Beyond seek and destroy: How to generate allelic series using genome editing tools

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations

Copernicus Cal/Val Solution - D3.1 Recommendations for R&D activities on Instrumentation Technologies

The Document identifies the gaps in instrumentation technologies for pre-flight characterisation, onboard calibration and Fiducial Reference Measurements (FRM) used for calibration and validation (Cal/Val) activities for the current Copernicus missions. It also addresses the measurement needs for future Copernicus missions and gives a prioritised list of recommendations for R&D activities on instrumentation technologies. Four types of missions are covered based on the division used in the rest of the CCVS project: optical, altimetry, radar and microwave and atmospheric composition. It also gives an overview of some promising instrumentation technologies in each measurement field for FRM that could fill the gaps for requirements not yet met for the current and future Copernicus missions and identifies the research and development (R&D) activities needed to mature these example technologies. The Document does not provide an exhaustive list of all the new technologies being developed but will give a few examples for each field to show what efforts are being made to fill the gaps. None of the examples is promoted as the best possible solutions. The selection is based on the authors' knowledge during the preparation of the Document. The information included is mainly collected from the deliverables of work packages 1 and 2 in the CCVS project. The new technologies are primarily from the interviews with various measurement networks and campaigns carried out in tasks 2.4 and 2.5. Reference documents can be found in section 1.3

Influence of the 20-kDa protein from Bacillus thuringiensis ssp. israelensis on the rate of production of truncated Cry1C proteins

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