From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas

Abstract

In the last years, tremendous progress has been achieved in the field of gene editing in plants. By the induction of single site-specific double-strand breaks (DSBs), the knockout of genes by non-homologous end joining has become routine in many plant species. Recently, the efficiency of inducing pre-planned mutations by homologous recombination has also been improved considerably. However, very little effort has been undertaken until now to achieve more complex changes in plant genomes by the simultaneous induction of several DSBs. Several reports have been published on the efficient induction of deletions. However, the induction of intrachromosomal inversions and interchromosomal recombination by the use of CRISPR/Cas has only recently been reported. In this review, we want to sum up these results and put them into context with regards to what is known about natural chromosome rearrangements in plants. Moreover, we review the recent progress in CRISPR/Cas-based mammalian chromosomal rearrangements, which might be inspiring for plant biologists. In the long run, the controlled restructuring of plant genomes should enable us to link or break linkage of traits at will, thus defining a new area of plant breeding.

Keywords: CRISPR/Cas; Chromosomal rearrangements; Genome engineering; Inversion; Translocation.

Fig. 1

Different mechanisms for the development of CRs. Different DNA molecules and different repair mechanisms can be involved in the development of CRs. (A) If the induction of a DSB occurs in a segment that is homologous to a second DSB on the same chromosome, recombination may occur via a HR-based mechanism called nonallelic homologous recombination (NAHR). The product of this HR-based recombination shown here is an inversion of the DNA stretch between the repeats. (B) If the occurrence of a DSB leads to HR-based strand invasion, an MI process may occur when the invaded strand has homologies to two different donor molecules. Such an intermediate can be dissolved by certain resolvases, which can lead to a translocation between the two invaded donor molecules. (C) The integration of a T-DNA can lead to the formation of a translocation by the independent integration of the two ends of the T-DNA into two DSBs on different chromosomes. As a result, two independent chromosomes can then be linked by a T-DNA. (D) Due to the presence of two DSBs within a chromosome, the area between the two DSBs can be inversely integrated into the genome through NHEJ-based repair

Fig. 2

The targeted generation of CRs through the induction of multiple DSBs. Induced intra-chromosomal rearrangements are characterized by break induction on the same chromosome. (A) If the sequence between the two DSBs is removed, a deletion arises. If the excised DNA is reintegrated in reverse orientation, an inversion is induced. (B) If two DSBs are induced on different chromosomes, both chromosome arms can be exchanged in a reciprocal translocation. (C) Furthermore, by inducing (one) or two DSBs at an identical position in homologues, crossovers can be achieved by HR or NHEJ, respectively. (D) By inducing pairs of DSBs on different chromosomes, the sequences between the DSBs can be exchanged between both chromosome arms

Fig. 3

Inversion induction to generate or separate linkage groups. Since the recombination between rearranged areas of the genome is suppressed in heterozygotes, it is possible to generate new linkage groups via the induction of an inversion. When two unlinked genes are located at different locations of the chromosome arm, it is likely that they are separated via CO formation during meiosis. With the induction of an inversion involving both genes, it is possible to link them, and thus ensure that they are inherited together. On the other hand, it should also be possible to reverse inversions that were previously formed and thus eliminate linkage groups caused by this inversion. As a result, it should be possible to separate both genes by a CO event during meiosis