Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats

Schindele, Angelina; Gehrke, Fabienne; Schmidt, Carla Adriana Pizarro; Röhrig, Sarah; Dorn, A.; Puchta, Holger

doi:10.1038/s41467-022-29130-w

Cited by 15 publications

(23 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Thus far, multiple endogenous genes have been constitutively activated in stable transgenic plants using both CRISPR‐based and engineered TALE activators, but tissue‐specific expression has not been reported (Lowder et al., 2018; Morbitzer et al., 2010; Pan et al., 2021; Xiong et al., 2021). However, inducible and constitutive knockouts using tissue‐specific promoters to drive Cas9 (or estradiol‐inducible transcription factors) have been reported and in principle similar approaches could be used for tissue specific activation of multiple endogenous genes (Decaestecker et al., 2019; Feder et al., 2020; Schindele et al., 2022; Wang et al., 2020). An alternative would be to use either endogenous or synthetic transcription factors to induce orthogonal synthetic promoter sequences that are not present in the host genome, similar to the approach we used here for dTALEs in rice.…”

Section: Discussionmentioning

confidence: 99%

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

Danila

Schreiber

Ermakova

et al. 2022

Plant Biotechnology Journal

View full text Add to dashboard Cite

In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems-dTALE1-STAP1 and dTALE2-STAP2-can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

show abstract

Section: Discussionmentioning

confidence: 99%

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

Danila

Schreiber

Ermakova

et al. 2022

Plant Biotechnology Journal

View full text Add to dashboard Cite

show abstract

“…Previously established pUbi‐SaCas9‐ITS2 and pUbi‐SaCas9‐ADH1 (Schindele et al ., 2022) were used as the source DNA to generate the inducible CRISPR‐Kill as well as the ADH1 control construct. An Eco RI restriction enzyme digestion enabled the removal of PcUbi4‐2.…”

Section: Methodsmentioning

confidence: 99%

“…While most genetic cell ablation approaches are based on the tissue‐specific expression of bacterial toxins (Goldman et al ., 1994; Day et al ., 1995; Weijers et al ., 2003), the application of the CRISPR/Cas system as a programmable site‐specific nuclease (Jinek et al ., 2012) has recently enabled another method for tissue‐specific cell elimination in Arabidopsis. The CRISPR‐Kill system relies on massive double‐strand break (DSB) induction in functional repetitive regions of DNA, namely the 45S ribosomal DNA (rDNA) tandem repeats or centromeric satellites (Schindele et al ., 2022). Targeting the internal transcribed spacer 2 (ITS2) of up to 750 45S rDNA tandem repeats per haploid genome resulted in a strong lethal effect when expressing the Staphylococcus aureus Cas9 (SaCas9) nuclease (Steinert et al ., 2015) constitutively (Schindele et al ., 2022).…”

Section: Introductionmentioning

confidence: 99%

“…The CRISPR‐Kill system relies on massive double‐strand break (DSB) induction in functional repetitive regions of DNA, namely the 45S ribosomal DNA (rDNA) tandem repeats or centromeric satellites (Schindele et al ., 2022). Targeting the internal transcribed spacer 2 (ITS2) of up to 750 45S rDNA tandem repeats per haploid genome resulted in a strong lethal effect when expressing the Staphylococcus aureus Cas9 (SaCas9) nuclease (Steinert et al ., 2015) constitutively (Schindele et al ., 2022). Moreover, localized effects such as prevention of organogenesis could be achieved by using tissue‐specific promoters.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

An inducible CRISPR‐Kill system for temporally controlled cell type‐specific cell ablation in Arabidopsis thaliana

et al. 2023

Self Cite

View full text Add to dashboard Cite

Summary The application of the CRISPR/Cas system as a biotechnological tool for genome editing has revolutionized plant biology. Recently, the repertoire was expanded by CRISPR‐Kill, enabling CRISPR/Cas‐mediated tissue engineering through genome elimination by tissue‐specific expression. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), CRISPR‐Kill relies on the induction of multiple double‐strand breaks (DSBs) in conserved repetitive genome regions, such as the rDNA, causing cell death of the targeted cells. Here, we show that in addition to spatial control by tissue‐specific expression, temporal control of CRISPR‐mediated cell death is feasible in Arabidopsis thaliana. We established a chemically inducible tissue‐specific CRISPR‐Kill system that allows the simultaneous detection of targeted cells by fluorescence markers. As proof of concept, we were able to eliminate lateral roots and ablate root stem cells. Moreover, using a multi-tissue promoter, we induced targeted cell death at defined time points in different organs at select developmental stages. Thus, using this system makes it possible to gain new insights into the developmental plasticity of certain cell types. In addition to enabling tissue engineering in plants, our system provides an invaluable tool to study the response of developing plant tissue to cell elimination through positional signaling and cell‐to‐cell communication.

show abstract

“…In fact, given that the centromere is largely repetitive sequences up to megabases in size, it provides many potential targets for manipulation with genome editing tools for chromosome engineering. Recently, the editing of centromere tandem repeats proved to be a valuable tool for organ‐specific cell elimination in Arabidopsis (Schindele et al., 2022 ). Hence, centromere editing made possible by the advent of genome editing tools, will be an important strategy in the generation of novel chromosomes, which will in turn accelerate karyotype change for crop improvements.…”

Section: Introductionmentioning

confidence: 99%

Centromeres: From chromosome biology to biotechnology applications and synthetic genomes in plants

Zhou

Liu

Guo

et al. 2022

Plant Biotechnology Journal

View full text Add to dashboard Cite

Centromeres are the genomic regions that organize and regulate chromosome behaviours during cell cycle, and their variations are associated with genome instability, karyotype evolution and speciation in eukaryotes. The highly repetitive and epigenetic nature of centromeres were documented during the past half century. With the aid of rapid expansion in genomic biotechnology tools, the complete sequence and structural organization of several plant and human centromeres were revealed recently. Here, we systematically summarize the current knowledge of centromere biology with regard to the DNA compositions and the histone H3 variant (CENH3)-dependent centromere establishment and identity. We discuss the roles of centromere to ensure cell division and to maintain the three-dimensional (3D) genomic architecture in different species. We further highlight the potential applications of manipulating centromeres to generate haploids or to induce polyploids offspring in plant for breeding programs, and of targeting centromeres with CRISPR/Cas for chromosome engineering and speciation. Finally, we also assess the challenges and strategies for de novo design and synthesis of centromeres in plant artificial chromosomes. The biotechnology applications of plant centromeres will be of great potential for the genetic improvement of crops and precise synthetic breeding in the future.

show abstract

Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats

Cited by 15 publications

References 41 publications

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice

An inducible CRISPR‐Kill system for temporally controlled cell type‐specific cell ablation in Arabidopsis thaliana

Centromeres: From chromosome biology to biotechnology applications and synthetic genomes in plants

Contact Info

Product

Resources

About