Cre-assisted fine-mapping of neural circuits using orthogonal split inteins

Luan, Haojiang; Kuzin, A.; Odenwald, Ward F.; White, Benjamin H.

doi:10.7554/elife.53041

Cited by 6 publications

(5 citation statements)

References 83 publications

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“…Upon translation, these peptides associate with one another, self-excise, and seamlessly trans-splice the two adjacent polypeptide chains to which they are fused ( 19 ). Split-inteins have been successfully exploited to generate split proteins used in other expression systems ( 20 , 21 ), and we sought to use them here to reconstitute wild-type Gal4 from two functionally inert fragments.…”

Section: Resultsmentioning

confidence: 99%

split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80

Ewen-Campen

Luan

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila . However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of “split-intein Gal4” in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type–specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm (“Two Against Background” or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.

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Section: Resultsmentioning

confidence: 99%

split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80

Ewen-Campen

Luan

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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show abstract

“…Upon translation, these peptides associate with one another, self-excise, and seamlessly trans-splice the two adjacent polypeptide chains to which they are fused (19). Split inteins have been successfully exploited to generate split proteins used in other expression systems (20, 21), and we sought to use them here to reconstitute wildtype Gal4 from two functionally inert fragments.…”

Section: Resultsmentioning

confidence: 99%

split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80

Ewen-Campen

Luan

et al. 2023

Preprint

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The split-Gal4 system allows for intersectional genetic labeling of highly specific cell-types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a new split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is fully repressible by Gal80. We demonstrate the potent inducibility of split-intein Gal4 in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell-type specific genetic drivers based on in silico predictions generated by single cell RNAseq (scRNAseq) datasets, and we describe a new algorithm (Two Against Background or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.

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“…To date, no other methods have emerged that meet this requirement. The recently developed SpaRCLIn method has been proposed as an alternative to Split Gal4, but its efficacy and promise remain to be demonstrated (Luan et al, 2020).…”

Section: Resultsmentioning

confidence: 99%

“…Red, 24B10 immunolabeling. Panel (A) from Luan et al (2020); panel (C) adapted from Lacin and Truman (2016); panels (D-F) from Ting et al (2011). neuroscience.…”

Section: Neurodevelopmentmentioning

confidence: 99%