High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50–100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.

High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple GFP insertions, 60 – 100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.

Transgenic expression of Nix converts genetic females into males and allows automated sex sorting in Aedes albopictus

Aedes albopictus is a major vector of arboviruses. Better understanding of its sex determination is crucial for developing mosquito control tools, especially genetic sexing strains. In Aedes aegypti, Nix is the primary gene responsible for masculinization and Nix-expressing genetic females develop into fertile, albeit flightless, males. In Ae. albopictus, Nix has also been implicated in masculinization but its role remains to be further characterized. In this work, we established Ae. albopictus transgenic lines ectopically expressing Nix. Several were composed exclusively of genetic females, with transgenic individuals being phenotypic and functional males due to the expression of the Nix transgene. Their reproductive fitness was marginally impaired, while their flight performance was similar to controls. Overall, our results show that Nix is sufficient for full masculinization in Ae. albopictus. Moreover, the transgene construct contains a fluorescence marker allowing efficient automated sex sorting. Consequently, such strains constitute valuable sexing strains for genetic control.

PDMPO: a specific silicon or silica, pH sensitive fluorescent probe?

To understand Si behavior in soils and plants, we evaluated the use of PDMPO that was supposed to be a Si-specific, pH-dependent fluorescence marker. We found that the PDMPO fluorescence is neither Si-specific nor pH dependent.

Integration of a FT expression cassette into CRISPR/Cas9 construct enables fast generation and easy identification of transgene-free mutants in Arabidopsis

The CRISPR/Cas9 genome editing technique has been widely used to generate transgene-free mutants in different plant species. Several different methods including fluorescence marker-assisted visual screen of transgene-free mutants and programmed self-elimination of CRISPR/Cas9 construct have been use to increase the efficiency of genome edited transgene-free mutants isolation, but the overall time length required to obtain transgene-free mutants has remained unchanged in these methods. We report here a method for fast generation and easy identification of transgene-free mutants in Arabidopsis. By generating and using a single FT expression cassette-containing CRISPR/Cas9 construct, we targeted two sites of the AITR1 gene. We obtained many early bolting plants in T1 generation, and found that about two thirds of these plants have detectable mutations. We then analyzed T2 generations of two representative lines of genome edited early bolting T1 plants, and identified plants without early bolting phenotype, i.e., transgene-free plants, for both lines. Further more, homologues aitr1 mutants were successful obtained for both lines from these transgene-free plants. Taken together, these results suggest that the method described here enables fast generation, and at the mean time, easy identification of transgene-free mutants in plants.