Single-Copy Knock-In Loci for Defined Gene Expression in<i>Caenorhabditis elegans</i>

Silva-García, Carlos Giovanni; Lanjuin, Anne; Heintz, Caroline; Dutta, Sneha; Clark, Nicole M.; Mair, William

doi:10.1534/g3.119.400314

Cited by 69 publications

(77 citation statements)

References 16 publications

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“…45 such as Hygromycin B resistance (Radman et al 2013) or a self-excising cassette (SEC) 70 (Dickinson et al 2015;Kasimatis et al 2018). More specialized CRISPR strategies, such as the 71 SKI LODGE method, facilitates tissue-specific expression by splitting the coding and promoter 72 element (Silva-García et al 2019). A particular advantage of this latter strategy is that it 73 introduces modular transgene integration, allowing for a more straightforward integration into a 74 backbone of standard genetic elements that are pre-integrated within a safe harbor location.…”

Section: Abstract: 24mentioning

confidence: 99%

Rapid self-selecting and clone-free integration of transgenes into engineered CRISPR safe harbor locations inCaenorhabditis elegans

Stevenson

Moerdyk-Schauwecker

Jamison

et al. 2020

Preprint

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24Precision genome editing for model organisms has revolutionized functional analysis and 25 validation of a wide variety of molecular systems. To date, the capacity to insert transgenes into 26 the model nematode Caenorhabditis elegans has focused on utilizing either transposable 27 elements or CRISPR-based safe harbor strategies. These methods require laborious screening 28 processes that often result in false positives from heritable extrachromosomal arrays or rely on 29 co-CRISPR markers to identify likely edited individuals. As a result, verification of transgene 30 insertion requires anti-array selection screening methods or extensive PCR genotyping 31 respectively. These approaches also rely on cloning plasmids for the addition of transgenes. 32Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered 33 insertion locations containing a synthetic guide RNA target and a split-selection system to 34 eliminate false positives from array formation, thereby providing integration-specific selection. 35This approach allows the experimenter to confirm an integration event has taken place without 36 molecular validation or anti-array screening methods, and is capable of producing integrated 37 transgenic lines in as little as five days post-injection. To further increase the speed of generating 38 transgenic lines, we also utilized the C. elegans native homology-based formation of extra-39 chromosomal arrays to assemble transgenes in-situ, removing the cloning step. We show that 40 complete transgenes can be made and inserted into our split-selection safe harbor locations 41 starting from PCR products, providing a clone-free and molecular-validation-free strategy for 42 single-copy transgene integration. Overall, this combination of approaches provides an 43 economical and rapid system for generating highly reproducible complex transgenics in C. 44 elegans. 45 46 3 Introduction: 47

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Section: Abstract: 24mentioning

confidence: 99%

Rapid self-selecting and clone-free integration of transgenes into engineered CRISPR safe harbor locations inCaenorhabditis elegans

Stevenson

Moerdyk-Schauwecker

Jamison

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…These approaches include generalized methods for transgenic cargo insertion, such as those using a selective marker like Hygromycin B resistance (Chen et al 2013) or a self-excising cassette (SEC) (Dickinson et al 2015;Kasimatis et al 2018). More specialized CRISPR strategies, such as the SKI LODGE method, facilitates tissue-specific expression by splitting the coding and promoter element (Silva-García et al 2019). A particular advantage of this latter strategy is that it introduces modular transgene integration, allowing for a more straightforward integration into a backbone of standard genetic elements that are pre-integrated within a safe harbor location.…”

Section: Introductionmentioning

confidence: 99%

Rapid Self-Selecting and Clone-Free Integration of Transgenes into Engineered CRISPR Safe Harbor Locations inCaenorhabditis elegans

Stevenson¹,

Moerdyk-Schauwecker²,

Jamison³

et al. 2020

G3 Genes|Genomes|Genetics

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Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert single-copy transgenes into the model nematode Caenorhabditis eleganshas focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require plate-level screening processes to avoid selecting heritable extrachromosomal arrays or rely on co-CRISPR markers to identify knock-in events. As a result, verification of transgene insertion requires anti-array selection screening methods and PCR genotyping. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native microhomology-based recombination, to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.

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“…Our current strategy to express rab-27 only in GABA neurons used an expression construct that contained the rab-3 3'UTR, while our previous efforts used the unc-54 3'UTR. The unc-54 UTR sequence can itself drive expression in the posterior gut because it contains regulatory and coding sequence for the intestinal gene aex-5 (Silva-García et al 2019). We hypothesized that a requirement for intestinal expression accounts for the different effects of the UTR.…”

Section: Introductionmentioning

confidence: 99%

rab-27acts in an intestinal secretory pathway to inhibit axon regeneration inC. elegans

Lin-Moore

Oyeyemi

Hammarlund

2020

Preprint

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Injured axons must regenerate to restore nervous system function, and regeneration is regulated in part by external factors from non-neuronal tissues. Many of these extrinsic factors act in the immediate cellular environment of the axon to promote or restrict regeneration, but the existence of long-distance signals regulating axon regeneration has not been clear. Here we show that the Rab GTPase rab-27 inhibits regeneration of GABAergic motor neurons in C. elegans through activity in the intestine. Re-expression of RAB-27, but not the closely related RAB-3, in the intestine of rab-27 mutant animals is sufficient to rescue normal regeneration. Several additional components of an intestinal neuropeptide secretion pathway also inhibit axon regeneration, including NPDC1/cab-1, SNAP25/aex-4, and KPC3/aex-5. Together these data indicate that RAB-27-dependent neuropeptide secretion from the intestine inhibits axon regeneration, and point to distal tissues as potent extrinsic regulators of regeneration.

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Single-Copy Knock-In Loci for Defined Gene Expression inCaenorhabditis elegans

Cited by 69 publications

References 16 publications

Rapid self-selecting and clone-free integration of transgenes into engineered CRISPR safe harbor locations inCaenorhabditis elegans

Rapid self-selecting and clone-free integration of transgenes into engineered CRISPR safe harbor locations inCaenorhabditis elegans

Rapid Self-Selecting and Clone-Free Integration of Transgenes into Engineered CRISPR Safe Harbor Locations inCaenorhabditis elegans

rab-27acts in an intestinal secretory pathway to inhibit axon regeneration inC. elegans

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