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Chapter 6

Biological Sciences 55-237 Chapter 6: Microbiology Topic 6 Part 2


Department
Biological Sciences
Course Code
BIOL 2070
Professor
tanyanoel
Chapter
6

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Microbiology Topic 6 Part 2
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
Bacterial immune system and revolutionary gene editing tool
Genome integrity and foreign DNA
Plasmids an other mobile elements considered selfish genetic elements
While HGT can occasionally provide adaptive genes to bacteria, foreign DNA is often
not beneficial to recipient
It can often be tempting to think of HGT as being adaptive in bacteria
While it can be in some situations, much of the time foreign DNA may not provide a
benefit and may often be detrimental to the recipient
Bacterial defenses against bacteriophages: Restriction enzymes/endonucleases
Restriction-modification systems: bacteria use restriction enzymes to cleave foreign
DNA, and modification enzymes (e.g. methyltransferases) to distinguish their own
DNA from foreign DNA in the cytoplasm
Restriction enzymes cut at specific sites, typically within palindromes
Bacteria are at risk of infection by bacteriophages, which is one way that foreign
DNA can enter the cell
As bacteria are single-celled, they are not capable of the types of immune response
systems in plants and animals; however they do have some adaptations that protect
them
A common one is restriction modification system
When viral DNA is infected into a bacterial cell, the DNA can be the target of
restriction endonucleases
These enzymes recognize particular sequences and cut the DNA
The host chromosome is protected by methylation of these sequences
We make use of restriction enzymes in molecular biology
They cut isolated DNA in a very targeted way
Bacterial defenses against bacteriophages: CRISPR & Cas
CRISPR and CRISPR-association Cas proteins provide a sort of immunity against
invading nucleic acids:
Storage of foreign sequences in CRISPR region of bacterial chromosome can be used
to recognize same/similar incoming sequences
Cas proteins (& CRISPR RNA) involved in surveillance/storage, recognition and
destruction of foreign DNA
Another defense mechanism that is more adaptive
Based on clustered regularly interspaces short palindromic repeats (called CRISPRs)
and the CRISPR-associated (Cas) proteins that work with them
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The CRISPR-Cas system can integrate nucleic acid fragments form invading mobile
elements (such as viruses and plasmids) into the CRISPR region of the bacterial
chromosome
The entire CRISPR regions is transcribed and cleaved into short mature CRISPR
RNAs or crRNAs
These crRNAs specifically guide Cas proteins to complementary invading nucleic
acid targets
In this way, the CRISPR-Cas system can provide the host with acquired and heritable
resistance
The CRISPR system is common among the Bacteria and Archaea studied so far with
three types of CRISPR systems identified
CRISPR is also considered to be a type of RNA interference but it is quite different
than the systems that have been observed in eukaryotes
The mechanism by which the CRISPR-Cas system works involves different stages
The first stage called adaptation or immunization is the recognition and integration
of nucleic acids from foreign invaders that are integrated as new spacers in the
CRISPR region by Cas proteins
Spacers are the foreign sequences in the CRISPR region of the bacterial chromosome
separated by repeats
In the CRISPR processing or biogenesis stage, the entire CRISPR is transcribed as a
precursor or pre crRNA, which is then cleaved by a dedicated nuclease enzyme
This process results in mature crRNA that remain associated with a Cas protein
complex
In the interference or immunity stage, the crRNA guides the Cas complex to known
invading nucleic acids to destroy the invading DNA with Cas nuclease activity
CRISPR/Cas gene editing systems
Cas9 nuclease and a guide RNA (gRNA) including crRNA and trans-activating
CRISPR RNA (tracrRNA) can allow gene editing in target cells/organisms
o Delete, introduce, change genes
Rapid, inexpensive, easy to use
Relatively new system
o Troubleshooting/optimization
o Concerns about safety, ethics
Like many other systems and components of bacteria, the CRISPR-Cas system has
been adapted for other uses
Using a type II system present in some bacteria, they Cas9 enzyme and a guide
molecule can be used to target a specific DNA sequence and edit it to disrupt genes
or insert a desired sequence
This is considered to be a revolutionary gene editing system that is relatively
inexpensive, rapid, and easy to use
It has already been used in plants, yeast, zebra fish, and some types of human cells
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Potential for engineering organisms, ecosystems, radical gene therapy using this
system
Has been getting a lot of attention recently, but like many new technologies, people
have raised concerns about its use, particularly in areas of safety and ethics
Bacterial genetics and biotechnology
Mutations, mutants
Mutations are one of the ways that we see changes in the genomes of bacteria, along
with recombination
DNA in bacteria
At least one chromosome
Possibly one or more plasmids
Mutations can occur in DNA spontaneously, or they can be induced
Like all cellular organisms, you will find at least one chromosome (usually circular)
Many bacteria will have plasmids, which are extrachromosomal DNA
o Usually circular
o A single bacterium may have many different plasmids and can have several
copies of the same plasmid
Mutations can occur in any kind of DNA
The type of mutation can be spontaneous or induced
Spontaneous mutations occur through regular DNA replication processes
There are proofreading capabilities that can correct a lot of errors in replication, but
they do not always work
There are many mutations that occur as the result of some sort of exposure or event
that can lead to mutations in an organism
Mutations and mutants
In bacterial genetics, we often study mutants to understand functions/effects of
particular genes
Strains isolated form nature usually considered wild-type strains
o Wild type may refer to gene or entire organism
Mutations in some genes can be markers e.g., so we can tell if (GT has occurred
Mutations are really important in bacterial genetics
We often need to study mutants to be able to determine what happens if a gene is
altered
Wildtype- indicates that we are working with something isolated from nature
o We can talk about wild-type organisms or wild-type genes
One of the important things about mutations is that we can sometimes use them in
experiments
Some of these can act as markers
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