This document describes the construction of a high capacity adenoviral vector (HCAdV) devoid of all viral genes that can be used to deliver the CRISPR/Cas9 system for genome editing. Researchers developed an intermediate shuttle plasmid containing the CRISPR/Cas9 genes and guide RNAs that could then be inserted into the HCAdV genome. The CRISPR/Cas9 system utilizes the natural bacterial immune system to precisely cut DNA at targeted locations guided by guide RNAs. This vector aims to improve the efficiency of delivering genome edited DNA to cells for applications such as gene therapy.

1. Crispr /Cas 9 delivery with
one adenoviral vector devoid
of all viral genes
Published on : 07 December 2017
Published in : Nature
Eric Ehrke-Schulz, Maren Schiwon,
Theo Leitner,Stephen David

2. •Introduction
•Objectives
•Adenoviral vector
•Construction of HCAdV
•CRISPR/Cas9 technology
a) Introduction
b) Discovery
c) Natural method
d) Current editing with CRISPR tool
e) Overview of both methods
• Finally constructed HCAdV vector
• Results
• Summary
• Applications
• Limitations
• References
Index

3. Introduction
• CRISPR has swept through the scientific world in the last few
years and is now poised for commercial use. To put it simply,
like all the other techniques ,the DNA edited by this technique
has been ligated with plasmids and introduced into host cells
to complete the last basic step of genome editing. Many
plasmids have been used as vectors except “adenovirus”.
• This article focuses on delivery of the CRISPR-edited DNA into
the cells by using an adenoviral vector.
• Here , we are concerned about two things:
1) Construction of HCAdV vector
2) CRISPR/Cas9 technology

4. Objectives
• As described earlier , adenoviral vectors serve many
advantages over normal plasmids and are highly efficient.
Despite this, no attempts have been made to deliver the
CRISPR/Cas9 through HCAdV.
• So the main objective of this research was to construct a
high capacity adenoviral vector (HCAdV) that is devoid of
all viral genes and one which contains the CRISPR/Cas9
system for improved and highly efficient gene therapy.

5. Adenoviral vector
•Stability
•Specificity
•Identification
•Limited no. of vectors
•Immune response
•Gene deletion
•Difficult vector production

6. Figure : Structure of a typical adenovirus. It has an icosahedral structure
and is non-enveloped. It is coated with capsid and contains a double
stranded DNA genome.

7. Figure : A 3 dimensional structure of
adenovirus .

8. I. HCAdV Construction
• There are several novel techniques for the
construction of vectors like:
a) Golden Gate Method
b) Endonuclease gene cloning
c) Recombineering
d) AdEasy Protocol
e) Overlap Recombination
f) Intermediate method

9. Intermediate Method
• Construction of intermediate CRISPR/Cas9 shuttle
plasmids for subsequent cloning or recombineering into
HCAdV genomes:
1) The intermediate shuttle plasmid was constructed by insertion of a
synthetic DNA fragment into the PexK plasmid.
2) The synthetic DNA fragment is composed of a multiple cloning
sites(MCS) flanked by non coding random DNA sequences.
3) This MCS is surrounded by multiple recognition sites for homing
endonucleases1 .
4) This enables the cloning of any insert into the respective restriction
sites in the HCAdV genome.
5) Outside of the endonucleases1 were added recognition sites for the
restriction sites for enzyme Swa1 to release the synthetic DNA
fragment from the PexK plasmid.

10. Figure : Schematic representation of intermediate CRISPR/Cas9
shuttle plasmids for simple gRNA manipulation and multiplexing
and subsequent transfer of the customized CRISPR/Cas9
machinery into the HCAdV genome.

11. 6) Ecor1 restriction digestion was the performed ( for removal of the
original MCS).
7) The resulting shuttle plasmid(pShV) served as the intermediate shuttle
plasmid for the CRISPR/Cas9 incorporation into the HCAdV genome.
8) The next step involves insertion of the CRISPR/Cas9 system genes which
will then finish our objective of research of creating a vector for efficient
gene transfer of genome edited through the CRISPR/Cas9 technology.
9) gRNA customisation was performed and was inserted into the respective
restriction site within the plasmid (pShV) genome .
10) After the successful insertion of CRISPR/Cas9 casette into the shuttle
vector , last step involves the transfer of customised CRISPR/Cas9
transgenes into the HCAdV genomes.This process is called
recombineering.

12. Figure : Workflow for gRNA customization and multiplexing of the
CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are
separately inserted between the BsaI restriction enzyme sites . Step 2: Customized
gRNA expression units gRNA1 and gRNA2 are amplified. Step 3: gRNA1 and 2 are
inserted into the respective restriction enzyme site.

13. Figure : (C) Transfer of customized CRISPR/Cas9 transgenes into the
HCAdV genomes. This can be done by either endonuclease guided
cloning or by recombining simply.

14. II. CRISPR technology
Introduction:
• This stands for Clustered Regularly Interspaced Short
Palindromic Sequences.
• The CRISPR/Cas9 technology is based on a natural system
used by bacteria to protect themselves from viral infections.
Discovery:
• The discovery of clustered DNA repeats occurred
independently in three parts of the world. The first
description of what would later be called CRISPR is
from Osaka University researcher Yoshizumi Ishino and his
colleagues in 1987. The function of the interrupted clustered
repeats was not known at the time.

15. Major contribution:
Jennifer Anne Doudna (an American biochemist) has been a
leading figure in what is referred to as the "CRISPR revolution"
for her fundamental work and leadership in developing CRISPR-
mediated genome editing. In 2012 Doudna and Emmanuelle
Charpentier were the first to propose that CRISPR/Cas9--
enzymes from bacteria that control microbial immunity--could
be used for programmable editing of genomes,[which is now
considered one of the most significant discoveries in the history
of biology.
• In 1993 researchers of Mycobacterium tuberculosis in the
Netherlands published two articles about a cluster of
interrupted direct repeats (DR) in this bacterium.

16. CRISPR/Cas
9
Components
Cas9 nuclease
protein.
gRNA
The concerned
Genome.
PAM
sequence
Desired
sequence

17. Description of components.
• DNA to be edited: The concerned genome to be edited has to
be sequenced so as to know the exact location/ portion of
nucleotides where the defected gene is present. This is the basic
and the most important step in genome editing procedure and
needs to be performed with precision.
• Desired gene : Once the defected portion of nucleotides is
known, they can be simply silenced , deactivated or replaced
by a good/desired gene instead.

18. • Cas9 : This stands for CRISPR associated protein9 . It is a RNA
guided DNA endonuclease protein . It has a crystalloid structure.
Originally isolated from bacteria, it memorizes and performs site
directed DSBs in the DNA to be edited..Its functions by recognizing a
PAM sequence and makes cuts just upstream of it. It is almost always
associated with the sgRNA which guides it.
• PAM sequence: This stand for Protospacer adjacent motif
(PAM) and is a 2-6 base pair DNA sequence immediately
following the DNA sequence targeted by the Cas9nuclease
.Cas9 will not successfully bind to or cleave the target DNA
sequence if it is not followed by the PAM sequence.PAM is a
component of the invading virus or plasmid, but is not a
component of the CRISPR locus.

19. Figure : Diagrammatic representation of the working structure in
CRISPR/Cas9 tool. The figure shows (1) The genomic DNA to be
edited.
(2) The guide RNA (3) The Cas9 enzyme forming a complex with the
sgRNA .

20. .
•gRNA: This stands for Guide RNA and is actually composed of two disparate
RNAs that associate to form the guide- the CRISPR RNA( cRNA) and the trans-
activating RNA( tracrRna).These two RNAs are naturally occuring. It is generally 20
nucleotide long and is made chemically once the DNA sequence is known. Its
primary function is to mediate cleavage of DNA through Cas9.
Figure : Exemplified structure of a typical gRNA . Nucleotides 1 to 32 represents the
naturally occurring crRNA while the nucleotides 37 to 100 represents the naturally
occurring tracrRNA .

22. 1)The original CRISPR/Cas9 system is a natural method used by E.coli and
several other bacterium as protection against bacteriophages.
2)Bacteriophages attack randomly on E.coli
3)When the bacterium detects the presence of viral DNA it produces two types of
short RNA , one of which matches a portion of the sequence of the invading virus.
4) These two RNAs form a complex with a protein called Cas9. The RNAs
together are called the gRNA.
5) Cas9 is a nuclease ( ability to cut DNA) present within the bacterial cell. When
the matching sequence produced by the bacterium finds its complementarity in
the viral DNA it starts moving towards it .
6) As the gRNA is complexed with the Cas9 protein, its also moves with it .The
complex will lock itself unto a short sequence known as the PAM.
7)The Cas9 will unzip the vDNA and match it to its target RNA.
8)When the match is complete, the Cas9 protein cuts the vDNA disabling the
virus. Thus the bacterium is protected against the bacteriophage
The Natural Method Used By E.coli

23. Figure : Schematic representation showing infection by bacteriophage and
subsequent CRISPR/Cas9 pathway followed by the bacterium ( here E. coli) as a
method to protect the cell from viral infection. The process of CRISPR/Cas9 sets in
as soon as the cell detects the presence of viral DNA within it.

24. Figure : Schematic representation showing infection by bacteriophage and
subsequent CRISPR/Cas9 pathway followed by the bacterium ( here E. coli) as a
method to protect the cell from viral infection. The process of CRISPR/Cas9 sets in
as soon as the cell detects the presence of viral DNA within it.

25. Modified/ Current Method For Gene
Editing
Researchers studying this system realized that it could be
engineered to cut not just viral DNA but any DNA sequence at a
precise location by changing the gRNA to match the DNA portion.
Principle:
The gRNA is designed specifically so as to match the
defected portion of DNA . The Cas9 protein cuts the DNA and
a good gene is then ligated into the genome. This forms the
basic principle of genome editing through the CRISPR/Cas9
technology.

26. Figure: Current method of genome editing by insertion
of donor (good gene) into the cut portion .

27. Explanation/Procedure
The current genome editing done through this technique is a
slight modification of the natural system described earlier.
1) The DNA to be edited is sequenced so as to know the
defected portion of the gene which needs to be corrected.
2)The PAM sequence now needs to be inserted just upstream
of the location that needs to be cleaved.
3)The gRNA is synthesized accordingly.(~ 15 to 20 nt)
4)The Cas9 nuclease is modified so that it cuts the DNA
specifically at the concerned location.
5) All this can be done in cultured cells , in test tubes where
the gRNA will form a complex with the endonuclease.

28. 6) Once this is done, the natural method of CRISPR/Cas9 sets in.
The gRNA guides the nuclease and this complex binds onto it
7) Nuclease identifies the PAM sequence on the DNA and cuts
the DNA selectively at a site just upstream of the PAM ( which is
actually the recognition sequence for Cas9 nuclease).
8)The mutated/defected portion of DNA causing the disease is
now destructed.Cas9 complex is removed.
9 )Within the same culture/cell a good gene is then inserted in
place of that sequence and the genome is corrected.
10 )Now, this corrected/edited DNA can now be inserted into
stem cells, the host, bone marrow cells, embryonic cells with the
help of vectors.

29. Figure : Processing of the CRISPR/Cas9 system in a cell.

30. Figure : Workflow model of CRISPR/Cas9 tool. The tool can be used in many
different ways including the two given above (1) The defected gene is cleaved
and the ends are joined by ligase .such as gene is said to be deactivated or
silenced (2) Instead of gene silencing, a good gene can be inserted in place of
the deleted gene. Thus the gene is edited .

31. Overview

32. Constructed vector

33. Transfer of HCAdv vector

34. Summary

36. Applications
One of the most common application of CRISPR/Cas9 is in
silencing of genes. Once, the dsB is made repair is done by NHEJ
and this leads to permanent termination of gene and hence the
defected gene is silenced.
Another common application is genome editing.
It promises the cure of sensitive inherited disorders such as
Sickle cell anemia.
CRISPR/Cas9 has been used to modify the gene responsible for
β-Thalassaemia( potentially fatal blood disease) by modifying
the HBB gene.

37. Germline changes to avoid/ prevent genetic disorders
To study the role and mechanisms of action of specific genes
or gene pathways.
Correcting dominant and recessive mutations.
Production of disease resistant plants and ones which are
highly nutritious.
Another , not so famous use lies in transcriptional activation
or repression i.e. CRISPRa and CRISPRi. Tere have been many
research groups who have developed ribonucleoprotein complex
that interfere o activate transcription process and consequently
either the concerned protein is not translated.

38. Production of Gene-edited CRISPR mushroom having prolonged
usage time .
Crispr has been used in humane genome editing where Chinese
scientists have genetically modified human embryos to reduce
miscarriages.
Used in Gene Knock-Out Procedures.
Used in casFISH (Labelling of DNA) : DNA fluorescent in situ
hybridization is widely accepted technique for labeling specific DNA
sequences. However, The standard protocol requires many harmful
chemicals and thus a non-toxic, cost effective FISH protocol was
made using Cas9.
Lactose intolerance or gluten intolerance can be checked with
the help of this technology.

39. Limitations
•Non-Specificity
•Toxicity
•Limited research
•Delivery
•Immuno-rejection
•Side effects
•Improper Target recognition
•Ethical issues

40. References
•www.nature.com/ scientificreports
•Genome editing techniques for gene and cell therapy : Maedar et
al
•www.biotechnologynotees.com/Notes on genomic DNA editing.
•Crispr/cas9 technology( Article by laboratory of Neuronal
communication)
•www.youtube.com/ Crispr/Cas9 by Shomu’s Biology
•www.youtube.com/genome editing with CRISPR-Cas9 / Mc-
Govern Institute
•www.biologydiscussion.com/ Genome editing