pairing between a

2. Expression. The CRISPR array is expressed as a pre-crRNA and then processed

SLIDE: CRISPR Systems Can Be Designed to Introduce Targeted DNA Breaks in

SLIDE: Targeted Mutagenesis Through Non-Homologous End Joining

● Mutations can be introduced into a target by designing a gRNA to recognize

SLIDE: Targeted Mutagenesis Through Homologous Recombination

● Precise DNA sequence alterations can be created by introducing a break with

SLIDE: Gene Editing and the Food Supply

SLIDE: Gene Editing and Human Health

● One of the first therapeutic applications of gene editing will be to combat

SLIDE: Future Prospects

● There are many applications of CRISPR reagents besides gene editing.

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