Scribd
Upload
103 views16 pages

Crispr Technique

The CRISPR-Cas9 system is a genome editing tool that originated from a bacterial immune system. It allows DNA to be cut at specific locations determined by a guide RNA. Bacteria use this system to defend against viruses by integrating viral DNA into their own, then using CRISPR RNAs and Cas9 to cut invading viruses. Researchers have adapted this system to edit genomes in other organisms. While powerful, CRISPR-Cas9 has limitations like off-target effects. A safer alternative called NICER uses Cas9 nickase to induce multiple nicks in the DNA rather than breaks, minimizing unintended mutations during repair.

Uploaded by

Ammar Abbas
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
103 views16 pages

Crispr Technique

The CRISPR-Cas9 system is a genome editing tool that originated from a bacterial immune system. It allows DNA to be cut at specific locations determined by a guide RNA. Bacteria use this system to defend against viruses by integrating viral DNA into their own, then using CRISPR RNAs and Cas9 to cut invading viruses. Researchers have adapted this system to edit genomes in other organisms. While powerful, CRISPR-Cas9 has limitations like off-target effects. A safer alternative called NICER uses Cas9 nickase to induce multiple nicks in the DNA rather than breaks, minimizing unintended mutations during repair.

Uploaded by

Ammar Abbas
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
  • Title Page: Displays the title and authors of the document, indicating the focus on the Crispr/Cas9 system.
  • Introduction: Introduces the CRISPR-Cas9 system, describing its role in adaptive immunity in prokaryotes and its genomic presence.
  • CRISPR Structure: Illustrates the structure and arrangement of CRISPR sequences, highlighting repeats and spacers.
  • Mechanism Details: Explains the components of the CRISPR-Cas9 system and how it interacts with DNA, emphasizing components like PAM.
  • CRISPR Arrays and Function: Details the natural function of CRISPR arrays in bacterial immune defense strategies.
  • CRISPR-Cas9 Principle: Describes the stages of CRISPR-Cas9 activity, including acquisition, expression, and interference phases.
  • CRISPR Components: Depicts the components required for CRISPR functionality, focusing on tracrRNA and crRNA interaction mechanisms.
  • DNA Repair Mechanism: Explores the DNA repair mechanisms post-Cas9 cleavage, including NHEJ and HDR pathways.
  • Genome Editing Potential: Highlights CRISPR-Cas9 as a genome editor, focusing on its precision and efficiency in genetic modifications.
  • Research and Development: Discusses the development of guide RNA technology and its specificity in genome targeting.
  • CRISPR Applications: Outlines the current and prospective applications of CRISPR/Cas9 across various fields such as medicine and agriculture.
  • CRISPR Limitations: Examines the challenges and inefficiencies in CRISPR technology, including delivery issues and inaccuracy.
  • CRISPR vs. Alternatives: Compares CRISPR/Cas9 with newer methods to address the drawbacks of unintended DNA changes.
  • The NICER Method: Introduces the NICER method as a safer alternative to CRISPR-Cas9 using multiple nicks and homologous repair.

Crispr / Cas9 system

Prepared by
Ammar H. Abbas
Supervised by
Dr. Raya ALSaade
Introduction
 CRISPR- Cas9 : is clustered, regularly interspaced, short
palindromic repeat and the associated Cas9 protein is
naturally an adaptive immunity mechanism in
prokaryotes.
 CRISPRs are found in approximately 40% of sequenced
bacterial genomes and 90% of sequenced archaea.
Clustered Regularly Interspaced Short Palindromic Repeats
Repeat Spacer Spacer Spacer

Bacteriophage
 Cas 9 is an endonuclease, first identified from Streptococcus
pyogenes bacteria. It’s genes are often located next to
CRISPR repeat-spacer arrays.
 Specificity of CRISPR-Cas9 depends on the presence of a
sequence-specific Protospacer Adjacent Motif (PAM) and
target sequence (20 bases).
 Absence of PAM in host genome enable to avoid self-
cleavage.
• CRISPR-Cas9 was adapted from a naturally occurring genome editing
system that bacteria use as an immune defense.
• When infected with viruses, bacteria capture small pieces of the viruses'
DNA and insert them into their own DNA in a particular pattern to create
segments known as CRISPR arrays.
• The CRISPR arrays allow the bacteria to "remember" the viruses (or
closely related ones). If the viruses attack again, the bacteria produce
RNA segments from the CRISPR arrays that recognize and attach to
specific regions of the viruses' DNA.
• The bacteria then use Cas9 or a similar enzyme to cut the DNA apart,
which disables the virus.
The Principle

Three Stages of CRISPR-Cas9


 Stage I: Acquisition- DNA fragments of invading phages are incorporated into the host
CRISPR locus as spacers between repeats.

 Stage II: Expression/Biogenesis- Cas proteins are expressed, the CRISPR array
containing acquired spacers is transcribed into pre-crRNA, and then cleaved into
mature crRNAs.

 Stage III: Cleavage/Interference- Cas proteins recognize the appropriate target with
the guidance of the crRNA and mediate the cleavage of the invading genome to
generate double strand brakes (DSB) at 3 bp upstream of PAM sequence.
1
2
Phage Phage

Phage genes
CRISPR RNAs
Phage genes

Transcription
Repeat Repeat Repeat Repeat Repeat Repeat Repeat Repeat Repeat Repeat Repeat Repeat

CRISPR region CRISPR region


Components of
Crispr system
Double Strand Break is
generated, then
What???

Nonhomologous DNA end joining (NHEJ) homology-directed repair

insertions/deletions (indels) single-stranded oligodeoxynucleotide (ssODN)


CRISPR-Cas9 – a highly specific genome editor

Knock genes out – disable them

Precisely replace genes or insert


new ones

Increase or decrease expression of


genes

Faster, cheaper, more accurate, more efficient.


Genome editing
• In 2012, researchers demonstrated that RNAs could be
constructed to guide a Cas nuclease (Cas9 was the first
used) to any DNA sequence. The so-called guide RNA can
also be made so that it will be specific to only that one
sequence, improving the chances that the DNA will be cut
at that site and nowhere else in the genome. Further testing
revealed that the system works quite well in all types of
cells, including human cells.
CRISPR/Cas9 Current and Prospective Application
Gene Therapy and
Other Therapeutic

Gene Function Study Agriculture


GMO food

Current and
Disease Vector Control Biofuel Industry
Prospective
Application

Enhancement Gene
Organ Transplantation Edition

Animal Disease
Model

12
Limitations
CRISPR/Cas is an extremely powerful tool, but it has important
limitations. It is:

• difficult to deliver the CRISPR/Cas material to mature cells in


large numbers, which remains a problem for many clinical
applications. Viral vectors are the most common delivery
method.
• not 100% efficient, so even the cells that take in CRISPR/Cas
may not have genome editing activity.
• not 100% accurate, and “off-target” edits, while rare, may
have severe consequences, particularly in clinical applications.
What is better than Crispr?

• Traditional CRISPR/Cas9 editing uses guide RNAs. The guide RNAs target a
specific section of the DNA and the Cas9 enzyme initiates a break in the
double-stranded DNA structure at this location. This break is key for initiating
changes to the DNA. However, cellular repair of double-strand breaks can
lead to unintended DNA mutations, as well as the integration of exogenous
DNA to the human genome, which raises safety concerns for clinical
applications of CRISPR/Cas9 technology.
A safer alternative
• To minimize these unintended mutations, the Osaka University-led
research team proposed NICER.
• multiple NIcks (MNs) induced by Cas9 nickase and a
homologous Chromosome as an Endogenous Repair template.
• Because the NICER method does not involve DNA double-strand breaks
or the use of exogenous DNA, this technique appears to be a safe
alternative to conventional CRISPR/Cas9 methods. NICER may represent
a novel approach for the treatment of genetic diseases caused by
heterozygous mutations.
NICER

Crispr / Cas9 system Prepared by Ammar H. Abbas Supervised by Dr. Raya ALSaade
Introduction CRISPR- Cas9 : is clustered, regularly interspaced, short palindromic repeat and the associated Cas9 protein i
Clustered Regularly Interspaced Short Palindromic Repeats Bacteriophage Repeat Spacer Spacer Spacer
Cas 9 is an endonuclease, first identified from Streptococcus pyogenes bacteria. It’s genes are often located next to CRI
• CRISPR-Cas9 was adapted from a naturally occurring genome editing system that bacteria use as an immune defense. • When i
The Principle Three Stages of CRISPR-Cas9  Stage I: Acquisition- DNA fragments of invading phages are incorporated into the
Repeat Repeat Repeat Repeat Repeat Repeat Phage CRISPR region Phage genes 1 2 Repeat Repeat Repeat Repeat Repeat Repeat Phage
Components of Crispr system
Double Strand Break is generated, then What??? insertions/deletions (indels)
CRISPR-Cas9 – a highly specific genome editor Faster, cheaper, more accurate, more efficient. Knock genes out – disable them

You might also like