Summary CRISPR editing
Application lecture 1: Genome editing tools to study the
biological function(s) of genes 17-
10-2022 Bart van de Sluis
In 2001, the Human Genome Project was finished. It was an international project in which for the first
time, the complete human genome (3x109 base pairs) was deciphered. These base pairs contain about
20.000 genes in total, with about 0.1% base pair diversity between individuals. Diversity arises through
mutations, but sometimes mutations can also cause disease. To analyze these mutations and with that the
diseases, biomedical and translational research is done in cellular and animal models. The first gene was
targeted in 1989 by Zijlstra et al, which was the beta 2 microglobulin. The research was published in
Nature. Later, in 2007, the Nobel prize in physiology went to Capecchi, Evans, and Smithies, for their
discoveries of principles of introducing specific gene
modifications in mice using embryonic stem cells.
Gene targeting in mice
For gene targeting in mice, first embryonic stem cells
(ESCs) are cultivated from mouse pre-implantation
embryos. A targeting vector is constructed, and this
contains pieces of DNA that are homologous to the
target gene, but also DNA that changes the target
gene and allows for selection (e.g. a fluorescent
marker). The ESCs are then transfected with these
vectors, and the machinery for homologous
recombination allows the recombination of the target
gene. In the culture, the cells can be selected for their
markers, and these are incubated further to create the wanted ESCs. Sometimes the
targeting vector is randomly integrated in another gene than the gene of interest. After
selection for the right ESCs, there is injection of the ESCs into blastocysts (like IVF), and
the blastocysts are put into a surrogate mother.
When knocking out the Commd1 gene in mice, embryos are not
viable: the knockout is embryonic lethal. Therefore, a conditional
knockout mouse is generated, using the Cre-LoxP system, which
allows the gene to be knocked out in specific tissues only. LoxP is
inserted around the Commd1 gene. If this mouse is crossed with
another mouse, which contains the Cre recombinase (only active in
specific tissues), the LoxP sites are cut and the Commd1 gene is
removed (knocked out).
Gene targeting in this way takes more than 1 year (including
breeding and crossbreeding), and its success rate is not very high. Another approach for genome
modification, and one that can very specifically do so, is the CRISPR/Cas9 method. This method takes less
long and has a higher success rate.
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,CRISPR/Cas9
The CRISPR/Cas9 method
uses a Cas9 protein, which is
an endonuclease and can cut
sites in the genome. Where
the Cas9 cuts depends on
the sgRNA, which contains a
crRNA and a tracrRNA. The
sgRNA targets a specific
sequence in the genome, where the Cas9 protein then makes a double stranded cut. What happens after a
cut, is that the cells try to repair the DNA again, using either Nonhomologous end joining (NHEJ) or
homology-directed repair (HDR). NHEJ is very error prone, and therefore often used for knocking out
genes, whereas HDR is more often used to induce mutations.
CRISPR/Cas9 applications
CRISPR/Cas9 has a lot of potential applications:
1. Addition of fluorescent tags to endogenous genes, to analyze expression. Cas9 can cut specifically in
front of a gene, and then a plasmid can be added that contains the wanted sequence.
2. Genome editing in zygotes to generate mouse models, for example creating knockout mice, mice
with a single substitution, or floxed mice (conditional knockouts using LoxP).
3. Genome editing in somatic cells, which is used to study disease related genes (for example in
cancers, immune disorders, or neurodegenerative diseases). Hepatic PTEN-deficiency (liver cancer)
was examined using the Cre-Lox system to knockout PTEN and look at the effect on the liver of a
mouse.
4. CRISPR/Cas9 screens in transplant models, which allows for example
discovery of genes that lead to cancer
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, Theory lecture 1: The history of CRISPR genome engineering
18-10-
2022 Kai Yu Ma
CRISPR is about 30 years old, with the first discovery and description between 1987 and 1993 (depending
on who you ask). It was first used in mammalian cells in 2013.
What is CRISPR?
There were three independent events that played a big role in the discovery of CRISPRs.
1. In Spain, Francisco Mojica studied the genome of the extreme salt tolerant archael microbes
Haloferax Mediterrannei. He discovered patterns in the sequence that are repeated over the whole
genome (palindromes). This had also been reported in other microbes before. RNA transcripts of
these sequences were also detected. The repeats were eventually called CRISPRs: clustered
regularly interspaced palindromic repeats. Later it was discovered that associated genes (cas,
CRISPR associated genes) encode for helicases and nucleases and interact with these CRISPR
sequences. By looking at the spacers between these repeats, he discovered that there is an overlap
of genomes of viruses. The function of the CRISPR loci remained unclear.
2. In France, Gilles Vergnaud, who worked for the Ministery of Defense, wanted to trace the source of
pathogens based on genetic differences among strains. They were afraid that biological weapons
would be used against the west. Therefore, they looked at Y. pestis samples from a plague outbreak
during the Vietnam war. There were a lot of overlapping CRISPR sequences in the isolates, which
may represent memory of past aggressions. Spacer sequences corresponded to prophage
sequence.
3. Philippe Horvath worked for a yogurt producing company, with the mission to overcome the
frequent phage infections that plagued the industrial cultures used in dairy fermentation. He
hypothesized that CRISPR is an adaptive immune system. He isolated phage-resistant bacteria from
a phage-sensitive thermophilus strain, and knocked out the CRISPR loci, after which the bacteria
were not phage-resistant anymore. So, this is functional proof that components of the CRISPR locus
are crucial for phage defense and that this defense is encoded in the DNA sequence.
So, the conclusion after these independent events is that CRISPR is an adaptive immune system.
How does CRISPR work?
There are again a few people that were involved in the discovery of the mechanisms that are used in the
CRISPR system.
1. John van der Oost took out a CRISPR locus of E.coli and
knocked out each cas gene, and looked at the total RNA.
For CasD and CasE there were no fragments found when
they were the only ones deleted, meaning that they are
involved in cleavage of the fragments. The spacer
sequences are flanked by repeat sequences that seemed
to form loops, forming pre-crRNAs. crRNAs (on both
strands) are crucial for mediating the antiviral response.
2. Marraffini and Sontheimer discovered that CRISPR
interferes with sequences from non-phage origins. Normally, transconjugants can be formed by
transfer of resistance genes upon contact of cells. However, by giving CRISPR to one of the parents,
no transconjugant is formed (because one of the plasmids is destroyed). So, CRISPR interferes with
horizontal gene transfer, and only targets DNA because RNA is too inefficient.
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