Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions

Bieluszewski, Tomasz; Szymanska-Lejman, Maja; Dziegielewski, Wojciech; Zhu, Longfei; Ziolkowski, Piotr A

doi:10.1007/978-1-0716-2253-7_13

Cited by 3 publications

(2 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CRISPR-Cas9 technique was used to generate desirable genome modifications in ChP and in L er . It was based on designing a pair of sgRNAs to induce a more efficient mutagenesis 78 , 79 . Constructs carrying cassettes with sgRNAs cloned under AtU3C or AtU6-26 promoters and Cas9 under ICU2 promoter were introduced to plants via Agrobacterium-mediated transformation.…”

Section: Methodsmentioning

confidence: 99%

“…Constructs carrying cassettes with sgRNAs cloned under AtU3C or AtU6-26 promoters and Cas9 under ICU2 promoter were introduced to plants via Agrobacterium-mediated transformation. Constructs used for the transformation of L er plants additionally contained a seed-expressed dsRed reporter marker for easier preselection of transformants 78 , 79 . Transformants were genotyped for the desired modifications and in the next generation construct-free plants with the heritable modifications were sequenced to confirm the length and the exact positions of the generated deletions.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The effect of DNA polymorphisms and natural variation on crossover hotspot activity in Arabidopsis hybrids

et al. 2023

Self Cite

View full text Add to dashboard Cite

In hybrid organisms, genetically divergent homologous chromosomes pair and recombine during meiosis; however, the effect of specific types of polymorphisms on crossover is poorly understood. Here, to analyze this in Arabidopsis, we develop the seed-typing method that enables the massively parallel fine-mapping of crossovers by sequencing. We show that structural variants, observed in one of the generated intervals, do not change crossover frequency unless they are located directly within crossover hotspots. Both natural and Cas9-induced deletions result in lower hotspot activity but are not compensated by increases in immediately adjacent hotspots. To examine the effect of single nucleotide polymorphisms on crossover formation, we analyze hotspot activity in mismatch detection-deficient msh2 mutants. Surprisingly, polymorphic hotspots show reduced activity in msh2. In lines where only the hotspot-containing interval is heterozygous, crossover numbers increase above those in the inbred (homozygous). We conclude that MSH2 shapes crossover distribution by stimulating hotspot activity at polymorphic regions.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The effect of DNA polymorphisms and natural variation on crossover hotspot activity in Arabidopsis hybrids

et al. 2023

Self Cite

View full text Add to dashboard Cite

show abstract

MSH2 stimulates interfering and inhibits non-interfering crossovers in response to genetic polymorphism

Dluzewska,

Dziegielewski,

Szymanska-Lejman

et al. 2023

Nat Commun

View full text Add to dashboard Cite

Meiotic crossovers can be formed through the interfering pathway, in which one crossover prevents another from forming nearby, or by an independent non-interfering pathway. In Arabidopsis, local sequence polymorphism between homologs can stimulate interfering crossovers in a MSH2-dependent manner. To understand how MSH2 regulates crossovers formed by the two pathways, we combined Arabidopsis mutants that elevate non-interfering crossovers with msh2 mutants. We demonstrate that MSH2 blocks non-interfering crossovers at polymorphic loci, which is the opposite effect to interfering crossovers. We also observe MSH2-independent crossover inhibition at highly polymorphic sites. We measure recombination along the chromosome arms in lines differing in patterns of heterozygosity and observe a MSH2-dependent crossover increase at the boundaries between heterozygous and homozygous regions. Here, we show that MSH2 is a master regulator of meiotic DSB repair in Arabidopsis, with antagonistic effects on interfering and non-interfering crossovers, which shapes the crossover landscape in relation to interhomolog polymorphism.

show abstract

CRISPR/Cas-mediated in planta gene targeting: current advances and challenges

Guzmán-Benito

Achkar

Bologna

et al. 2023

Journal of Experimental Botany

View full text Add to dashboard Cite

We can use gene targeting (GT) to make modifications at a specific region in a plant's genome and create high-precision tools for plant biotechnology and breeding. However, its low efficiency is a major barrier to its use in plants. The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas-based site-specific nucleases capable of inducing double-strand breaks in desired loci resulted in the development of novel approaches for plant GT. Several studies have recently demonstrated improvements in GT efficiency through cell-type-specific expression of Cas nucleases, the use of self-amplified GT-vector DNA, or manipulation of RNA silencing and DNA repair pathways. In this review, we summarize recent advances in CRISPR/Cas-mediated GT in plants and discuss potential efficiency improvements. Increasing the efficiency of GT technology will help us pave the way for increased crop yields and food safety in environmentally friendly agriculture.

show abstract

Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions

Cited by 3 publications

References 35 publications

The effect of DNA polymorphisms and natural variation on crossover hotspot activity in Arabidopsis hybrids

The effect of DNA polymorphisms and natural variation on crossover hotspot activity in Arabidopsis hybrids

MSH2 stimulates interfering and inhibits non-interfering crossovers in response to genetic polymorphism

CRISPR/Cas-mediated in planta gene targeting: current advances and challenges

Contact Info

Product

Resources

About