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.

Propositional Kernels

The pervasive presence of artificial intelligence (AI) in our everyday life has nourished the pursuit of explainable AI. Since the dawn of AI, logic has been widely used to express, in a human-friendly fashion, the internal process that led an (intelligent) system to deliver a specific output. In this paper, we take a step forward in this direction by introducing a novel family of kernels, called Propositional kernels, that construct feature spaces that are easy to interpret. Specifically, Propositional Kernel functions compute the similarity between two binary vectors in a feature space composed of logical propositions of a fixed form. The Propositional kernel framework improves upon the recent Boolean kernel framework by providing more expressive kernels. In addition to the theoretical definitions, we also provide an algorithm (and the source code) to efficiently construct any propositional kernel. An extensive empirical evaluation shows the effectiveness of Propositional kernels on several artificial and benchmark categorical data sets.

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.

Two Probabilistic Models for Quick Dissimilarity Detection of Big Binary Data

The task of data matching arises frequently in many aspects of science. It can become a time consuming process when the data is being matched to a huge database consisting of thousands of possible candidates, and the goal is to find the best match. It can be even more time consuming if the data are big (> 100 MB). One approach to reducing the time complexity of the matching process is to reduce the search space by introducing a pre-matching stage, where very dissimilar data are quickly removed. In this paper we focus our attention to matching big binary data. In this paper we present two probabilistic models for the quick dissimilarity detection of big binary data: the Probabilistic Model for Quick Dissimilarity Detection of Binary vectors (PMQDD) and the Inverse-equality Probabilistic Model for Quick Dissimilarity Detection of Binary vectors (IPMQDD). Dissimilarity detection between binary vectors can be accomplished quickly by random element mapping. The detection technique is not a function of data size and hence dissimilarity detection is performed quickly. We treat binary data as binary vectors, and hence any binary data of any size and dimension is treated as a binary vector. PMQDD is based on a binary similarity distance that does not recognize data and its exact inverse as containing the same pattern and hence considers them to be different. However, in some applications a specific data and its inverse, are regarded as the same pattern, and thus should be identified as being the same; IPMQDD is able to identify such cases, as it is based on a similarity distance that does not distinguish between data and its inverse instance as being dissimilar. We present a comparative analysis between PMQDD and IPMQDD, as well as their similarity distances. We present an application of the models to a set of object models, that show the effectiveness and power of these models

Advances in assembling gRNA/Cas9 constructs in genome editing of plants

This chapter reviews the principles of CRISPR cloning in binary vectors and the different methods and elements employed, including the nucleases alternative to Cas9. It pays special attention to modular cloning strategies and multiplexing tools as well as the engineering of expanded Cas activities. Finally, the chapter includes a case study of the cloning of a nine gRNA multiplex construct and the analysis of its transformants in tobacco plants.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

Representations of Generalized Self-Shrunken Sequences

Output sequences of the cryptographic pseudo-random number generator, known as the generalized self-shrinking generator, are obtained self-decimating Pseudo-Noise (PN)-sequences with shifted versions of themselves. In this paper, we present three different representations of this family of sequences. Two of them, the p and G-representations, are based on the parameters p and G corresponding to shifts and binary vectors, respectively, used to compute the shifted versions of the original PN-sequence. In addition, such sequences can be also computed as the binary sum of diagonals of the Sierpinski’s triangle. This is called the B-representation. Characteristics and generalities of the three representations are analyzed in detail. Under such representations, we determine some properties of these cryptographic sequences. Furthermore, these sequences form a family that has a group structure with the bit-wise XOR operation.