Transcript Bioteh_Klonesana un in vivo inhenierija_2015

BIOTEHNOLOĢIJA III :
REKOMBINANTU BIOTEHNOLOĢIJA
JAUNĀ
BIOTEHNOLOĢIJA
I. Muižnieks, 2015. g. pavasaris
Jaunās klonēšanas un in vivo gēnu inženierijas
metodes
Klonēšanas metodes:
Gibsona savākšanās
In vivo gēnu inženierijas metodes:
Zn-pirkstu nukleāzes
TALEN nukleāzes
CRISP/Cas sistēma
Sekvenēšanas metožu attīstība
W-H Chen, Z-J Qin, J Wang,G-P Zhao. The MASTER (methylation-assisted tailorable ends rational) ligation
method for seamless DNA assembly. Nucleic Acids Research, 2013, 1–9, doi:10.1093/nar/gkt122
H. Kim & J-S Kim. A guide to genome engineering with programmable nucleases
Nature Reviews Genetics 15, 321–334, (2014) doi:10.1038/nrg3686
a | Nuclease-induced double-strand breaks (DSBs) can lead to sequence insertion, nucleotide correction or change (red box)
through homology-directed repair (HDR) in the presence of a donor DNA or a single-strand oligodeoxynucleotide (ssODN), both of
which contain homology arms. DSBs can also be repaired through error-prone non-homologous end-joining (NHEJ), which does
not require donor DNA or ssODN and consequently often leads to small insertions and deletions (indels). Typical indel sequences
and the number of inserted (+3 and +1) or deleted (−2, −4 and −10) bases are shown.
b | When two DSBs are generated in cis on a single chromosome by programmable nucleases, the flanking region can be deleted
or inverted.
c | When two DSBs are generated on two different chromosomes, chromosomal translocations can be induced.
Jaunās pieejas augu genoma modifikācijai
Zn-pirkstu nukleāzes
Genes VII, Lewin B., 2005, p. 276/382
Jaunās pieejas augu genoma modifikācijai
Zn-pirkstu nukleāzes
Modificēta FokI endonukleāze
Zinc-finger nucleases as gene therapy agents, D Carroll, 2008
A schematic representation of a zinc-finger nuclease (ZFN) pair
a | Each ZFN is composed of a zinc-finger protein (ZFP) at the amino terminus and the FokI
nuclease domain at the carboxyl terminus. In the zinc-finger motif consensus, X represents any
amino acid. Target sequences of ZFN pairs are typically 18–36 bp in length, excluding spacers.
b | A computer model structure of a ZFN pair bound to DNA is shown. Each zinc-finger is shown in
shades of pink in ribbon (left) and space-filling (right) representations. The grey region represents
the linker between the DNA-binding and catalytic domains. The FokI catalytic domains are shown in
blue and purple at the centre using space-filling representations.
Part b is modified, with permission, from Ref. 191 © (2011) Genetics Society of America.
Jaunās pieejas augu genoma modifikācijai
TALE faktori
Transcription activator like effectors (TALEs) (Boch et
al. 2009; Moscou and Bogdanove 2009).
TALEs are produced by plant pathogens in the genus
Xanthomonas, which deliver the proteins to plant cells
during infection
Proteīna-DNS
mijiedarbības
specifiskums
TALE efektorā
Christian M. et al., Targeting DNA Double-Strand Breaks with TAL Effector Nucleases, Genetics
186: 757–761 (October 2010)
A schematic representation of
a transcription activator-like
effector nuclease (TALEN)
pair.
a | Each TALEN is composed of
transcription
activator-like
effectors (TALEs) at the amino
terminus and the FokI nuclease
domain
at
the
carboxyl
terminus. Each TALE repeat is
comprised of 33–35 amino acids
and recognizes a single base
pair through the amino acids at
positions 12 and 13, which is
called the repeat variable
diresidue (RVD; shown in red).
Target sequences of TALEN
pairs are typically 30–40 bp in
length, excluding spacers.
b | In the TALE–DNA co-crystal
structure, the RVDs in TALE
interact with DNA in the major
groove. The amino-terminal
repeats (designated as 0 and −1
in the box) contact 5′ thymine.
Part b is modified, with permission, from Ref.
73 © (2012) American Association for the
Advancement of Science.
Jaunās pieejas augu genoma modifikācijai
CRISP-Cas
CRISPR (clustered regularly interspaced short palindromic repeat)
Cas (CRISPR-associated) genes,
CRISPR-based adaptive immune systems Terns and Terns, 2011
Jaunās pieejas genoma modifikācijai
CRISP-Cas
Mali P. et al. RNA-Guided Human Genome Engineering via Cas9. Science, V339, p. 824, 2013
Schematic representations of RNA-guided
engineered nucleases (RGENs).
a | An RGEN is comprised of CRISPR
(clustered
regularly
interspaced
short
palindromic repeat)-associated protein 9
(Cas9), a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), which form the
dualRNA–Cas9.
b | Alternatively, an RGEN can contain Cas9
and a single-chain guide RNA (sgRNA). The
guide sequence in the crRNA (part a) or
sgRNA (part b) is complementary to a 20-bp
target DNA sequence known as protospacer,
which is next to the 5′-NGG-3′ (where N
represents any nucleotide) sequence known
as protospacer adjacent motif (PAM). Grey
dots indicate weak bonding.
c | Target DNA cleaved by an RGEN yielding
blunt ends is shown.
d | A three-dimensional model of Cas9
complexed with DNA is shown. Part d
courtesy of D. W. Taylor (University of
California, Berkeley, USA), J. A. Doudna
(University of California, Berkeley, USA) and
M. Jinek (University of Zurich, Switzerland).
J. J. Day (2014) New approaches to manipulating the epigenome.
www.dialogues-cns.org
DNS sekvenēšanas metožu attīstība
DNS sekvenēšana ar daļēji specifiskas ķīmiskās degradācijas palīdzību
Walter Gilbert,
1932
Andrejs Mirzabekovs,
1937 -2002
http://nationaldiagnostics.com/article_info.php/articles_
id/20
Frederick Sanger, 1918
www.nwfsc.noaa.gov/.../figur
es/moranfig4.htm
METODES
Manuāla
sekvenēšana
Analīzes metodes
Klasiskā pieeja: fragmentu klonēšana, subklonēšana, sekvenēšana
http://seqcore.brcf.med.umich.edu/doc/
JAC, BAC, PAC, PUC
Bioinformātikas idejas: nejauša klonēšana (shotgun), sekvenēšana
http://img4.wikia.nocookie.net
K. Venters, B.Klintons, F. Kolins, 2000, cilvēka genoma projekta finiša taisnē
http://www.vfa-bio.de/static/
Resekvenēšanas metodes
“Resekvenēšana”, vai genoma sekvenēšana nto reizi,
vai genoma daļas sekvenēšana organismam, kam
viena genoma sekvence jau zināma (vai pat
radniecīgam organismam) ir vieglāka un lētāka nekā
de novo sekvenēšana.
Vairākas firmas piedāvā liela apjoma, ātrdarbīgas
paralēlās resekvenēšanas platformas.
454 Life Sciences
(http://www.454.com/enabling-technology/the-system.asp)
Solexa (Illumina)
(http://www.illumina.com/pages.ilmn?ID=203)
PERSONISKIE GENOMA PROJEKTI
Samuel Levy, et al. (Craig Venter)
The Diploid Genome Sequence of an Individual Human
PLoS BIOLOGY October 2007 | Volume 5 | Issue 2113 10 | e254
David A. Wheeler, et al. (James Watson)
The complete genome of an individual by massively parallel DNA
sequencing
Nature 452, 872-876 (17 April 2008)
Jeffrey M. Kidd, et al.
Mapping and sequencing of structural variation from eight human
genomes
Nature 453, 56-64 (1 May 2008)
Analīzes metodes
Analīzes metodes
Solex – Illumina tehnoloģija
Analīzes metodes
Solex – Illumina tehnoloģija
Analīzes metodes
Solex – Illumina tehnoloģija
Solex – Illumina
JONU PUSVADĪTĀJU SEKVENĒŠANA –ION TORRENT TECHNOLOGIES
January 10, 2012
Life Technologies Benchtop Ion Proton Sequencer will sequence human genomes in one
day for less than $1000 by yearend and Illumina will have a competing sub-$1000 per
human genome sequencer by yearend
The Ion Proton™ Sequencer is ideal for sequencing both exomes — regions in the
DNA that code for protein — and human genomes. The Ion Proton™ I Chip, ideal for
sequencing exomes, will be available mid-2012. The Ion Proton™ II Chip, ideal for
sequencing whole human genomes, will be available about six months later. In
addition, the Ion Proton™ OneTouch™ system automates template prep and a standalone Ion Proton™ Torrent Server performs the primary and secondary data analysis.
Nanopore DNA sequencing technique promises entire genome
in minutes or your money back
https://www.sciencenews.org/sites/
http://www.futuretimeline.net/blog/images/1137.jpg
Rick Merritt DNA Chip Will Plug Into Handsets 6/9/2014
Sekvenēšanas perspektīvas