Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

Background Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis thaliana mutants. Low entry, reliable assembly of CRISPR/Cas9 vectors and efficient mutagenesis is necessary to enable a maximum of researchers to break through the genetic redundancy within plant multi-gene families and allow for a plethora of gene function studies that have been previously unachievable. It will also allow routine de novo generation of mutations in ever more complex genetic backgrounds that make introgression of pre-existing alleles highly cumbersome. Results To facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating mutations at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either a FastRed, FastGreen or FastCyan fluorescent seed selection cassette. FastRed and FastGreen binary vectors can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) from A. thaliana or the constitutive promoter from Petroselinum crispum (PcUBi4-2). Available enzymes are “classical” Cas9 codon-optimized for A. thaliana and a recently reported, intron-containing version of Cas9 codon-optimized for Zea mays, zCas9i. We observed the highest efficiency in producing knockout phenotypes by using intron-containing zCas9i driven under egg-cell specific pEC1.2 promoter. Finally, we introduced convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in some of the T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system. Conclusion A rapid, simple and flexible CISPR/Cas9 cloning system was established that allows assembly of multi-guide RNA constructs in a robust and reproducible fashion, by avoiding generation of very big constructs. The system enables a flexible, fast and efficient screening of single or higher order A. thaliana mutants.

Screening and validation of genome-edited animals

The emergence of an array of genome-editing tools in recent years has facilitated the introduction of genetic modifications directly into the embryo, increasing the ease, efficiency and catalogue of alleles accessible to researchers across a range of species. Bypassing the requirement for a selection cassette and resulting in a broad range of outcomes besides the desired allele, genome editing has altered the allele validation process both temporally and technically. Whereas traditional gene targeting relies upon selection and allows allele validation at the embryonic stem cell modification stage, screening for the presence of the intended allele now occurs in the (frequently mosaic) founder animals. Final confirmation of the edited allele can only take place at the subsequent G1 generation and the validation strategy must differentiate the desired allele from a range of unintended outcomes. Here we present some of the challenges posed by gene editing, strategies for validation and considerations for animal colony management.

Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis mutants. To facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating large deletions at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either FastRed, FastGreen or FastCyan fluorescent seed selection cassette. Both, FastRed and FastGreen binary vectors, can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors at the same time. Our approach provides fast and flexible cloning, avoids very big constructs and enables screening different order mutants in the same generation. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the dark, non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) or ubiquitous promoter (PcUBi4-2). Available enzymes are classical Cas9, a recently reported, intron-optimized version or Cpf1 (Cas12a). Finally, we have taken care to introduce convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in the final T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.

Development of a Transposon Mutagenesis System in the Oral Spirochete Treponema denticola

ABSTRACT Here, we report successful transposon mutagenesis in the oral spirochete Treponema denticola. A modified Himar1 transposon, including a new antibiotic selection cassette for T. denticola, generated mutations affecting cell division, transport, and chemotaxis, among other processes. This random mutagenesis system should facilitate research on the biology and pathogenesis of this spirochete, which is associated with human periodontal diseases.

308 INFLUENCE OFA MATRIX ATTACHMENT REGION ON THE EXPRESSION OF BICISTRONIC VECTORS TRANSFECTED IN MAMMALIAN CELLS CULTURED IN VITRO

We investigated the effect of the 52 MAR (matrix attachment region) sequence of chicken lysozyme gene as a possible insulator for the expression of our transgenes in somatic cells to be used for nuclear transfer. With the preliminary purpose to demonstrate a possible positive effect (position or copy number) on the long-term combined expression during in vitro culture, we have created a bicistronic ubiquitous expression vector with (MAR+) or without (MAR–) MAR. The main structure of our constructs is composed of the pCAGGS promoter driving the expression of a reporter gene (enhanced green fluorescent protein, EGFP) followed by a loxable selection cassette (loxP-PGKneo). The MAR region was inserted before the selection cassette. After KpnI digestion, the resulting linearized vectors were purified and subsequently used to transfect adult porcine fibroblast cell lines using the Nucleofector system (Amaxa, Cologne, Germany). Fibroblasts were cultured in DMEM/M199 medium (1:1) + 10% FCS supplemented with 5 ng mL–1 of basic fibroblast growth factor (bFGF). In every experiment, 1 � 106 cells were transfected with 2.5 µg of linearized plasmid and selected for 3 weeks with medium supplemented with 400 µg mL–1 of Geneticin (G418 sulfate, Calbiochem, La Jolla, CA). On Day 8 of G418 selection, we analyzed 150 colonies for each treatment, using fluorescence microscopy with fluorescein isothiocyanate filters. Colonies were classified according to size (large) and cell morphology (small cells without signs of aging). In addition, colonies were classified for uniform GFP expression (uniform), patchy GFP expression (variegated), and no GFP expression (negative). Resistant colonies derived from MAR+ and MAR– vectors, respectively, had 36 (24%), 42 (28%), and 56 (37%) v. 79 (53%), 58 (39%), and 29 (19%) uniform, variegated, and negative GFP. Differences were significant for variegated and negative in MAR+ v. MAR– (chi square, P < 0.05). Thirty-six MAR+ and 42 MAR– colonies uniformly expressing GFP were transferred to 24-well plates and subjected to G418 selection until Day 22, when 7 MAR+ and 15 MAR– clones were still growing in culture. Four of seven MAR+ (57%) and 7/15 MAR– (47%) uniformly expressed high levels of GFP. In conclusion, we found that significantly fewer colonies expressed GFP with the MAR+ vector; however, within the GFP-expressing clones, expression was more uniform. Therefore, we did not find a beneficial effect of MAR sequences on expression in somatic cells during in vitro culture; however, further work is needed to investigate their effect after nuclear transfer and/or on the next generation of cloned transgenic animals.