Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system

Abstract

During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild-type and in the non-homologous end-joining (NHEJ) mutant ku70-1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild-type, most junctions are formed by microhomology-mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg-cell-specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will.Results of PCR-based genotyping to identify inversion events in the T1 generation. For each approach up to 200 T1 plants were screened and there has been at least one heritable inversion event for each line.

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

“…Using this two‐step screening procedure, the time and effort required for identification of individuals with the desired genomic modification can be kept to a minimum while still assuring success. We recently applied a similar protocol for identifying successfully SaCas9‐induced heritable chromosomal inversions in Arabidopsis (Schmidt et al ., ).…”

A modular cloning toolkit for genome editing in plants

“…that could be used as an alternative to the most commonly utilised SpCas9. SaCas9, for instance, has proven to be an efficient tool for generating gene knockouts in a number of plant species [13][14][15][16] and, in addition, has been shown to increase HDR efficiencies in plants [15]. Due to SaCas9 and StCas9 having longer than SpCas9 PAM motifs they are also likely to be more specific when it comes to DNA target recognition.…”

“…SpCas9 [12] with the 'NGG' protospacer adjacent motif (PAM) is the most commonly used Cas9 variant for genome editing applications in various organisms, including plants. SaCas9 ('NNGRRT' PAM) and StCas9 ('NNRGAA' PAM) are less common but have also been successfully used in rice, tobacco [13] and Arabidopsis [14][15][16]. ScCas9 (pFH76), SpCas9-NG (pFH32) and SpCas9-derived xCas9 (pFH22) are characterised by broadened PAM motif requirements: 'NNG' for ScCas9 [17], and 'NG' for SpCas9-NG [18] and xCas9 [19].…”

Quilting plant chromosomes with CRISPR/Cas9

“…Now, new work by Schmidt et al . () shows that in addition to deletions and mutations, the CRISPR/Cas9 system can be applied to create heritable chromosomal inversions in plant genomes.…”

“…To tackle chromosomal inversions, Schmidt et al . () focused on four loci on three different Arabidopsis chromosomes. To induce inversions, guide RNAs were designed to generate two DSBs flanking each locus (see Figure).…”