About CSIRO’s HairpinRNAi
HairpinRNAi – a CSIRO invention
Figure 1. Conventional hairpinRNAi constructs |
In 1995, CSIRO was first to recognise the role of double-stranded RNA as the trigger for gene silencing, now popularly known as “RNA Interference” or “RNAi”.
When a plant with an antisense construct directed against a virus sequence was crossed with a plant containing a sense construct against the same sequence, only the progeny that acquired both sense and antisense constructs were immune to virus infection (Waterhouse et al., 1998).
The key CSIRO invention was a construct with linked sense and antisense or inverted repeat sequences (Figure 1). When transcribed, a self complementary RNA is produced that forms a ‘hairpin’ structure by folding back on itself (Waterhouse et al., 1998).
Hairpin RNA with a double-stranded ‘stem’ is a more effective trigger of RNAi than separate sense plus antisense RNAs (Figure 2). This was proved by using constructs containing sequences from a reporter gene (GUS) and a viral gene (Potato Virus Y).
Figure 2. Double-stranded “stem”
is responsible
|
The preferred version of hairpinRNAi uses an intron to separate the inverted repeat sequences, making the construct stable in bacteria and increasing silencing efficiency in plants (Figure 3) (Smith et al., 2000).
CSIRO’s hairpinRNAi technology has been shown to be highly efficient and effective in silencing hundreds of genes and is proving to be very valuable in functional genomics and novel trait development in plants.
Figure 3. Efficiency of induction of RNAi by different gene constructs and the predicted structure of RNA transcribed from the transgenes.
(a) RNAi efficiency measured for potato virus (PVY) and Δ12-desaturase genes as the percentage of independent transgenic plants immune to PVY and the percentage of plants with enzyme activity reduced by more than 20% compared with wild type, respectively. In the predicted structures of RNA transcripts, right- and left-pointing arrows represent sense and antisense orientation of sequences, respectively; small vertical arrows represent splice-junction sequences remaining after the intron has been spliced out. (hpRNA, hairpin RNA; n, number of independent transformants; GUS, b-glucuronidase)
(b) Design of intron-containing hairpin constructs. (OCS, octopine synthase; NOS, nopaline synthase)
Waterhouse PM et al.,
1998, PNAS, 95, 13959-13964
Smith NA et al., 2000,
Nature 407, 319-320
