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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
