Gene Drives in Plants
CRISPR/Cas9-based gene drives have so far been described mainly in insects and mice. CRISRPR/Cas-GDs in plants have not yet been developed, as there are several biological hurdles to overcome at the molecular, cellular, organismal and population levels:
- The activation of the HDR repair is necessary for a CRISPR/Cas-based gene drive (GD) to incorporate the gene drive construct and other genetic elements at a specific target sequence in the genome, thereby increasing the inheritance of the GD construct. However, HDR is only marginally activated in plants and works inefficiently. In plants, another mechanism known as NHEJ predominantly takes over the repair of the CRISPR/Cas-induced DNA double-strand break (DSB) at the target sequence. Due to the low efficiency of HDR in plants, CRISPR/Cas-GDs are not considered to be very promising. A feasibility study describing a CRISPR/Cas-based GD in plants has been lacking so far.
- Plants have different reproductive strategies and differ in their life cycles, which makes the development of plant gene drives particularly difficult.
- A CRISPR/Cas-GD is based on the recognition of a specific target sequence to be cut by the gene scissors. If changes to this target sequence occur either naturally or through gene scissor activity, resistance develops, and the drive is not passed on.
Summary of the results of the publication
The study by Zhang, et al (2021) is the first scientific publication to describe a CRISPR/Cas9-GD in the model plant Arabidopsis thaliana. Different CRISPR/Cas9 constructs were tested to find the ideal time when the gene scissors are formed during meiosis and when HDR works most efficiently. The first step was to develop Arabidopsis plants carrying the GD construct in their genome. The target sequence of the gene drive is located in the CRY1 gene, which is disrupted by the insertion of the GD construct. Thus, the CRY1 gene is no longer expressed in these plants.
Plants with the GD construct found to have the highest efficiency were subsequently crossed with Arabidopsis plants that have a different genetic background and their progeny were examined. The GD construct cannot be passed on via pollen, only via the embryo. Furthermore, it was found that in some plants HDR does not work very precisely and errors can occur in adjacent DNA regions. The GD construct was only incorporated into the genome as desired in 3 out of a total of 195 plants. Self-pollination showed that the inheritance of the GD construct deviated slightly from the usual Mendelian inheritance ratio i.e., 25% of the progeny would be expected to have two copies of the gene drive. However, analysis of the progeny showed that this was the case in 37% of the plants. It appears that the gene drive is activated in heterozygous plants, thus causing an increase in the proportion of homozygous plants.
In addition, it was proven that in approx. 40 % of the plants, NHEJ repair led to a change in the target gene sequence resulting in a loss of function of the CRY1 gene. This result shows that resistance can develop in this still very simple and not very efficient gene drive. The gene drive can no longer insert itself into “resistant gene copies”.
What is the significance of these results?
Basically, the results of the study confirm once again that HDR works imprecisely and inefficiently in plants. How efficiently this gene drive spreads in later generations and larger populations cannot currently be predicted. However, given the low efficiency of this gene drive, it is very unlikely that entire wild populations can be altered.
The GD constructs tested were developed specifically for the model plant Arabidopsis. For many other agriculturally relevant plants, there are far fewer genetic elements and tools with which gene drives could be developed and tested. Further studies are needed to create new GD constructs that preferentially activate HDR.
Zhang T, Mudgett M, Rambabu R, Abramson B, Dai X, Michael TP, Zhao Y (2021) Selective inheritance of target genes from only one parent of sexually reproduced F1 progeny in Arabidopsis. Nature Communications 12 (1):3854. doi:10.1038/s41467-021-24195-5