In the role of God

Myths and realities of synthetic biology

© Vasyl Mykytyuk, 2014

You always have a choice.

And you make it every day

“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

Monday 31 March 2014

HOW REAL IS IT?

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)

one from 277 attempts

Somatic nuclear transfer technique

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

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.

5 June 2014 Young Gie Chung

Human Somatic Cell Nuclear Transfer Using Adult Cells

Somatic cell nuclear transfer (SCNT) Vs Induced pluripotent stem cells (iPSCs)

Usage in therapeutic cloning

To be continued…

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

2012

Jun 25, 2013

In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translationMichael C Jewett, Brian R Fritz, Laura E Timmerman and George M Church

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

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

Are you satisfied with your genetic code?

A semi-synthetic organism with an expanded genetic alphabetDenis 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)

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

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

Genome editing methods

Made by Vasyl Mykytyuk

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)

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).

Engineered homing endonucleases

Zinc-finger Nucleases

(ZFNs)

Transcription activator-like

Effectornucleases(TALENs)

CRISPR/Cas9

TALENs RVD-base alphabet

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

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

Clustered

Regularly

Interspaced

Short

Palindromic

Repeats

Fig. 1. Overview of the four CRISPR/cas systems present in Streptococcus thermophilus DGCC7710. Foreach system, gene organization is depicted on the top, with cas genes in gray, and the repeat-spacerarray 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. Bottomline, consensus repeat sequence. L1 to L4, leader sequences. The predicted secondary structure of theCRISPR3 repeat is shown on the right. S. thermophilus CRISPR2, CRISPR3, and CRISPR4 systems arehomologous to the CRISPR systems of Staphylococcus epidermidis (20), Streptococcus mutans (19), andE. coli (28), respectively.

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

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

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)

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

All-in-one pSpCas9(sgRNA) plasmid for gRNA and Cas9 delivery

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

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.