16 research outputs found
Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing
Engineering CRISPR/Lb Cas12a for highly efficient, temperatureâtolerant plant gene editin
Increasing deletion sizes and the efficiency of CRISPR /Cas9âmediated mutagenesis by SunTag âmediated TREX1 recruitment
Previously, it has been shown that mutagenesis frequencies can be improved by directly fusing the human exonuclease TREX2 to Cas9, resulting in a strong increase in the frequency of smaller deletions at the cut site. Here, we demonstrate that, by using the SunTag system for recruitment of TREX2, the mutagenesis efficiency can be doubled in comparison to the direct fusion in Arabidopsis thaliana. Therefore, we also tested the efficiency of the system for targeted deletion formation by recruiting two other 3âČ-5âČ exonucleases, namely the human TREX1 and E. coli ExoI. It turns out that SunTag-mediated recruitment of TREX1 not only improved the general mutation induction efficiency slightly in comparison to TREX2, but that, more importantly, the mean size of the induced deletions was also enhanced, mainly via an increase of deletions of 25âbp or more. EcExoI also yielded a higher amount of larger deletions. However, only in the case of TREX1 and TREX2, the effect was predominately SunTag-dependent, indicating efficient target-specific recruitment. Using SunTag-mediated TREX1 recruitment at other genomic sites, we were able to obtain similar deletion patterns. Thus, we were able to develop an attractive novel editing tool that is especially useful for obtaining deletions in the range from 20 to 40âbp around the cut site. Such sizes are often required for the manipulation of cis-regulatory elements. This feature is closing an existing gap as previous approaches, based on single nucleases or paired nickases or nucleases, resulted in either shorter or longer deletions, respectively
From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas
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
Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a
Nicotiana tabacum is a nonâfood herb that has the potential to be utilized as bioâfactory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for preâdesigned genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of siteâspecific doubleâstrand breaks to enhance homologous recombinationâmediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Casâbased nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperatureâinsensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacteriumâmediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without TâDNA integration. Thus, we were not only able to obtain CRISPR/Casâbased heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops