Download Lezione 23 - 24 martedì 10 maggio 2011

Lezione 23 - 24
martedì 10 maggio 2011
corso vettori biologici II
Biotec industriali
ore 14:00 -16:00 aula 6A
Efficient design and assembly of custom TALEN and
other TAL effector-based constructs for DNA targeting
(Nucleic Acids Research, 2011, 1ﾐ11 doi:10.1093/nar/gkr218)
TALENs are important new tools for genome engineering. Fusions of transcription
activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI
nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined
by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We
present a method and reagents for efficiently assembling TALEN constructs with
custom repeat arrays. We also describe design guidelines based on naturally
occurring TAL effectors and their binding sites. Using software that applies these
guidelines, in nine genes from plants, animals and protists, we found candidate
cleavage sites on average every 35 bp. Each of 15 sites selected from this set was
cleaved in a yeast-based assay with TALEN pairs constructed with our reagents.
We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in
Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for
making custom TAL effectors and one for TAL effector fusions to additional proteins
of interest. Using the former, we constructed de novo a functional analog of
AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through
the non-profit repository AddGene and a web-based version of our software is
freely accessible online.
Nuovo metodo di assemblaggio
A Modular Cloning System for Standardized Assembly of
Multigene Constructs (PLoS One. 2011 Feb 18;6(2):e16765)
The field of synthetic biology promises to revolutionize biotechnology
through the design of organisms with novel phenotypes useful for
medicine, agriculture and industry. However, a limiting factor is the ability
of current methods to assemble complex DNA molecules encoding
multiple genetic elements in various predefined arrangements. We
present here a hierarchical modular cloning system that allows the
creation at will and with high efficiency of any eukaryotic multigene
construct, starting from libraries of defined and validated basic modules
containing regulatory and coding sequences. This system is based on the
ability of type IIS restriction enzymes to assemble multiple DNA
fragments in a defined linear order. We constructed a 33 kb DNA
molecule containing 11 transcription units made from 44 individual basic
modules in only three successive cloning steps. This modular cloning
(MoClo) system can be readily automated and will be extremely useful for
applications such as gene stacking and metabolic engineering.
The Golden Gate assembly method
(and MoClo)
The Golden Gate method (Engler 2008, Engler 2009, Engler
2011) offers standardized, quasi-scarless, multi-part DNA
assembly, and is an excellent choice for combinatorial library
construction (see also a variation of the Golden-gate protocol
for use with j5).
The Golden Gate method relies upon the use of type IIs
endonucleases, whose recognition sites are distal from their
cut sites. Although there are several different type IIs
endonucleases to choose from, the example below uses BsaI
(equivalent to Eco31I) (the Golden Gate method only uses a
single type IIs endonuclease at time).
Golden Gate assembly of partA with a linearized
destination vector:
QuickTime™ e un
decompressore TIFF (Non compresso)
sono necessari per visualizzare quest'immagine.
Golden Gate assembly put together
QuickTime™ e un
decompressore TIFF (Non compresso)
sono necessari per visualizzare quest'immagine.
Golden gate combinatory library
QuickTime™ e un
decompressore TIFF (Non compresso)
sono necessari per visualizzare quest'immagine.
Cloning versus syntetic
Generation of families of construct variants using golden
gate shuffling. Methods Mol Biol. 2011;729:167-81.
Current standard cloning methods based on the use of restriction enzymes and ligase are
very versatile, but are not well suited for high-throughput cloning projects or for assembly
of many DNA fragments from several parental plasmids in a single step. We have
previously reported the development of an efficient cloning method based on the use of
type IIs restriction enzymes and restriction-ligation. Such method allows seamless
assembly of multiple fragments from several parental plasmids with high efficiency, and
also allows performing DNA shuffling if fragments prepared from several homologous
genes are assembled together in a single restriction-ligation. Such protocol, called Golden
Gate shuffling, requires performing the following steps: (1) sequences from several
homologous genes are aligned, and recombination sites defined on conserved
sequences; (2) modules defined by the position of these recombination sites are amplified
by PCR with primers designed to equip them with flanking BsaI sites; (3) the amplified
fragments are cloned as intermediate constructs and sequenced; and (4) finally, the
intermediate modules are assembled together in a compatible recipient vector in a onepot restriction-ligation. Depending on the needs of the user, and because of the high
cloning efficiency, the resulting constructs can either be screened and analyzed
individually, or, if required in larger numbers, directly used in functional screens to detect
improved protein variants. Engler C, Marillonnet S.
Un nuovo codice genetico !!!
The most distinctive characteristic of TAL effectors is a central repeat domain
containing between 1.5 and 33.5 repeats that are usually 34 residues in length
(the C-terminal repeat is generally shorter and referred to as a “half repeat”).[2]
A typical repeat sequence is
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG, but the residues at the 12th
and 13th positions are hypervariable (these two amino acids are also known as
the repeat variable diresidue or RVD).
The experimentally validated code between RVD sequence and target
DNA base can be expressed as (asparagina-isoleucne) NI = A, (istidinaaspart ac)HD = C, (asparagina-glycine)NG = T, (aspar-asparag)NN = G
or A, and (aspar-serine)NS = A, C, G, or T. Further studies has shown
that the RVD (asparag-lysine)NK can target G. Target sites of TAL
effectors also tend to include a T flanking the 5’ base targeted by the
first repeat, but the exact mechanism of this recognition is not known.
Sintetizzare nuovi effettori TAL
This simple code between amino acids in TAL effectors and DNA bases in their
target sites might be useful for protein engineering applications. Numerous groups
have design artificial TAL effectors capable of recognizing new DNA sequences in a
variety of experimental systems. Such engineered TAL effectors have been used to
create artificial transcription factors that can be used to target and activate
endogenous genes in tomato, Arabidopsis thaliana, and human cells. Engineered
TAL effectors can also be fused to the cleavage domain of FokI to create TAL
effector nucleases (TALENs) that show activity in a yeast-based assay, at
endogenous yeast genes, in a plant reporter assay, at an endogenous plant gene,
and at endogenous human genes. The human HPRT1 gene has been targeted at
detectable, but unquantifiable levels and TALENs containing the FokI cleavage
domain fused to a different portion of the TAL DNA binding domain have been used
to target the endogenous NTF3 and CCR5 genes in human cells with efficiencies
of up to 25%. Engineered TAL effectors can be constructed using either
conventional gene synthesis or modular assembly. TAL effector nuclease
constructs are also commercially available from a European biotechnology
company. Such nucleases share some properties with zinc finger nucleases and
may be useful for genetic engineering and gene therapy applications
Talen rec system
One of the pairs of TALENs targeting the human HPRT1
gene was subcloned into the mammalian expression vector pCDNA3.1(-)
(Invitrogen) using XhoI and AflII. These enzymes excise the entire TALEN from
pTAL3 or pTAL4 and place the coding sequence under control of the CMV
(cytomegalovirus) promoter. The resulting plasmids were introduced into
HEK293T cells by transfection using Lipofectamine 2000 (Invitrogen) following
the manufacturer’s protocol. Cells were collected 72 h after transfection and
genomic DNA isolated and digested with Hpy188I, which cuts in the spacer
sequence of the TALEN target site. After digestion, a chromosomal fragment
encompassing the target site was amplified by PCR. Upon completion, the
reactions were incubated for 20 min at 72 C with 4 ml of Taq DNA poly-merase.
PCR products then were digested with Hpy188I and cloned in a TOPO TA
vector (Invitrogen) Independent clones containing the full-length PCR product
were sequenced to evaluate mutations at the cleavage site.