Enhancing gene editing and gene targeting efficiencies in <i>Arabidopsis thaliana</i> by using an intron‐containing version of <scp>tt<i>Lb</i>Cas12a</scp>

Schindele, Patrick; Merker, Laura; Schreiber, Tom; Prange, Anja; Tissier, Alain; Puchta, Holger

doi:10.1111/pbi.13964

Cited by 20 publications

(14 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Notably, the GT alleles were inherited in the next generations (Figure 4 and Figure S16), indicating that the GT alleles were stably fixed into the tomato’s genome. This observation validates our KUDN-based GT tools and is consistent with other reports of GT in plants (Cermak et al, 2015; Dahan-Meir et al, 2018; Merker et al, 2020a; Merker et al, 2020b; Schindele et al, 2023; Wolter and Puchta, 2019). More importantly, the GT tools did not induce off-targeting activities in other genome sites (Figure S20).…”

Section: Discussionsupporting

confidence: 92%

“…Previously, we established a GT system based on a geminiviral replicon and the CRISPR-LbCas12a nuclease (Vu et al, 2020) that was improved by using the temperature-tolerant (ttLbCas12a) variant (Huang et al, 2021; Merker et al, 2020b; Schindele et al, 2023; Vu et al, 2021a) and further enhanced by chemical treatments for cNHEJ suppression (Vu et al, 2021a). However, it is still challenging to conduct GT experiments without allele-associated markers for selecting GT events, which limits the applications of GT in tomato and other plants.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Redirecting DNA repair for efficient CRISPR-Cas-based gene targeting in tomato

Van Vu,

Nguyen,

Kim

et al. 2024

Preprint

View full text Add to dashboard Cite

The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical nonhomologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented various approaches to shift the repair pathway preference toward HR by using a dominant-negative KU80 mutant protein (KUDN) to disrupt the initiation of cNHEJ and enhance DSB end resection through nucleases. Our results show from 1.71- to 3.55-fold improvement of the GT efficiency at the callus stage and a more remarkable, up to 9.84-fold, increase in GT efficiency at two specific tomato loci, SlHKT1;2 and SlEPSPS1, when we screened transformants obtained from the KUDN-mediated cNHEJ suppression approach. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated GT at an additional locus, SlCAB13. These findings provide a promising method for more efficient and precise plant breeding in the future.

show abstract

Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Redirecting DNA repair for efficient CRISPR-Cas-based gene targeting in tomato

Van Vu,

Nguyen,

Kim

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…T2 plants are then screened for imazapyr‐resistant seedlings that represent GT events. The highest ipGT frequencies could be obtained by the use of the highly efficient temperature‐tolerant LbCas12a (Merker et al., 2020; Schindele et al., 2023; Schindele & Puchta, 2020).…”

Section: Resultsmentioning

confidence: 99%

Deficiency of both classical and alternative end‐joining pathways leads to a synergistic defect in double‐strand break repair but not to an increase in homology‐dependent gene targeting in Arabidopsis

Merker,

Feller,

Dorn

et al. 2024

The Plant Journal

Self Cite

View full text Add to dashboard Cite

SUMMARYIn eukaryotes, double‐strand breaks (DSBs) are either repaired by homologous recombination (HR) or non‐homologous end‐joining (NHEJ). In somatic plant cells, HR is very inefficient. Therefore, the vast majority of DSBs are repaired by two different pathways of NHEJ. The classical (cNHEJ) pathway depends on the heterodimer KU70/KU80, while polymerase theta (POLQ) is central to the alternative (aNHEJ) pathway. Surprisingly, Arabidopsis plants are viable, even when both pathways are impaired. However, they exhibit severe growth retardation and reduced fertility. Analysis of mitotic anaphases indicates that the double mutant is characterized by a dramatic increase in chromosome fragmentation due to defective DSB repair. In contrast to the single mutants, the double mutant was found to be highly sensitive to the DSB‐inducing genotoxin bleomycin. Thus, both pathways can complement for each other efficiently in DSB repair. We speculated that in the absence of both NHEJ pathways, HR might be enhanced. This would be especially attractive for gene targeting (GT) in which predefined changes are introduced using a homologous template. Unexpectedly, the polq single mutant as well as the double mutant showed significantly lower GT frequencies in comparison to wildtype plants. Accordingly, we were able to show that elimination of both NHEJ pathways does not pose an attractive approach for Agrobacterium‐mediated GT. However, our results clearly indicate that a loss of cNHEJ leads to an increase in GT frequency, which is especially drastic and attractive for practical applications, in which the in planta GT strategy is used.

show abstract

“…Linker-fused exonuclease level 0 modules were combined with other level 0 modules (promoter, endonuclease (Cas9 or Cas12a) and terminator) to assemble transcriptional units (level 1). For SpCas9 and ttLbCas12a the intronized version have been used (Grutzner et al, 2021; Schindele et al, 2023). Cas9 single guide RNAs (sgRNAs) were designed according to Grützner et al (Grutzner et al, 2021), whereas the flip extension sgRNA scaffold and the Arabidopsis U6-26 t67 terminator were used as template for PCR-based sgRNA amplification (Castel et al, 2019; Chen et al, 2013; Dang et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

Efficient scar-free knock-ins of several kilobases by engineered CRISPR/Cas endonucleases

Schreiber,

Prange,

Schäfer

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. Using a transient assay inNicotiana benthamianawe identified two groups of exonucleases, respectively from the Herpes Virus and from the bacteriophage T7 families, hat confer up to 38-fold increase in HDR frequencies when fused to Cas9. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. InArabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins. Our results open perspectives for the routine production of knock-in and gene replacement events in plants.One-Sentence SummaryFusions of CRISPR endonucleases to specific 5′-exonucleases leads to significant increase in scar-free multikilobase knock-ins.

show abstract

Enhancing gene editing and gene targeting efficiencies in Arabidopsis thaliana by using an intron‐containing version of ttLbCas12a

Cited by 20 publications

References 7 publications

Redirecting DNA repair for efficient CRISPR-Cas-based gene targeting in tomato

Redirecting DNA repair for efficient CRISPR-Cas-based gene targeting in tomato

Deficiency of both classical and alternative end‐joining pathways leads to a synergistic defect in double‐strand break repair but not to an increase in homology‐dependent gene targeting in Arabidopsis

Efficient scar-free knock-ins of several kilobases by engineered CRISPR/Cas endonucleases

Contact Info

Product

Resources

About