CRISPR
TECHNOLOG
Y
PRESENTED BY:-
JHALAK SINGHAL
[Link] BIOTECHNOLOGY(6TH SEM)
CONTENT
Introduction
History of CRISPR
Natural pathway of CRISPR
Mechanism of CRISPR in Biotechnology
Application
Advantages
INTRODUCTION
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats is genome
editing is based on natural immune response used by bacteria to defend
themselves against viruses and a type of adaptive immunity system in prokaryotes.
CRISPR system recognize and cleave complementary DNA sequences allowing
bacteria to destroy viral invaders.
CRISPR are repetitive DNA sequences found in bacteria.
CRISPR Cas system allows to alter the genetic codes of any given organism.
CRISPRs are short DNA sequences each about 30 bases that reads similarly
backwards and forwards thus palindromic and repeat every 35 bases.
A CRISPR array is composed of series of repeats interspaced by spacer sequences.
Palindromic
sequences
Cas gene
Spacer DNA
CRISPER BRIEF
4
PAM:- Protospacer Adjacent Motif, these are specific location of short
DNA sequences of ‘N’GG that are recognized by cas enzyme of
bacteria and marks as target site for CRISPR array
tracrRNA:-small RNA synthesized in bacteria that binds to CRISPR
region to form a complex. It helps in stabilization of cas9 protein with
CRISPR and protospacer complex.
PROTOSPACER:- the region that is found upstream of PAM site of
bacteriophage that is snip off by cas1 and cas2 enzyme that is
incorporated at 5’ DNA of bacteria in spacer region of CRISPR array
Cas 9:- is a protein that acts as endonuclease that cuts invading
phage dsDNA into small fragments that are incorporated in the CRISPR
array as a spacer.
SINGLE GUIDE RNA 6
HISTORY OF CRISPR
• Ishino [Link]. found CRISPR sequences in [Link]
1987
• CRISPR sequences are found to be common in
2000 other microbes
• Coined CRISPR name and defined Cas genes by
2002 Francisco Mojica
• First experimental evidence for CRISPR
2007 adaptive immunity by Barrangou [Link]
• First demonstration of cas9 genome
2013 engineering in eukaryotic cells by Zhang.
NATURAL PATHWAY OF CRISPR
• DNA INVASION: Viral DNA invades the bacterial cell.
• INVADING DNA IS INCORPORATED INTO CRISPR ARRAY: DNA fragments
from invading DNA are incorporated into CRISPR locus as spacers.
• PRE crRNA Transcription: The cell transcribes spacer group into pre
crRNA by RNA polymerase enzyme.
• Guide RNA formation: tracrRNA combined with CRISPR and protospacer
region to form a complex that binds with PAM site of bacteriophage.
• Cas9 activation: inactive cas9 protein produced by CAS gene present
upstream in CRISPR array binds to guide RNA.
• Target binding: the activated guide RNA binds to target DNA
• Target cleavage: the cas9 protein cleaves the invading DNA and
destroys it
9
CRISPR MECHANISM IN
BIOTECHNOLOGY
CRISPR technology in biotechnology begins in 2012 when Jennifer doudna and
Emmanuelle Charpentier proposed that bacterial CRISPR-cas9 system could
be used as programmable toolkit for genome editing in humans and other
animal species.
CRISPR-CAS system has been modified to edit genomes allowing existing genes
to be removed or new ones added.
STEP1:- Identify the target gene
STEP2:- Amplify the target gene using PCR
STEP3:- Ligate gene into CRISPR/CAS plasmid vector
STEP4:- Transform the vector into cell lines
STEP5:- Validate gene modification by PCR
COMPLEX FORMATION
CLEAVAGE OF DNA crRNA and tracrRNA are combined
by fusing them together by linkers
SEQUENCES AT to form single guide RNA(sgRNA).
DESIRED LOCATION The cas9 binds with sgRNA to form
effector molecule to cleave DNA.
CRISPR
TECHNOLOGY
BINDING OF COMPLEX READS DNA
COMPLEX WITH Reads DNA until finds
PAM SEQUENCE appropriate PAM sequence
11
After cleavage natural DNA repair mechanism are initiated.
The cleaved DNA can undergo repair via two routes:-
1)Homology directed repair (HDR)
2)Non homologous end joining (NHEJ)
APPLICATIONS OF CRISPR
TECHNOLOGY
CELL DIFFRENCIATION
CRISPR technology is used to improve cell differentiation and derive a variety of cell types
for transplantation.
GENE THERAPY
This technology is used to knock out mutant proteins and reduce their production. E.g.:
Huntington's disease
INSECT BORNE DISEASE
Insect borne diseases such as malaria is a health concern all over the world,. Researcher's
modify crispr technology to gene drive systems that spread disease resistant genes to
populations.
TYPE-1 DIABATIES
Insulin dependent diabetes is caused by autoimmune destruction of pancreatic β cells. Crispr
technology modifies human pluripotent stem cells and differentiate them into beta cells that produce
insulin and injected in patient.
DISEASE MODELS
Crispr simplifies creation of animals for research that mimic disease or shows what happens when gene
is mutated.
CANCER THERAPY
Crispr has been used in cancer cell lines to repair to delete mutated genes. The first approach was done in patient
with refractory lung cancer in which T cells are extracted and engineered by CRISPR technology to delete PD-1
genes that hinders in cancer elimination and administered into the bloodstream.
GENE ACTIVATION AND SILENCING
This technology is used to artificially regulate a certain target gene through modification of cas9 protein.
AGRICULTURE
Potential tool for developing virus resistance crop varieties and improving natural food production.
It is used to eradicate herbicide resistant weeds and pests, used to develop abiotic and biotic resistant
traits in plants, increase shelf life, improve nutritional content in plants.
E.g.:-
CORN: Targeted mutagenesis
RICE: Targeted mutagenesis
SWEET ORANGE: Targeted gene modification
SOYABEEN: Gene coding
BIOFULES
It helps in improving production of biofuels by manipulating the metabolic pathways of
organisms.
COVID-19
Corona disease caused by severe acute respiratory syndrome coronavirus-2
is the most deadly pandemic in all over the world. This technology is used
for diagnosis and elimination of SARS-CoV-2 as it effectively cleaves RNA
sequences of SARS-CoV-2 in lung epithelial cells.
17
ADVANTAGES OF CRISPR
1)High potency and specificity and efficiency
2)Broad applicability
3)Simple editing tool
4)Multifunctional as it can repair, delete and insert
5)Ability to target multiple DNA sites simultaneously.
6) Faster than older methods
7)Much precise than other editing tools .
8)Less expensive and require less training than other techniques.
THANKYOU
