Cis-regulatory elements used to control gene expression in plants

Biłas, Róża; Szafran, Katarzyna; Hnatuszko-Konka, Katarzyna; Kononowicz, Andrzej K.

doi:10.1007/s11240-016-1057-7

Cited by 210 publications

(129 citation statements)

References 129 publications

(185 reference statements)

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“…In addition, the reported profiles are consistent with the literature, as other authors have shown a prevalence of deletions with Cas12a in comparison with SpCas9 in mammalian cells (Kim et al ., ) and plants (Endo et al ., ). The distinctive mutation signature of Cas12a has interesting functional implications, considering, for example, that larger deletions are more prone to generate loss‐of‐function mutants and remove regulatory operators in promoter regions, two features that can be exploited in breeding practices (Biłas et al ., ). Furthermore, whereas DSB in Cas9 takes place at a position proximal to the PS sequence, Cas12a cleaves at a distal position, thus allowing target conservation after cleavage, which allegedly promotes larger deletions by MMEJ or gene insertions via homology direct repeat (HDR; Moreno‐Mateos et al ., ; Tóth et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Assessment of Cas12a‐mediated gene editing efficiency in plants

Bernabé‐Orts

Casas‐Rodrigo

Minguet

et al. 2019

Plant Biotechnology Journal

113

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Summary The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of −10 to −2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

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Section: Discussionmentioning

confidence: 99%

Assessment of Cas12a‐mediated gene editing efficiency in plants

Bernabé‐Orts

Casas‐Rodrigo

Minguet

et al. 2019

Plant Biotechnology Journal

113

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“…A possible reason for the partial discrepancy between RT-qPCR and gene reporter data is that the promoter region that was used in the constructs is incomplete. This is unlikely to be the case for six promoters, especially for those that are close To solve this problem, integration of insulators in the vector, at both ends of the transgene to prevent undesirable effects could be an answer (Hasegawa and Nakatsuji 2002;Biłas et al 2016). Indeed, insulators are sequences that stabilize gene expression by guaranteeing gene autonomy (Hasegawa and Nakatsuji 2002;Biłas et al 2016).…”

Section: A Variety Of Expression Patterns Was Detected For the Differmentioning

confidence: 99%

“…This is unlikely to be the case for six promoters, especially for those that are close To solve this problem, integration of insulators in the vector, at both ends of the transgene to prevent undesirable effects could be an answer (Hasegawa and Nakatsuji 2002;Biłas et al 2016). Indeed, insulators are sequences that stabilize gene expression by guaranteeing gene autonomy (Hasegawa and Nakatsuji 2002;Biłas et al 2016). Another option could be to insert the transgenes in a specific locus, preferentially one promoting high expression of genes (Abdel-ghany et al 2015).…”

Section: A Variety Of Expression Patterns Was Detected For the Differmentioning

confidence: 99%

“…However, generating a single T-DNA vector bearing several genes each placed under the same trichome-specific promoter can lead to a dilution effect since transcription might be limited by the amount of available transcription factors recruited by cis-elements present in this promoter (Biłas et al 2016). To prevent such dilution effect, genes coding for each enzyme could be placed downstream different trichome-specific promoters.…”

Section: Glandular Trichomesmentioning

confidence: 99%

“…For a T-DNA containing multiple expression cassettes, the expression of each of the transgenes can be subjected to mutual interference with neighboring ones, originating from competitive binding of certain transcription factors to the different promoters (Hasegawa and Nakatsuji 2002;Biłas et al 2016). Therefore, the insertion of an insulator at both ends of the construct as well as between the different genes within the construct can prevent the influence of both native genome regulatory sequences and regulatory sequences included in the construct, especially in the case of multigene constructs (Hasegawa and Nakatsuji 2002;Biłas et al 2016).…”

Section: Glandular Trichomesmentioning

confidence: 99%

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Identification of two new trichome-specific promoters of Nicotiana tabacum

Pottier

Laterre

Wessem

et al. 2019

Preprint

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Key message3 6 pRbcS-T1 and pMALD1, two new trichome-specific promoters of Nicotiana tabacum, were 3 7 identified and their strength and specificity were compared to those of previously described 3 8 promoters in this species. Abstract 4 1 Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and 4 2 derivatives in tall glandular trichomes, which actively synthesize and secrete specialized 4 3 metabolites. However, implementation of an entire biosynthetic pathway in glandular 4 4 trichomes requires the identification of trichome-specific promoters to appropriately drive the 4 5 expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR 4 6 analysis was carried out on wild-type N. tabacum plants to compare the expression pattern 4 7 and gene expression level of NtRbS-T1 and NtMALD1, two newly identified genes expressed 4 8 in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, and NtLTP1, which 4 9 were reported in the literature to be specifically expressed in glandular trichomes. The latter 5 0 were previously investigated separately, preventing any accurate comparison of their 5 1 expression level. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in 5 2 glandular trichomes like NtCYP71D16, NtCBTS2α, and NtCPS2, while NtLTP1 was also 5 3 expressed in other leaf tissues as well as in the stem. Transcriptional fusions of each of the six 5 4promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by 5 5

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