it is related to CRSIPR cas system

1. CRISPR PURIFICATION AND MULTIPLEXIBLE
CRISPR EXPRESSION SYSTEMS
SUBMITTED SUBMITTED
TO BY
DR. DIVYA BHATIA ISHA
MTech BIOTECH
2ND SEM, 252354003

2. CRISPR
• CRISPR stands for Clustered Regularly Interspaced Short Palindromic
Repeats.
• Its derived from a natural defense mechanism found in bacteria that
helps them find off virus by stopping snippets of viral DNA and using
them to recognize and destroy viral invaders in future.
• It is a gene editing technology.
• It was first discovered in archae and later in bacteria by Franciso Mojica,
a scientist at the University of Alicante in Spain

3. CRISPR PURIFICATION
• It involves isolating the CRISPR components mainly cas proteins and
guide RNAs from a mixture to obtain highly pure and active samples.
• CRISPR – it refers to specific DNA sequences in bacteria, that contain
short, repeated segments of DNA interspersed with spacer sequences
derived from past encounters with virus or foreign particles.
• Cas9 – it is a enzyme that acts as a molecular part of scissors. It can cut
DNA at specific locations determine by a short RNA
molecule(guideRNA).
• Guide RNA – it is designed to complementary to a target DNA
sequence within the genome.

4. TWO STEP METHOD TO OBTAIN HIGHLY
PURE CAS-9 NUCLEASE
• A. CELL LYSIS AND ISOLATION OF SOLUBLE PROTEINS
• The first step involves growing bacterial cells which express the desired
CRISPR components. Cells are typically grown in culture media under
conditions conducive to protein expression.
• After reaching the desired density, cells are harvested by centrifugation or
filtration and lysed to release the intracellular components.
• Most commonly method used for cell lysis are sonication, French press or
chemical lysis using detergents and enzymes.
• After cell lysis, cell debris or insoluble components or other impurities are
removed by centrifugation at high speed.

5. B. AFFINITY CHROMATOGRAPHY
• It is used to capture the CRISPR componets(cas protein or gRNA).
• For Cas proteins, commonly used affinity tags include polyhistidine (His-
tag), Strep-tag, or maltose-binding protein (MBP), which allows specific
binding to corresponding affinity resins.
• For gRNAs, purification can be achieved using hybridization-based
methods with complementary oligonucleotides immobilized on a solid
support.
• After binding the resin is washed to remove impurities and the purified
crispr components is eluted using a competitive agent that disrupts the
interaction between the tag and the affinity resin.
• The bound proteins or nucleic acids are then eluted under conditions
that disrupt the interaction between the target and the affinity resin.

6. C. CATIONIC EXCHANGE CHROMATOGRAPHY
• This is an optional step.
• It is used for those that are positively charged or have a net positive charge at
the ph and ionic conditions used in the purification process. It relies on the
reversible interaction between the positive charged molecules analytes and
negatively charged functional ligands immobilized on a solid support resin.
• The resin typically contains sulfonic acid and carboxylic acid group that are
negatively charged under the buffer conditions used in the purification process.
• The CRISPR samples contain positively charged components such as the cas
protein , is loaded onto the column. Under the equilibration conditions, the
positively charged CRISPR components bind to the negatively charged
functional groups on the resin via electrostatic interactions.
• After sample loading, the column is washed with a buffer solution to remove
unbound or weakly bound impurities. The washing step helps to increase the
purity of the bound CRISPR components while removing contaminants.

7. D. BUFFER EXCHANGE AND PROTEIN CONCENTRATION
• Eluted fractions often contain high concentrations of salts and other buffer components used during
purification, which can interfere with downstream applications. It is crucial for removing salts.
• Buffer exchange are performed using techniques such as dialysis, gel filtration chromatography, or
ultrafiltration.
• Buffer exchange methods aim to replace the elution buffer containing high concentrations of salts and
other unwanted components with a suitable storage or reaction buffer that is compatible with
downstream applications.
• Dialysis:
• Dialysis involves placing the purified sample in a semipermeable membrane bag or tubing and
immersing it in a large volume of the desired buffer solution.
• Small molecules such as salts and buffer components can diffuse across the membrane, while the larger
CRISPR components remain inside the bag.
• The buffer solution is changed periodically to facilitate the removal of contaminants until the desired
level of desalting is achieved.

8. • Gel Filtration Chromatography:
• Gel filtration chromatography, also known as size exclusion chromatography, separates
molecules based on their size.
• The purified CRISPR components are applied to a column packed with porous beads.
• As the sample passes through the column, smaller molecules are slowed down by
entering the pores of the beads, while larger molecules pass through more quickly.
• The elution buffer used for chromatography typically lacks salts and is compatible with
downstream applications, thus achieving buffer exchange during the process.
• Ultrafiltration:
• Ultrafiltration involves passing the purified sample through a semipermeable
membrane with a defined molecular weight cutoff (MWCO).
• The membrane retains molecules above a certain size (e.g., proteins, nucleic acids)
while allowing smaller molecules (e.g., salts, small solutes) to pass through.
• The purified sample is concentrated and simultaneously exchanged into a desired
buffer by applying pressure or centrifugation.

9. MULTIPLEXIBLE CRISPR EXPRESSION SYSTEMS
• It refers to a versatile platform that allows for the simultaneous expression of
multiple CRISPR components, such as Cas proteins and guide RNAs, to achieve
precise genome editing or other applications.
• Some key features and considerations for a multiplexible CRISPR expression
system:
• Modularity: The system should be modular, allowing for the independent
expression of different CRISPR components. This allows researchers to mix and
match various Cas proteins and guide RNAs according to their experimental
needs.
• Scalability: The system should be scalable to accommodate the expression of
multiple CRISPR components simultaneously. This enables more complex
genome editing tasks, such as multiplex gene knockout or targeted gene
activation/repression.
• Flexibility: Flexibility in the choice of expression vectors, promoters, and
regulatory elements is crucial for adapting the system to different cell types,
organisms, or experimental conditions.

10. • Efficiency: The system should maintain high editing efficiency even when
multiple CRISPR components are expressed simultaneously. This requires careful
optimization of expression levels and stoichiometry of the components.
• Specificity: Ensuring high specificity of genome editing is essential to minimize
off-target effects, especially in multiplex editing scenarios where the likelihood
of off-target events may increase.
• Delivery: Consideration should be given to the method of delivering CRISPR
components into target cells or organisms. This could involve viral vectors, lipid
nanoparticles, electroporation, or other delivery methods depending on the
application and target cell type.
• Characterization: Thorough characterization of the system is necessary to
understand its performance characteristics, including editing efficiency,
specificity, and off-target effects, across different experimental conditions.

11. WORKING OF MULTIPLEXABLE EXPRESSION SYSTEMS
• A multiplexible CRISPR expression system works by utilizing the CRISPR-Cas
gene editing technology to target and modify specific genes within an
organism's genome.
• Designing CRISPR RNA (crRNA):
• The process begins with the design of crRNAs, which are short RNA
sequences that guide the CRISPR-associated protein (Cas) to the target DNA
sequence.
• Each crRNA is designed to be complementary to a specific region of the
target gene's DNA sequence.
• Expression of CRISPR Components:
• The system includes components for expressing both the crRNA and the Cas
protein within the target organism.
• This is typically achieved using plasmids or viral vectors that carry the genetic
sequences encoding these components.

12. • The crRNA and Cas protein sequences are placed under the control of specific promoters, which
regulate their expression levels and timing.
• Delivery of CRISPR Components:
• The CRISPR components are delivered into the target cells or organisms using appropriate
methods such as plasmid transfection, viral transduction, or direct injection.
• Once inside the cells, the expression machinery of the cell reads the genetic information
carried by the plasmids or viral vectors, leading to the production of crRNA and Cas protein.
• Formation of CRISPR-Cas Complex:
• Inside the cell, the crRNA molecules combine with the Cas protein to form a functional
CRISPR-Cas complex.
• Each crRNA within the complex is specific to a particular target gene sequence.
• Targeting and Binding to DNA:
• The CRISPR-Cas complex scans the cell's genome to find DNA sequences that match the
crRNA.
• When the complex encounters a sequence complementary to the crRNA, it binds to the
target DNA through base pairing interactions.
• .

13. • DNA Cleavage and Modification:
• Once bound to the target DNA, the Cas protein within the CRISPR-Cas complex cuts the
DNA at a specific location, known as the protospacer adjacent motif (PAM).
• This cleavage triggers DNA repair mechanisms within the cell, leading to the introduction of
changes in the target gene's sequence.
• Depending on the desired outcome, these changes can include gene knockout, gene
insertion, or precise nucleotide substitutions.
• Multiplexing:
• In a multiplexible CRISPR system, multiple crRNAs targeting different genes or genomic loci
are expressed simultaneously or sequentially.
• Each crRNA directs the Cas protein to a specific target sequence, allowing for the editing of
multiple genes in a single experiment.
• Verification and Analysis:
• After gene editing, the modified cells or organisms are typically subjected to verification
and analysis to confirm the desired genetic changes.
• This may involve techniques such as polymerase chain reaction (PCR), DNA sequencing, and
functional assays to assess the efficacy and specificity of the CRISPR-mediated
modifications.

14. REFERENCES
• Wikipedia
• N.Rajagopalan, Kagale, Pankaj Bhowmik and Halim SongA Two-Step
Method for Obtaining Highly Pure Cas9 Nuclease for Genome Editing,
Biophysical, and Structural Studies