D 1.

3 – Mutation & Gene editing (AHL)

“How do gene mutations occur?”

“What are the consequences of gene mutation?”

Learning Objectives

Gene knockout as a technique Students are not required to know details of techniques. Students should
D1.3.8 for investigating the function of appreciate that a library of knockout organisms is available for some species
AHL a gene by changing it to make it used as models in research.
inoperative
Use of the CRISPR sequences Students are not required to know the role of the CRISPR–Cas system in
D1.3.9 prokaryotes. However, students should be familiar with an example of the
and the enzyme Cas9 in gene
AHL successful use of this technology.
editing
Hypotheses to account for Conserved sequences are identical or similar across a species or a group of
D1.3.10 species; highly conserved sequences are identical or similar over long periods of
conserved or highly conserved
AHL evolution. Two hypotheses for the mechanism are functional requirements for
sequences in genes the gene products and slower rates of mutation.
Gene knockout
….as a technique for investigating the function of a gene by changing it to make it inoperative

ttps://www.snexplores.org/article/explainer-why-scientists-sometimes-knock-out-genes
To find out the function
of a gene or its base
sequence, targeteted
knockout of genes by
deletions or insertions
are carried out.
Gene knockout
….as a technique for investigating the function of a gene by changing it to make it inoperative
DNA is prepared with a base
sequence that allows it to be
inserted into the genome of
embryonic mouse cells as a
replacement for a target gene,
which is therefore deleted.

https://www.genome.gov/genetics-glossary/Knockout
Gene knockout is often carried out in mice as model organism. The
phenotype of the mice containing the engineered genes is then investigated
to find out which traits have been changed by deletion of the target gene. A
whole library of knock-out strains can be generated that way.
 Gene knockout
https://www.researchgate.net/figure/Generating-a-knockout-mouse-model-Generation-of-a-knockout-mouse-model-involves-the_fig1_345318767

….as a technique for investigating the function of a gene by changing it to make it inoperative
Isolation of
embryonic stem cells

In mice or other model

organism, one specific gene is Replacement of gene X with a suitable
sequence to delete gene X
knocked out (typically by
inserting a deletion) to Isolation of knocked out diploid cells
which contain one knocked-out gene
investigate the function. and one normal gene.
Thousands of different strains of
Insertion of knock-out stem cells into a blastocyst
mice, each lacking one specific
gene, have been created to Implantation of blastocyst into surrogate mother to produce
baby (heterozygous) mice with cells from two strains.
establish “gene libraries” to be
used in research. Adult knock-out
heterozygous mice

Knock-out mice crossbred with wild-type

homozygous mice to produce 50% of
mice with the knock-out gene only.
Examples:
Interbreeding of
Mice which lack the gene for heterozygous mice –
p53, a cell cycle regulator 25% of offspring will be
homozygous
protein have been created to
investigate the risk of bone and Selection of homozygous
knock-out mice
other cancers in mammals.
Gene knockout
….as a technique for investigating the function of a gene by changing it to make it inoperative
Using in vivo model organism such as Drosophila melanogaster (fruit fly) or
Caenorhabditis elengans (nematode) is extremely valuable, because their
effects can predict similar effects in closely related organisms.

https://www.cell.com/trends/genetics/fulltext/S0168-9525%2820%2930253-5
Use of the CRISPR sequences and the enzyme Cas9
…in gene editing
CRISPR-Cas9 is In nature, the main two
gene editing based elements for this system are:
on a natural system • CRISPR regions within the
that exists in many genome
• The enzyme Cas9
species of
prokaryotes, which
use it as a defense
against viral
attacks.

https://www.news-medical.net/news/20230111/Newly-discovered-CRISPR-immune-system-shuts-down-infected-cells-to-thwart-infection.aspx
Use of the CRISPR sequences and the enzyme Cas9
…in gene editing

https://1drv.ms/v/s!Au8ZKE_EDcrQhLUCQ7rivmsGGNYsKg?e=IEB1mq
Use of the CRISPR sequences and the enzyme Cas9
…in gene editing
CRISPR is an acronym which stands for the following features:
Clustered - The sequences are grouped in one part of the bacterial genome
Regularly - The sequences are separated by other base sequences at regular
Interspersed

https://www.moleculardevices.com/applications/gene-editing-with-crispr-engineering
Short - intervals.
The number Theofnumber of spacers
base pairs differs (between
in a sequence 2 –– 120
is short (23 / CRISPR)
47 bp)
Palindromic - Each sequence has parts which read the same backwards as forwards
Repeats - The same base sequence occurs several times in the genome.

CRISPR repeat showing an

array of the cluster of five
repeats (R) interspersed
with four spacers, plus a
leader sequence that is
rich in AT base pairs. The
spacers are effectively viral
DNA which has been cut
previously, and then
inserted into the bacterial
DNA as “memory” for
future infections.
Use of the CRISPR sequences and the enzyme Cas9
…in gene editing

https://www.labiotech.eu/in-depth/crispr-cas9-review-gene-editing-tool/
When bacteria are infected by a virus, they use their CRISPR system
within the genome to cut up the invading DNA and insert pieces
(spacers) of it into their own genome as a memory of the infection. Two
sequences within the CRISPR system are transcribed into RNA:
Spacer sequences Repeat sequences
Bacteria transcribe the
The repeat sequences
spacers into RNA (the guide
are transcribed into
RNA) which forms a complex
RNA which form a
with Cas9 and is used to
loop and bind with the
search for viral base
CAS9 enzyme.
sequences in the target DNA.
Use of the CRISPR sequences and the enzyme Cas9
…in gene editing

https://www.labiotech.eu/in-depth/crispr-cas9-review-gene-editing-tool/
1 Cas9 moves along target
DNA molecules (e.g the ones
of a virus which entered a
bacterium) and brings the
DNA adjacent to the variable
base sequence of the spacer
in the guide RNA.

3 Cas9 contains two

2
endonucleases
(“scissors”). If the target
If matching (viral) DNA is
sequence is recognized,
encountered the spacer
the endonucleases cut
(guide) RNA, the RNA-
the DNA strands to
Cas9 complex binds to it.
destroy the foreign DNA.
Use of the CRISPR sequences and the enzyme Cas9
…in gene editing
The precise specifity of the CRISPR
system offers opportunities for
gene editing. This can come in
useful in the treatment of genetic
diseases. If finding the affected
base sequence of an individual in
the genome it could be cut out and https://1drv.ms/v/s!Au8ZKE_EDcrQhLR0OdcZpg_UdZGNCg?e=LiVfgf

replaced with the desired sequence.

Use of the CRISPR sequences and the enzyme Cas9
…in gene editing
Gene editing to treat genetic diseases
requires a method for finding a base
sequence in the genome that is the target,
and replacing it with a desired sequence.

https://1drv.ms/v/s!Au8ZKE_EDcrQhLR1ZOuC-QmP5qlY7g?e=c4VP3i

One of the first diseases which shows potential to be treated by CRISPR is

sickle cell anemia. In experimental treatments the gene on the human
chromosome 11 responsible for the disease has been edited. Bone marrow
(stem) cells, so-called hematopoietc stem cells, are removed from patients
and successfully repaired using the CRISPR/Cas9 system.
Conserved or highly conserved sequences in genes
Conserved sequences are identical or similar in nucleic acids (DNA or
RNA) across species or a group of species. Highly conserved sequences
are identical or similar over long periods of evolution.

The HACNS1 gene is a highly conserved

squence across a wide range of animals,
which implies that it has stayed mostly
unchanged over millions of years. However,
the rate of change in human evolution
from primate ancestor is muct faster – 13
human specific base substitutions in this
gene have taken place.

Most conserved sequences are in protein coding areas of the genome, which are sequences
with a known function. If there are no changes over a long period of evolutionary time
these genes might have even stayed unchanged during speciation events when groups
diverge. This is why some squences are found in all mammals or even all species.
Conserved or highly conserved sequences in genes

Read the article here to learn

how the conserved sequence
has evolutionized over time!
https://www.nationalgeographic.com/science/article/did-a-gene-enhancer-humanise-our-thumbs
Conserved or highly conserved sequences in genes
The protein cytochrome c (cyt c) and its corresponding gene are highly conserved,
meaning that cyt c has changed little over evolutionary time. Cytochrome C is involved
in aerobic respiration and is therefore part of every organism that depends on oxygen.
Pigs, cows and sheet have identical cyt c molecules – amino acid for amino acid.
Conserved or highly conserved sequences in genes

https://www.ensembl.org/index.html