The heat is on: a simple method to increase genome editing efficiency in plants
Küçük Resim Yok
Tarih
2022
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Bmc
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Background Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. Results Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions. Conclusions Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.
Açıklama
Anahtar Kelimeler
CRISPR, Plant biotechnology, Genome engineering, Heat-shock, Base editing, Homology-directed repair, Arabidopsis, Mutagenesis, Crispr/Cas9, Expression, Nucleases, Gene, Dna, Recombination, Endonuclease, Temperature
Kaynak
Bmc Plant Biology
WoS Q Değeri
Q1
Scopus Q Değeri
Q1
Cilt
22
Sayı
1
