NUCLEIC ACID

AMPLIFICATION TECHNIQUES
&CRISPR

Dr Mohammed Alshehri
• Recombinant DNA Technology
– Intentional modification of organisms’ genomes for
practical purposes
– Three goals
– Eliminate undesirable phenotypic traits
– Combine beneficial traits of two or more organisms
– Create organisms that synthesize products humans
need
Figure 8.1 Overview of recombinant DNA technology
Bacterial cell
DNA containing
gene of interest

Bacterial Plasmid
chromosome

Isolate plasmid. Gene of interest

Enzymatically cleave
DNA into fragments.

Isolate fragment
with the gene of
interest.

Insert gene into plasmid.

Insert plasmid and gene into

bacterium.

Culture bacteria.

Harvest copies of
Harvest proteins
gene to insert into
coded by gene
plants or animals

Eliminate Create Produce vaccines,

undesirable beneficial antibiotics,
phenotypic
traits of traits
MDufilho
combination hormones, or
enzymes
The Tools of Recombinant DNA Technology

• Mutagens
– Physical and chemical agents that produce
mutations
– Scientists utilize mutagens to
– Create changes in microbes’ genomes to change
phenotypes
– Select for and culture cells with beneficial
characteristics
– Mutated genes alone can be isolated
The Tools of Recombinant DNA Technology

• The Use of Reverse Transcriptase to

Synthesize cDNA
– Isolated from retroviruses
– Uses RNA template to transcribe molecule
of cDNA
– Easier to isolate mRNA molecule for desired
protein first
– mRNA of eukaryotes has introns removed
– Allows cloning in prokaryotic cells
The Tools of Recombinant DNA Technology

• Synthetic Nucleic Acids

– Molecules of DNA and RNA produced in cell-
free solutions
– Uses of synthetic nucleic acids
– Elucidating the genetic code
– Creating genes for specific proteins
– Synthesizing DNA and RNA probes to locate
specific sequences of nucleotides
– Synthesizing antisense nucleic acid molecules
The Tools of Recombinant DNA Technology

• Restriction Enzymes
– Bacterial enzymes that cut DNA molecules only at
restriction sites
– Categorized into two groups based on type of cut
– Cuts with sticky ends
– Cuts with blunt ends
Figure 8.2 Actions of restriction enzymes-overview
The Tools of Recombinant DNA Technology

• Vectors
– Nucleic acid molecules that deliver a gene into
a cell
– Useful properties
– Small enough to manipulate in a lab
– Survive inside cells
– Contain recognizable genetic marker
– Ensure genetic expression of gene
– Include viral genomes, transposons, and
plasmids
Figure 8.3 Producing a recombinant vector mRNA for human
growth hormone (HGH)
Antibiotic Restriction
resistance site
gene
Reverse
transcription

cDNA for HGH

Plasmid (vector)

Restriction Restriction
enzyme enzyme

Sticky ends

Gene for human

growth hormone

Ligase

Recombinant plasmid

Introduce recombinant
plasmid into bacteria.

Bacterial Recombinant
chromosome plasmid

Inoculate bacteria
on media containing
antibiotic.
Bacteria containing
the plasmid with
HGH gene survive
MDufilho because they also
have resistance gene.
The Tools of Recombinant DNA Technology

• Gene Libraries
– A collection of bacterial or phage clones
– Each clone in library often contains one gene of
an organism’s genome
– Library may contain all genes of a single
chromosome
– Library may contain set of cDNA
complementary to mRNA
Figure 8.4 Production of a gene library-overview

Genome

Isolate genome
or organism.

Generate fragments using

restriction enzymes.

Insert each fragment

into a vector.

Introduce vectors
into cells.

Culture recombinant cells;

descendants are clones.
CRISPR CAS : A NEW GENOME
EDITING TOOL
Introduction

• Genome editing, or genome editing with engineered

nucleases (GEEN) is a type of genetic engineering in which
DNA is inserted, replaced, or removed from a genome using
artificially engineered nucleases, or "molecular scissors”.

• The nucleases create specific double-strand breaks (DSBs)

at desired locations in the genome and harness the cell’s
endogenous mechanisms to repair the induced break by
natural processes of homologous recombination (HR) and
non-homologous endjoining (NHEJ)
• CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeat) has opened new era in
biotechnology.

• Poised to transform developmental biology with single

solution to many problems.

• Provides simple, easy, cost effective and efficient

access to manipulate virtually any part of the genome
of any organism.

• Widely accepted by academics and research

organizations- led to CRISPR Craze.
Why genome editing?

• To understand the function of a gene or a protein, one interferes with

it in a sequence-specific way and monitors its effects on the organism.
• In some organisms, it is difficult or impossible to perform site-specific
mutagenesis, and therefore more indirect methods must be used,
such as silencing the gene of interest by short RNA interference
(siRNA).
• But sometime gene disruption by siRNA can be variable or
incomplete.
• Nucleases such as CRISPR can cut any targeted position in the
genome and introduce a modification of the endogenous sequences
for genes that are impossible to specifically target using conventional
RNAi.
HISTORY
 CRISPR – Cas systems

• These are the part of the Bacterial immune system which

detects and recognize the foreign DNA and cleaves it.
i. THE CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeats) loci
ii.Cas (CRISPR- associated) proteins can target and cleave
invading DNA in a sequence – specific manner.

• A CRISPR array is composed of a series of repeats

interspaced by spacer sequences acquired from invading
genomes.
 Components of CRISPR

1) Protospacer adjacent motif (PAM)

2) CRISPR-RNA (crRNA)
3) trans-activating crRNA (tracrRNA)
Different CRISPR-Cas system in Bacterial Adaptive
Immunity

• Class 1- type I (CRISPR-Cas3) and

type III (CRISPRCas10)
uses several Cas proteins and the
crRNA Class

• 2- type II (CRISPR-Cas9) and type

V (CRISPRCpf1)
employ a large single-component
Cas-9 protein in conjunction with
crRNA and tracerRNA.
Different Cas proteins and their function
What is CRISPR-cas9 system?

– Clustered regularly interspaced short palindromic

repeats
– segments of prokaryotic DNA containing,repetitive
base sequences.
– These play a key role in a bacterial defence system,
– form the basis of a genome editing technology
known as CRISPR-Cas9 that allows permanent
modification of genes within organisms.
– CRISPRs are found in approximately 40% of
sequenced bacterial genomes and 90% of
sequenced archaea
Structure of cas9 protein
Key component
What makes CRISPR system the ideal genome
engineering technology
General protocol for CRISPR
 Conclusion…

• Genome editing tools provide new strategies for genetic manipulation

in plants and are likely to assist in engineering desired plant traits by
modifying endogenous genes.
• Genome editing technology will have a major impact in applied crop
improvement and commercial product development .
• CRISPR will no doubt be revolutionized by virtue of being able to
make targeted DNA sequence modifications rather than random
changes.
• In gene modification, these targetable nucleases have potential
applications to become alternatives to standard breeding methods to
identify novel traits in economically important plants and more
valuable in biotechnology as modifying specific site rather than whole
gene.