Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion

Nandy, Soumen; Zhao, Shan; Pathak, Bhuvan; Manoharan, Muthusamy; Srivastava, Vibha

doi:10.1186/s12896-015-0212-2

Cited by 29 publications

(14 citation statements)

References 59 publications

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“…Inducible expression systems can be argued as more versatile transient expression systems, provided they generate low or undetectable background expression and high induced expression. Heat-shock promoters meet these criteria as they have been successfully used in applications where their proper regulation was critical, e.g., controlling Cre-lox recombination or nuclease activity for marker excision (Khattri et al, 2011;Lloyd et al, 2005;Nandy et al, 2015;Nandy and Srivastava, 2012;Zhang et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice

Nandy

Pathak

Zhao

et al. 2018

Preprint

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Transient expression of CRISPR/Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat-shock inducible CRISPR/Cas9 system for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using a soybean heatshock protein gene promoter and the rice U3 promoter to express Cas9 and sgRNA, respectively, we developed the heat-shock (HS) inducible CRISPR/Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted by transforming rice with HS-CRISPR/Cas9 vectors, and the presence of targeted mutations was determined before and after the HS treatment. We found only a low rate of targeted mutagenesis before HS, but an increased rate of mutagenesis after HS treatment among the transgenic lines. Specifically, only ~11% of transformants showed characteristic insertions-deletions at the ambient room temperature, but a higher percentage (~45%) of callus lines developed mutations after a few days of HS treatment. Analysis of regenerated plants harboring HS-CRISPR/Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS, which was detectable by Sanger sequencing after several weeks of HS treatments. Most importantly, the HS-induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutant lines that independently segregated from Cas9. Taken together, this work shows that HS-CRISPR/Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome-wide off-target effects and improving the precision of genome editing. Significance Statement:A method for the temporal control on gene editing based on the use of heat-shock induced expression of CRISPR/Cas9 is described, which was efficient in producing heritable mutations in the rice genome. We assume this method will be useful for targeting essential genes and improving the precision of CRISPR/Cas9.

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Section: Discussionmentioning

confidence: 99%

Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice

Nandy

Pathak

Zhao

et al. 2018

Preprint

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“…This CTL approach is different from previously reported transgene stacking using RMCE (Ow, 2011;Nandy et al, 2015;Srivastava and Thomson, 2016;Chen and Ow, 2017). CRISPR-Cas-enabled gene targeting allows precise positioning of trait genes within a small, preselected region on a chromosome.…”

Section: Discussionmentioning

confidence: 79%

Complex Trait Loci in Maize Enabled by CRISPR-Cas9 Mediated Gene Insertion

et al. 2020

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Modern maize hybrids often contain biotech and native traits. To-date all biotech traits have been randomly inserted in the genome. Consequently, developing hybrids with multiple traits is expensive, time-consuming, and complex. Here we report using CRISPR-Cas9 to generate a complex trait locus (CTL) to facilitate trait stacking. A CTL consists of multiple preselected sites positioned within a small well-characterized chromosomal region where trait genes are inserted. We generated individual lines, each carrying a site-specific insertion landing pad (SSILP) that was targeted to a preselected site and capable of efficiently receiving a transgene via recombinase-mediated cassette exchange. The selected sites supported consistent transgene expression and the SSILP insertion had no effect on grain yield. We demonstrated that two traits residing at different sites within a CTL can be combined via genetic recombination. CTL technology is a major step forward in the development of multi-trait maize hybrids.

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“…Inducible expression systems can be argued as more versatile transient expression systems, provided they generate low or undetectable background expression and a high‐induced expression. Heat‐shock promoters meet these criteria as they have been successfully used in applications where their proper regulation was critical, for example , controlling the Cre‐ lox recombination or the nuclease activity for marker excision (Khattri, Nandy, & Srivastava, ; Lloyd, Plaisier, Carroll, & Drews, ; Nandy & Srivastava, ; Nandy, Zhao, Pathak, Manoharan, & Srivastava, ; Zhang et al., ).…”

Section: Discussionmentioning

confidence: 99%

Heat‐shock‐inducible CRISPR/Cas9 system generates heritable mutations in rice

et al. 2019

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Summary Transient expression of CRISPR /Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat‐shock‐inducible CRISPR /Cas9 for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using the soybean heat‐shock protein gene promoter and the rice U3 promoter to express Cas9 and sg RNA , respectively, we developed the heat‐shock ( HS )‐inducible CRISPR /Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted in rice, and the presence of targeted mutations was determined before and after the HS treatment. Only a low rate of targeted mutagenesis was detected before HS (~16%), but an increased rate of mutagenesis was observed after the HS treatment among the transgenic lines (50–63%). Analysis of regenerated plants harboring HS ‐ CRISPR /Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS , which was detectable by Sanger sequencing after a few weeks of HS treatments. Most importantly, the HS ‐induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutations that independently segregated from the Cas9 gene. Additionally, off‐target mutations were either undetectable or found at a lower rate in HS ‐ CRISPR /Cas9 lines as compared to the constitutive‐overexpression CRISPR /Cas9 lines. Taken together, this work shows that HS ‐ CRISPR /Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome‐wide off‐target effects and improving the precision of genome editing.

show abstract

Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion

Cited by 29 publications

References 59 publications

Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice

Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice

Complex Trait Loci in Maize Enabled by CRISPR-Cas9 Mediated Gene Insertion

Heat‐shock‐inducible CRISPR/Cas9 system generates heritable mutations in rice

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