Transcript Hot New Technologies 2015

Hot New Technologies 2015
CRISPR-Cas
• CRISPR – clustered regularly interspersed
short palindromic repeat
• Cas9 - CPISPR-associated
• Very new
• Edit or modulate specific DNA sequences
• Applicable to any organism for which there is
sequence available, not just model species
• Cas9 is an RNA-guided DNA endonuclease innate to prokaryotic
immune systems.
• Cas9 can be targeted to specific genomic sequences by
engineering a separately encoded guide RNA with which it
forms a complex.
• As only a short RNA sequence must be synthesized to confer
recognition of a new target, CRISPR/Cas9 is a relatively cheap
and easy to implement technology that has proven to be
extremely versatile.
• Together with other sequence-specific nucleases, CRISPR/ Cas9
is a game-changing technology that is poised to revolutionise
basic research and plant breeding.
• The transition of the CRISPR-Cas system from
biological phenomenon to genome
engineering tool came about when it was
shown that the target DNA sequence could be
reprogrammed simply by changing 20
nucleotides in the crRNA and that the
targeting specificity of the crRNA could be
combined with the structural properties
tracrRNA in a chimeric single guide RNA
(gRNA) (2 component system).
• In August 2013, 5 reports were published
discussing the first CRISPR/Cas-based genome
editing in plants.
• CRISPER craze!
Considerations
• Potential off target effects
• How to get constructs into plant cells (will
depend on plant species
– Protoplast transfection
– Agroinfiltration
– Stable transgenic plants – Agro or biolistics
– Cell penetrating peptides
Transgene-free mutated plants
• Transient expression of the nuclease components
– Agroinfiltration
– Viral vectors
– Cell penetrating peptides
• Delivery of the components as functional gRNA
and Cas9 protein
• Incorporation of the gRNA and Cas9 transgenes
on a separate chromosome to the targeted locus
so that they may be removed by segregation
Transgene-free mutated plants
• Selection?
– By phenotype
– Selectable markers
– Scorable markers
Transgene effects
• “Conventional” transformation integrates
transgenes randomly – could cause
unintended cosequences
• The potentiall to introduce transgenes at a
specific and predetermined chromosomal
positional should all but eliminate the risk of
such unpredictable events.
G. GRULLÓN/SCIENCE
Chloroplast Engineering
Insecticidal RNA
• Chloroplasts lack the cellular RNAi
machinery and therefore, long dsRNA produced in
these organelles are protected from being cleaved
by Dicer.
• Thus, beetles that fed on the chloroplasttransformed plants ate almost entirely long dsRNA,
whereas beetles that ate nuclear-transformed plants
consumed mostly siRNAs.
• Beetles feed on chloroplast transformed potato
plants died.
Horizontal Gene Transfer