Summary of the technology
Insertion of new CRISPR spacer units is very infrequent in most species. Detection of these events usually requires large screenings of CRISPR clusters of a high number of clones.
In order to decrease the number of clones to be tested, it is possible to select adapted cells when such acquisition changes the immunity pattern (i.e. enables for the degradation of target molecules). This causes a bias against detection of insertions of other sequences and cannot be executed in cells with silenced CRISPR immunity.
In this sense, the availability of a selectable tool for readily detecting spacer insertion independently of its consequences over the degradation of target genetic elements is highly advantageous compared to the methods currently in use.
Description of the technology
CRISPR structures are a component of a recently discovered prokaryotic immune system. CRISPR structures are arranged in clusters and interspaced by unique spacer sequences, which serve as a guide for the recognition and restriction of infectious nucleic acids. The research group of Molecular Microbiology of the University of Alicante has developed a novel method that enables detection of spacer integration in artificial CRISPR structures, called insertion modules. The main advantage of this technology is that it makes possible the positive selection of cells that have acquired a new spacer without relying on the immunity these spacers may confer. A spacer insertion in these artificial modules brings about a switch in the reading frame of an out-of-frame reporter gene, rendering a functional protein. Ensuing protein activity identifies adapted cells where an insertion has taken place. The method at hand can be used in industrial sectors related to genetics and biotechnology as well as research in microbiology and molecular biology. The research group is looking for companies acquiring this invention for licensing agreement or technical cooperation.
The present invention makes reference to a method for obtaining and detecting spacer insertions in artificial structures (insertion modules) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) located within a transcriptional unit which includes the sequence of a reporter gene with a displaced reading frame. A new insertion restores the reading frame of the reporter gene, yielding a functional protein (Figure 1). This protein’s activity enables insertion detection and the selection of adapted cells in a way that is independent of the immunization the new spacer could provide. The inserted CRISPR spacer unit encompasses a number of nucleotides that is not multiple of three. The spacer in the insertion module, when it is made up of two CRISPR, contains a cryptic STOP codon that circumvents selection of duplications generated by homologous recombination between repeats (Figure 1). The insertion modules contain a sequence known as leader, which enables the intervention of the acquisition mechanism. The acquisition assays begin with a culture of cells carrying the artificial module in a plasmid. A volume from this culture is spread in a solid culture medium that allows for the selection of the activity provided by the reporter gene. As the tested reporter gene confers resistance to chloranfenicol, the selection is conducted in medium containing this antibiotic. After incubation, grown colonies allegedly contain an insertion. Then, a PCR (Polymerase Chain Reaction) is used to discard false positives and plasmids are purified and sequenced. The analysis of inserted spacers is useful for the study of the acquisition process. The spacers can be used to bestow specific immunity against foreign genetic elements containing a sequence recognizable by the system, i.e. akin to the spacer sequence and adjacent to a 2-3 nucleotides motif (termed PAM) characteristic of the particular system at use. Explanation of Figure 1: Schematic representation of the integration reporter modules cloned in the pCSIR T plasmid and strategy used for specific detection of integration events. (A) The integration cassette in pCSIR-T consists of a leader and two CRISPR duplicons (CR) interspaced by a spacer (Sp-1) fused upstream to a fragment of lacZ-α gene (lacZα’) and, downstream, to the complete coding sequence of an out-of-frame cat gene (cat +2). The translation initiation codon of lacZ-α (white arrowhead) is indicated. Translation of transcripts generated from the lac promoter (Plac, denoted with an arrow) will stop at the leader (black arrowhead). Translation of the transcript generated from Plac will end at the leader in case of deletion (cat would remain out of frame, cat +1) (B), at the duplicated spacer (Sp-1) in case of CRISPR-spacer duplication (C) or at the end of the in frame cat coding sequence in case of new spacer (Sp-2) insertion (D).
Main advantages of its use
- Comparative studies among isolates from the same species.
- Development of Molecular Biology and Genetic Engineering tools. CRISPR-Cas systems are being optimized for genetic expression regulation and genome editing of prokaryotic and eukaryotic organisms (including the human species). It allows in vivo silencing or replacement of genes. Some of the applications are gene therapy or plant improvement for agri-food.
- Studies related to Microbial Ecology and Metagenomics.
- The main advantage of this technology is that it makes possible the positive selection of cells that have acquired a new spacer.
- To engineer bacteria of biotechnological interest, providing them with immunity against phages or plasmids conferring antibiotic resistance.
- CONSUMER RELATED: Food and Beverages.
- GENETIC ENGINEERING/MOLECULAR BIOLOGY: Recombinant DNA.
- INDUSTRIAL PRODUCTS: Other Industrial Products.
- MEDICAL/HEALTH RELATED: Therapeutic.
Additional information (attached documents)
Additional information (attached documents)