MULUNGUSHI UNIVERSITY

SCHOOL OF NATURAL AND APPLIED SCIENCES

NAME: VINWELL CHANGWE

STUDENT NUMBER: 202204922

COURSE: MOLECULAR BIOLOGY

COURSE CODE: BIO 321

PROGRAME: BACHELOR OF SCIENCE IN BIOLOGICAL SCIENCES (MICROBIOLOGY)

LECTERER: DR. P BWALYA

DATE GIVEN:

DUE DATE: 25TH OCTOBER, 2024

The CRISPR-Cas system is a defense mechanism found in many bacteria and archaea that
allows them to recognize and destroy foreign DNA, such as that from bacteriophages or
plasmids. The system is based on two main components:

1. CRISPR loci: DNA sequences in the bacterial genome that contain short, repetitive elements
(called repeats) interspersed with unique sequences (called spacers).

2. Cas (CRISPR-associated) proteins: Enzymes that perform various functions, including DNA
cutting.

The CRISPR-Cas mechanism operates in three main phases: Adaptation, Expression, and
Interference.

ADAPTATION (SPACER ACQUISITION)

When a bacterial cell encounters foreign DNA from a virus (phage) or a plasmid, it can capture a
small segment of this invading DNA and insert it into its own genome. These segments are added
to a special region called the CRISPR array as new spacers between the repetitive sequences.
This process is called spacer acquisition, and it effectively creates a "memory" of past
infections, enabling the bacterium to recognize and defend against the same invaders in the
future.

Cas1 and Cas2 proteins are usually involved in the acquisition process. They detect the foreign
DNA, cut a fragment, and insert it into the CRISPR array.

EXPRESSION (CRISPR RNA FORMATION)

Once a new spacer has been integrated into the CRISPR array, the entire array is transcribed into
a long RNA molecule known as pre-crRNA. This RNA is then processed into smaller fragments,
called crRNAs, each containing one spacer and part of the repeat sequence.

 The crRNA then binds to a tracrRNA (in some systems, like CRISPR-Cas9) to form a
guide RNA. This guide RNA directs the Cas proteins to the target DNA.
  The Cas9 protein or other Cas nucleases are responsible for binding to the crRNA. The
guide RNA will now recognize any sequence that matches the spacer sequence stored in
the crRNA.

INTERFERENCE (TARGET CLEAVAGE)

In the final phase, if the bacterium is re-infected by the same virus or foreign genetic element,
the CRISPR-Cas system is activated. The crRNA (guide RNA) directs the Cas protein to the
matching DNA sequence (called the protospacer) in the invading DNA. The Cas protein then
cuts the invading DNA at specific sites, effectively neutralizing the infection.

PAM Sequence: A key feature of CRISPR-Cas systems is that they recognize a short sequence
called the Protospacer Adjacent Motif (PAM). The PAM sequence, which is typically just a few
nucleotides long, is critical because it allows the Cas protein to distinguish between the
bacterium’s own DNA (the CRISPR array) and foreign DNA. The absence of a PAM sequence in
the bacterial genome protects it from self-destruction.

APPLICATION OF CRISPR-Cas IN MICROBIOLOGY

The CRISPR-Cas system's ability to target specific DNA sequences has made it a powerful tool
for various applications in microbiology, biotechnology, and genetic engineering.

GENOME EDITING

CRISPR-Cas9 has revolutionized the field of genome editing, allowing scientists to make precise
changes in the DNA of many organisms, including microbes. This is done by designing a guide
RNA that matches the sequence of the gene of interest. Cas9 is directed to that sequence, where
it makes a cut, allowing researchers to either delete, insert, or modify the target gene.

Gene Knockouts: CRISPR-Cas9 can be used to inactivate or knock out specific genes by
introducing small insertions or deletions that disrupt the gene’s function. This allows scientists to
study the role of individual genes in microbial cells.
Gene Replacements or Insertions: By providing a donor template along with the CRISPR system,
researchers can replace a gene or introduce new sequences at a specific site within the genome
(homology-directed repair).

ANTIMICROBIAL DEVELOPMENT

CRISPR-Cas systems are being developed as a novel class of antimicrobials that can specifically
target and kill pathogenic bacteria. Unlike traditional antibiotics, which often kill both harmful
and beneficial bacteria, CRISPR-based antimicrobials can be designed to precisely target
antibiotic-resistant bacteria without affecting the rest of the microbial community.

CRISPR-Cas Antimicrobials: Researchers are designing CRISPR-based systems that target

essential genes in antibiotic-resistant bacteria, leading to their selective destruction. This
approach could help combat antibiotic-resistant infections by providing a highly targeted method
of killing harmful bacteria.

PHAGE THERAPY ENHANCEMENT

Bacteriophages (viruses that infect bacteria) are being used to combat bacterial infections in
humans, animals, and plants. CRISPR-Cas can be applied to modify bacteriophages, enhancing
their specificity and effectiveness in targeting bacterial pathogens.

CRISPR-Enhanced Phage Therapy: By engineering phages with CRISPR-Cas systems,

scientists can ensure that the phages will only infect and destroy specific strains of bacteria,
leaving other beneficial bacteria unharmed. This has applications in agriculture, medicine, and
environmental microbiology.

MICROBUIME MANIPULATION

CRISPR-Cas systems are being used to engineer and control microbial communities. The ability
to precisely delete or modify specific genes in microbes enables scientists to modulate the
composition and function of microbial populations, with potential applications in:

Fermentation: Optimizing microbial communities to enhance the production of food products

(e.g., yogurt, cheese, beer) or biofuels through fermentation.
Gut Health: Using CRISPR to selectively eliminate harmful bacteria from the gut while
promoting the growth of beneficial microbes, potentially offering new treatments for
gastrointestinal disorders.

PATHOGEN DETECTION

CRISPR-based systems are being harnessed to develop highly sensitive and rapid diagnostic
tools for detecting pathogens. For instance, CRISPR-Cas12 and CRISPR-Cas13 systems are
being used to detect viral and bacterial infections by recognizing and cleaving specific nucleic
acids.

SHERLOCK and DETECTR: These CRISPR-based diagnostic tools allow for the detection of
viruses like COVID-19 or bacteria such as Mycobacterium tuberculosis. The detection process
can produce a visible signal (e.g., color change), making it a simple and effective tool for field
diagnostics.

BIOSENSING

CRISPR systems are also used in biosensors, devices that can detect the presence of specific
molecules or pathogens in various environments, including clinical samples, water, or food
products.

Environmental Monitoring: CRISPR-based biosensors can help monitor water quality by

detecting microbial contamination or harmful bacteria.

CONCLUSION

The CRISPR-Cas system is a versatile and powerful tool that has had a profound impact on
microbiology, biotechnology, and genetic engineering. Its ability to accurately and efficiently
target specific DNA sequences allows scientists to manipulate microbial genomes, fight
antibiotic-resistant bacteria, develop diagnostic tools, and even engineer microbial communities
for various biotechnological applications. The broad range of CRISPR applications continues to
grow as researchers develop new strategies for using this technology in science and medicine.
REFERENCES

1. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012).
Science, 337(6096), 816-821.

2. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.
A., & Horvath, P. (2007)

3. Wright, A. V., Nuñez, J. K., & Doudna, J. A. (2016). Biology and applications of CRISPR
systems: Harnessing nature's toolbox for genome engineering. Cell, 164(1-2), 29-44.

4. Bikard, D., Euler, C. W., Jiang, W., Nussenzweig, P. M., Goldberg, G. W., Duportet, X.,
Fischetti, V. A., & Marraffini, L. A. (2014). Exploiting CRISPR-Cas nucleases to produce
sequence-specific antimicrobials. Nature Biotechnology, 32(11), 1146-1150.

5. Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J.,
Verdine, V., Donghia, N., Daringer, N. M., Freije, C. A., & Myhrvold, C. (2017). Nucleic acid
detection with CRISPR-Cas13a/C2c2. Science, 356(6336), 438-442.

6. Koonin, E. V., Makarova, K. S., & Zhang, F. (2017). Diversity, classification and evolution of
CRISPR-Cas systems. Current Opinion in Microbiology, 37, 67-78.

7. Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9
for genome engineering. Cell, 157(6), 1262-1278.