Genome editing methods such as ZFNs, TALENs, and CRISPR/Cas9 use engineered nucleases to create targeted double-stranded breaks in DNA which are then repaired through endogenous cellular processes. These nucleases can be used to modify genomes through techniques like gene knockout, targeted mutation insertion/deletion/correction, and studying gene function. CRISPR/Cas9 uses a guide RNA and Cas9 nuclease to target specific DNA sequences for editing. The four main steps for CRISPR are: 1) selecting target sequences near a PAM site, 2) designing and cloning gRNA, 3) delivering Cas9 and gRNA into cells, and 4) DNA repair after cleavage results in gene modification

1. In the role of
God
Myths and realities of
synthetic biology
© Vasyl Mykytyuk, 2014

2. You always have
a choice.
And you make it
every day

3. “Synthetic biology is an emerging area of research that can broadly be
described as the design and construction of novel artificial biological
pathways, organisms or devices, or the redesign of existing natural
biological systems.” UK Royal society

5. Monday 31 March 2014

8. HOW REAL
IS IT?

13. Кого і коли клонували?
1970 р. жаба (Англія)
1985 р. кісткові риби(СРСР)
1996 р. вівця Доллі (Шотландія)
1998 р. корова (Японія)
1999 р. козел (США)
2001 р. кішка (США)
2002 р. кролик (Бельгія)
2003 р. олень, бик (бантенг - дикий бик,
тварина, яка вимерла) і мул (всі - в США)
2005 р. Собака ( Півд. Корея)
2009 р. верблюд (ОАЕ)
2011 р. Койот (Півд. Корея)

14. one from 277 attempts

16. Somatic nuclear transfer technique

17. One reason of low efficiency of somatic nuclear transfer is
abnormal reprogramming

18. The first hybrid human clone was
created in November 1998, by
Advanced Cell Technology. It was
created using SCNT - a nucleus was
taken from a man's leg cell and
inserted into a cow's egg from which
the nucleus had been removed, and
the hybrid cell was cultured, and
developed into an embryo. The
embryo was destroyed after 12 days.
On January 2008, Dr. Andrew French
and Samuel Wood of the
biotechnology company Stemagen
announced that they successfully
created the first five mature human
embryos using SCNT. In this case,
each embryo was created by taking a
nucleus from a skin cell (donated by
Wood and a colleague) and inserting
it into a human egg from which the
nucleus had been removed. The
embyros were developed only to the
blastocyst stage, at which point they
were studied in processes that
destroyed them.

20. 5 June 2014 Young Gie Chung
Human Somatic Cell Nuclear Transfer
Using Adult Cells

21. Somatic cell nuclear transfer (SCNT) Vs Induced pluripotent stem cells (iPSCs)
Usage in therapeutic cloning

22. To be
continued…

23. What is stored in DNA?
Or what can be stored?

24. 2012

25. Jun 25, 2013
In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation
Michael C Jewett, Brian R Fritz, Laura E Timmerman and George M Church

26. Експеримент Міллера-Юрі – одна з перших спроб створити
живу систему

27. Adam P. Johnson, H. James Cleaves, Jason P. Dworkin, Daniel P. Glavin, Antonio
Lazcano, and Jeffrey L. Bada.
The Miller Volcanic Spark Discharge Experiment. - Science, 2008

30. Are you
satisfied with
your genetic
code?

31. A semi-synthetic organism with an expanded genetic alphabet
Denis A. Malyshev, Kirandeep Dhami, Thomas Lavergne, Tingjian Chen, Nan Dai, Jeremy M. Foster,
Ivan R. Corrêa & Floyd E. Romesberg
Nature 509, 385–388 (15 May 2014)

32. Science 18 October 2013:
Genomically Recoded Organisms
Expand Biological Functions
Marc J. Lajoie, Alexis J. Rovner, Daniel B.
Goodman, Hans-Rudolf Aerni, Adrian D.
Haimovich, Gleb Kuznetsov, Jaron A.
Mercer, Harris H. Wang, Peter A. Carr,
Joshua A. Mosberg, Nadin Rohland, Peter
G. Schultz, Joseph M. Jacobson, Jesse
Rinehart, George M. Church, Farren J.
Isaacs

33. Total Synthesis of a Functional
Designer Eukaryotic
Chromosome
Narayana Annaluru et al.
Science 4 April 2014: 55-58.Published online
27 March 2014

36. Genome editing methods
Made by Vasyl Mykytyuk

37. 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."
• Gene knockout
• Targeted gene mutation(deletion/insertion/correction)
• Creating chromosome rearrangement
• Study gene function with stem cells
• Transgenic animals
• Endogenous gene labeling
• Targeted transgene addition
• Promoter study
• Targeted gene epigenetic change (DNA methylation, chromatin
modifications)

39. The nucleases create specific double-stranded break (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 nonhomologous end-joining
(NHEJ).

40. Engineered homing
endonucleases
Zinc-finger
Nucleases
(ZFNs)
Transcription
activator-like
Effector
nucleases
(TALENs)
CRISPR/Cas9

42. TALENs RVD-base alphabet
RVD(repeat-variable di-residues) – two amino acids which determine
specificity to one nitrogenous base

44. ZFNs&TALENs can be used for site-specific epigenetic
modification induction

45. Clustered
Regularly
Interspaced
Short
Palindromic
Repeats

47. Fig. 1. Overview of the four
CRISPR/cas systems present in
Streptococcus thermophilus
DGCC7710. For
each system, gene organization
is depicted on the top, with cas
genes in gray, and the repeat-
spacer
array in black. Below the gene
scheme, the repeat and spacer
(captured phage or plasmid
nucleic acid)
content is detailed as black
diamonds (T, terminal repeat)
and white rectangles,
respectively. Bottom
line, consensus repeat
sequence. L1 to L4, leader
sequences. The predicted
secondary structure of the
CRISPR3 repeat is shown on
the right. S. thermophilus
CRISPR2, CRISPR3, and
CRISPR4 systems are
homologous to the CRISPR
systems of Staphylococcus
epidermidis (20), Streptococcus
mutans (19), and
E. coli (28), respectively.

48. Fig. 2. Overview of the
CRISPR/Cas mechanism
of action. (A) Immunization
process: After insertion of
exogenous DNA from
viruses or plasmids, a Cas
complex recognizes foreign
DNA and integrates a
novel
repeat-spacer unit at the
leader end of the CRISPR
locus. (B) Immunity
process: The CRISPR
repeat-spacer
array is transcribed into a
pre-crRNA that is
processed into mature
crRNAs, which are
subsequently used as
a guide by a Cas complex
to interfere with the
corresponding invading
nucleic acid. Repeats are
represented as diamonds,
spacers as rectangles, and
the CRISPR leader is
labeled L.

52. CRISPR Design Tool (http://tools.genome-engineering.org)

54. Delivery of ZNFs, TALENs and CRISPR(gRNA, Cas9) in
vivo
Nuclease-encoded genes are delivered into cells by:
1. Transfection of plasmid DNA.
2. Viral vectors(adenovirus-mediated ZFN gene
delivery into T lymphocytes, Integrase-deficient
lentiviral vectors (IDLVs))
3. In vitro transcribed mRNA.
4. Direct transport (purified ZFN proteins are
capable of crossing cell membranes and inducing
endogenous gene disruption)

58. CRISPR Design Tool (http://tools.genome-engineering.org)
All-in-one pSpCas9(sgRNA) plasmid for gRNA and Cas9 delivery

60. 1. Genome engineering using the CRISPR-Cas9 system - 2013 Nature America
F Ann Ran1, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott & Feng Zhang
2. CRISPR/Cas, the Immune System of Bacteria and Archaea - Philippe Horvath and Rodolphe
Barrangou - Science 327, 167 (2010);
3. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering – Thomas Gaj,
Charles A. Gersbach, and Carlos F. Barbas III - Trends in Biotechnology July 2013, Vol. 31, No. 7

61. Figure 4 | Target selection and reagent preparation. (a) For S. pyogenes Cas9, 20-bp targets (highlighted in blue) must be followed at their 3′ends by 5′-NGG,
which can occur in either the top or the bottom strand of genomic DNA, as in the example from the human EMX1 gene. We recommend using the CRISPR
Design Tool (http://tools.genome-engineering.org) to facilitate target selection. (b) Schematic for co-transfection of the Cas9 expression plasmid (pSpCas9) and
a PCR-amplified U6-driven sgRNA expression cassette. By using a U6 promoter-containing PCR template and a fixed forward primer (U6-Fwd), sgRNA-
encoding DNA can be appended onto the U6 reverse primer (U6-Rev) and synthesized as an extended DNA oligo (Ultramer oligos from IDT). Note that the
guide sequence in the U6-Rev primer, designed against an example target from the top strand (blue), is the reverse complement of the 20-bp target sequence
preceding the 5′-NGG PAM. An additional cytosine (‘C’ in gray rectangle) is appended in the reverse primer directly 3′ to the target sequence to allow guanine as
the first base of the U6 transcript. (c) Schematic for scarless cloning of the guide sequence oligos into a plasmid containing Cas9 and the sgRNA scaffold
(pSpCas9(BB)). The guide oligos for the top strand example (blue) contain overhangs for ligation into the pair of BbsI sites in pSpCas9(BB), with the top and
bottom strand orientations matching those of the genomic target (i.e., the top oligo is the 20-bp sequence preceding 5′-NGG in genomic DNA). Digestion of
pSpCas9(BB) with BbsI allows the replacement of the Type II restriction sites (blue outline) with direct insertion of annealed oligos. Likewise, a G-C base pair
(gray rectangle) is added at the 5′ end of the guide sequence for U6 transcription, which does not adversely affect targeting efficiency. Alternate versions of
pSpCas9(BB) also contain markers such as GFP or a puromycin resistance gene to aid the selection of transfected cells.