CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity

Xu, Xiangru; Gantz, Valentino M.; Siomava, Natalia; Bier, Ethan

doi:10.7554/elife.30281

Cited by 33 publications

(35 citation statements)

References 41 publications

(57 reference statements)

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“…2) The gRNA-only gene drive (gRNA-GD) is based on CopyCat gRNA elements 19 that are capable of allelic conversion in the presence of a genetic source of Cas9. Since only the gRNA element is propagated in this case, its spread is regulated by the presence of separate, static Cas9 transgene 10,20,21 . The use of a full-GD is causing concern to the scientific community as an accidental release could spread unchecked 15 .…”

Section: Introductionmentioning

confidence: 99%

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

Amo

Bishop

C.³

et al. 2019

Preprint

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CRISPR-based gene drives spread through populations bypassing the dictates of Mendelian genetics, offering a population-engineering tool for tackling vector-borne diseases, managing crop pests, and helping island conservation efforts; unfortunately, current technologies raise safety concerns for unintended gene propagation. Herein, we address this by splitting the two drive components, Cas9 and gRNAs, into separate alleles to form a novel trans-complementing split-genedrive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This bi-component nature allows for individual transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a smallmolecule-controlled version to investigate the biology of component inheritance and use our system to study the maternal effects on CRISPR inheritance, impaired homology on efficiency, and resistant allele formation. Lastly, mathematical modeling of tGD spread in a population shows potential advantages for improving current gene-drive technologies for field population modification. Figure 1 -The trans-complementing gene-drive system (tGD) features simultaneous super-Mendelian inheritance of two transgenes.(A) Schematic of the tGD genetic arrangement with two elements that can be kept separated as different transgenic lines. The Cas9 transgene is inserted in the genomic location targeted by the gRNA-A, while a separate cassette expressing a tandem gRNA construct (gRNA-A, gRNA-B) is inserted at the location targeted by gRNA-B. Upon genetic cross, each gRNA combines with Cas9 to generate a double-strand DNA break at each locus on the wild-type allele. Each break is then repaired by homology-directed repair (HDR) pathway using the intact chromosome carrying the transgene as a template. (B) Outline of the genetic cross used to demonstrate tGD in fruit flies. F0 males carrying a DsRed-marked Cas9 transgene inserted in the yellow locus were crossed to females carrying a GFP-marked cassette containing two gRNAs (y1-e1) inserted in the ebony coding sequence. Trans-heterozygous F1 females (carrying both Cas9 and gRNAs) were crossed to wild-type males to assess germline transmission rates of the fluorophores marking the transgenes in the F2 progeny. The conversion event is indicated by the red and green triangles in the F1 females (C) Single F1 female germline inheritance output measured as GFP and DsRed marker presence in the F2 progeny. The black bar represents the inheritance average. The blue shading represents the deviation from the expected 50% "Mendelian" inheritance. Inheritance average, standard deviation, number of samples (n) and total number of flies scored in each experiment are represented over the graph in line with the respective data.

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Section: Introductionmentioning

confidence: 99%

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

Amo

Bishop

C.³

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…2The gRNA-only gene drive (gRNA GD) is based on CopyCat gRNA elements 19 that are capable of allelic conversion in the presence of a separate genetic source of Cas9. Since only the gRNA element is propagated in this case, its spread is regulated by the presence of a separate, static Cas9 transgene 10,[19][20][21] . The use of a full GD is causing concern to the scientific community as an accidental release could spread unchecked 15 .…”

mentioning

confidence: 99%

A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment

et al. 2020

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CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.

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“…Several CRISPR/Cas9-based methods have been used to investigate the function of enhancers in Drosophila, some of which are described in the previous section. For instance, Xu et al (2017) have used a split-drive configuration of a CRISPR/Cas9based gene drive system (dubbed as CopyCat) to replace the wing vein enhancer of the knirps (kni) gene in Drosophila with that of a mutant allele or the homologous enhancers of other dipteran species. Also, some next-generation CRISPR/Cas technologies, such as CRISPRa, have been successfully used in vivo using Drosophila (Ewen-Campen et al, 2017;Jia et al, 2018;Lin et al, 2015).…”

Section: Functional Analysis Of Enhancers Through Genome Editingmentioning

confidence: 99%

How to study enhancers in non-traditional insect models

Tomoyasu

Halfon

2020

Journal of Experimental Biology

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Transcriptional enhancers are central to the function and evolution of genes and gene regulation. At the organismal level, enhancers play a crucial role in coordinating tissue-and context-dependent gene expression. At the population level, changes in enhancers are thought to be a major driving force that facilitates evolution of diverse traits. An amazing array of diverse traits seen in insect morphology, physiology and behavior has been the subject of research for centuries. Although enhancer studies in insects outside of Drosophila have been limited, recent advances in functional genomic approaches have begun to make such studies possible in an increasing selection of insect species. Here, instead of comprehensively reviewing currently available technologies for enhancer studies in established model organisms such as Drosophila, we focus on a subset of computational and experimental approaches that are likely applicable to non-Drosophila insects, and discuss the pros and cons of each approach. We discuss the importance of validating enhancer function and evaluate several possible validation methods, such as reporter assays and genome editing. Key points and potential pitfalls when establishing a reporter assay system in non-traditional insect models are also discussed. We close with a discussion of how to advance enhancer studies in insects, both by improving computational approaches and by expanding the genetic toolbox in various insects. Through these discussions, this Review provides a conceptual framework for studying the function and evolution of enhancers in non-traditional insect models.

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CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity

Cited by 33 publications

References 41 publications

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment

How to study enhancers in non-traditional insect models

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