Journal: Nature Communications

Article Title: A multifunctional system for genome editing and large-scale interspecies gene transfer

doi: 10.1038/s41467-022-30843-1

Figure Lengend Snippet: The outer layer contains relevant features of each plasmid. The white inner layer (if applicable) represents the modular parts prior to assembly using MoCloFlex (MCF) or the CRISPR SWAPnDROP assembly system. The black inner layer (if applicable) shows the location of variable elements (orange) of the plasmids specific for each edit. Cas9 and meganuclease target sites are indicated by scissors. The sgRNA loci and the respective target sites share the same colour.

Article Snippet: Independent of the CRISPR/Cas9 methods, general approaches for modular cloning were developed to assemble larger constructs from small standardized parts, e.g. MoClo, MoCloFlex, Golden Mutagenesis and Marburg Collection – .

Techniques: Plasmid Preparation, CRISPR

Journal: bioRxiv

Article Title: Developing a Molecular Toolkit to ENABLE all to apply CRISPR/Cas9-based Gene Editing in planta

doi: 10.1101/2025.11.09.687425

Figure Lengend Snippet: Concept of the ENABLE® Gene Editing in planta toolkit. (a) The major components envisioned for the ENABLE gene editing toolkit to allow the user to clone a CRISPR vector for transient or stable plant transformation. (b) The 2-step Golden Gate cloning strategy involves subcloning of two CRISPR target sequences into the two vectors pGMF1 and pGMF2 via annealed oligos encoding the 20 bp target site, followed by assembly of the final T-DNA binary vector. (c) Four different CAS9 cassettes with different promoters, nuclear localization signals (NLS), tags, codon optimizations (Ara = Arabidopsis, wheat or human codon optimized) and terminators were tested in this study. (d) sgRNA expression via an Arabidopsis or rice polymerase III promoter was tested in this study.

Article Snippet: Due to the modular nature of the ENABLE® vectors and the use of the widely used MoClo syntax ( ; ), different nuclease modules as well as resistance markers from existing resources (e.g. ; ) could be easily ordered from platforms such as Addgene to replace the CAS9 cassette and/or selection marker cassettes in our toolkit if needed.

Techniques: CRISPR, Plasmid Preparation, Transformation Assay, Cloning, Subcloning, Expressing

Journal: bioRxiv

Article Title: Developing a Molecular Toolkit to ENABLE all to apply CRISPR/Cas9-based Gene Editing in planta

doi: 10.1101/2025.11.09.687425

Figure Lengend Snippet: Testing of the ENABLE® Gene Editing in planta toolkit in rice protoplasts. (a) Eight different CRISPR vectors were assembled to target the OsPDS1 gene with each containing a unique combination of CAS9 cassette (CasA – CasD) and sgRNA expression cassette (sgRNA expression via OsU6-2 promoter or AtU6-26 promoter). (b) The OsPDS1 gene. Black boxes indicate exons. The position of the two sgRNA target sites in Exon 2 is annotated. The PAM sequence is underlined. (c) Exemplary close-up from untransformed rice protoplasts (top) and a protoplast transformed with one of the eight CRISPR vectors containing an eGFP gene (CasA_OsU6-2p::OsPDS1_sgRNAs). (d) OsPDS1 PCR amplicon analysis of protoplast cultures transformed with different CRISPR constructs. Expected product size in non-edited rice cells: 498 bp, distance between sgRNA1 and sgRNA2 cut site: 97 bp. M = Marker. (e) NGS analysis reveals high proportions of modified reads in PCR amplicons derived from protoplast cultures transformed with CRISPR constructs expressing sgRNAs using the OsU6-2 promoter.

Article Snippet: Due to the modular nature of the ENABLE® vectors and the use of the widely used MoClo syntax ( ; ), different nuclease modules as well as resistance markers from existing resources (e.g. ; ) could be easily ordered from platforms such as Addgene to replace the CAS9 cassette and/or selection marker cassettes in our toolkit if needed.

Techniques: CRISPR, Expressing, Sequencing, Transformation Assay, Amplification, Construct, Marker, Modification, Derivative Assay

Journal: bioRxiv

Article Title: Developing a Molecular Toolkit to ENABLE all to apply CRISPR/Cas9-based Gene Editing in planta

doi: 10.1101/2025.11.09.687425

Figure Lengend Snippet: Testing of the ENABLE® Gene Editing in planta toolkit in stably transformed Arabidopsis plants. (a) Eight different CRISPR vectors were assembled to target the AtGL1 gene with each containing a unique combination of CAS9 cassette (CasA – CasD) and sgRNA expression cassette (sgRNA expression via OsU6-2 promoter or AtU6-26 promoter). (b) The AtGL1 gene. Black boxes indicate exons. The position of the two sgRNA target sites in Exon 2 is annotated. The PAM sequence is underlined. (c) Exemplary pictures from Arabidopsis plants with WT trichome patterning (left), full glabrousness (middle, transformed with CasB_AtU6-26p::AtGL1_sgRNAs construct) or chimeric phenotype (right, transformed with CasD_AtU6-26p::AtGL1_sgRNAs construct). (d) Results of visual inspection of trichome phenotype in independent primary Arabidopsis transformants. (e) Results of AtGL1 genotyping in independent primary Arabidopsis transformants.

Article Snippet: Due to the modular nature of the ENABLE® vectors and the use of the widely used MoClo syntax ( ; ), different nuclease modules as well as resistance markers from existing resources (e.g. ; ) could be easily ordered from platforms such as Addgene to replace the CAS9 cassette and/or selection marker cassettes in our toolkit if needed.

Techniques: Stable Transfection, Transformation Assay, CRISPR, Expressing, Sequencing, Construct