Enhancing <i>in planta</i> gene targeting efficiencies in Arabidopsis using temperature‐tolerant CRISPR/<i>Lb</i>Cas12a

Merker, Laura; Schindele, Patrick; Huang, Tsi-Shu; Wolter, Felix; Puchta, Holger

doi:10.1111/pbi.13426

Cited by 43 publications

(52 citation statements)

References 10 publications

(20 reference statements)

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“…Therefore, our group developed a temperature-tolerant version of LbCas12a (ttLbCas12a) to enhance cutting efficiencies at lower temperatures (Malzahn et al, 2019;. Just recently, we were able to demonstrate that ttLbCas12a outperformed the native enzyme in ipGT at 22°C by 2.4-fold and at 28°C by 1.7-fold (Merker, Schindele, Huang, Wolter, & Puchta, 2020).…”

Section: Introductionmentioning

confidence: 99%

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

2020

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CRISPR/Cas systems enable gene editing through the induction of site‐specific DNA double‐strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non‐homologous end joining (NHEJ)‐mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T‐rich regions. Most recently, we tested an improved, temperature‐tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants

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Section: Introductionmentioning

confidence: 99%

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

2020

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“…Several independent recent studies indicated that LbCas12a is indeed able to outperform Cas9 for GT in plants (Li et al 2020a;van Vu et al 2020;Wolter and Puchta 2019). GT efficiency could be further increased in Arabidopsis and tobacco using the temperature tolerant version of LbCas12a (Huang et al 2021;Merker et al 2020). The system used in Arabidopsis was the ''in planta GT'' approach.…”

Section: Improvement Of Gene Targetingmentioning

confidence: 99%

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

Huang

Puchta

2021

Transgenic Res

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In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.

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“…There have been several recent developments in the CRISPR technology that can be directly implemented in disease-resistant crop production: for example, generating gene-edited dicotyledonous plants through de novo meristem induction and eliminating time-consuming tissue culture steps [113], using temperature-tolerant CRISPR/ LbCas12a to increase the targeting and efficiency [114], enabling large DNA insertions (up to 2 kb) with precision in rice [115], and applying heat-inducible CRISPR system to increase the efficiency of gene targeting in maize [116]. Chromosome engineering in crops is another exciting recent development enabling controlled restructuring of plant genomes [117] and breaking genetic linkage via somatic chromosome engineering [118].…”

Section: Limitations and Future Prospectsmentioning

confidence: 99%

Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants

et al. 2020

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To meet increasing global food demand, breeders and scientists aim to improve the yield and quality of major food crops. Plant diseases threaten food security and are expected to increase because of climate change. CRISPR genome-editing technology opens new opportunities to engineer disease resistance traits. With precise genome engineering and transgene-free applications, CRISPR is expected to resolve the major challenges to crop improvement. Here, we discuss the latest developments in CRISPR technologies for engineering resistance to viruses, bacteria, fungi, and pests. We conclude by highlighting current concerns and gaps in technology, as well as outstanding questions for future research.

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Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature‐tolerant CRISPR/LbCas12a

Cited by 43 publications

References 10 publications

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants

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