FIELD OF THE INVENTION

The present invention refers to the genetic engineering field. Particularly, the present invention refers to a CRISPR-Cas system, suitable for gene edition, which comprises: a) at least a guide sequence capable of hybridizing to a target sequence in a eukaryotic cells, b) at least an effector protein, or a nucleic acid encoding the effector protein, with nuclease or nickase activity, and c) catalytic domain of a chromatin remodelling protein coupled to the effector protein.

STATE OF THE ART

Genome editing is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site specific locations.

Genome editing can be achieved by using CRISPR-Cas system, wherein a small piece of RNA with a short "guide" sequence attaches (binds) to a specific target sequence of DNA in a genome. The RNA also binds to the Cas enzyme. The modified RNA is used to recognize the DNA sequence, and the Cas enzyme cuts the DNA at the targeted location. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpfl) can also be used.

Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

Precisely, homology directed repair (HDR) is a mechanism used by the cells to repair DNA cuts. The most common form of HDR is homologous recombination. The HDR mechanism can only be used by the cell when there is a homologous piece of DNA present in the nucleus, mostly in G2 and S phase of the cell cycle. Thus, after double or single stranded DNA cuts in the genome, the cell machinery resolves the event by next homologous end joining, which has unpredictable and deleterious effects. To do precise gene editing this cut needs to be repaired by homologous recombination which is very inefficient.

Thus, there is an unmet need in the genetic engineering field to find strategies or systems which are able to efficiently repair the DNA cuts originated by CRISPR system. In other words, the objective of the present invention is to increase the efficiency of HDR mechanism in the context of gene editing.

The present invention solves this problem by providing a strategy for improving the efficiency of the HDR system after CRISPR-Cas nuclease activity on a DNA target. This has been achieved by coupling CRISPR-Cas to specific proteins as explained below.

DESCRIPTION OF THE INVENTION Brief description of the invention

The present invention refers to a CRISPR-Cas system, suitable for gene edition, wherein the efficiency of the HDR system after CRISPR-Cas nuclease activity on a DNA target has been improved.

The first embodiment of the present invention refers to a CRISPR-Cas system of the invention comprises: a) at least a guide sequence capable of hybridizing to a target sequence in a eukaryotic cells, b) at least an effector protein, or a nucleic acid encoding the effector protein, with nuclease or nickase activity, and c) catalytic domain of a chromatin remodelling protein coupled to the effector protein.

In a preferred embodiment, the CRISPR-Cas system further comprises an HDR template. The HDR template may consist of a DNA fragment of variable length with a right and left homology arm and a central region containing the desired edit to introduce in the genome. The HDR template used can be a ssDNA linear or circular, a dsDNA linear or circular or an AAV DNA genome.

In a preferred embodiment, the effector protein is cas9 or Cpf 1.

In a preferred embodiment, the chromatin remodelling protein is a member of the PRDM family.

In a preferred embodiment, the chromatin remodelling protein is a member of the chromatin remodelling protein is selected from the group comprising: PRDM9, PRDMl, PRDM2, PRDM3, PRDM 16 or PRDM6.

In a preferred embodiment, the chromatin remodelling protein member of the chromatin remodelling protein is PRDM9. In a preferred embodiment, the chromatin remodelling protein is coupled in N- or C- configuration.

In a preferred embodiment, the effector protein and/or the chromatin remodelling protein are wild type proteins. In a preferred embodiment, the effector protein and/or the chromatin remodelling protein are mutant proteins.

In a preferred embodiment, the system further comprises at least one aptamers linked to the guide sequence.

In a preferred embodiment, the system further comprises at least one linker, preferably selected from:

In a preferred embodiment, the system further comprises a MS2 loop linked to the guide sequence.

In a preferred embodiment, the system further comprises one or more HDR activator and/or one or more NHEJ (Non-homologous end joining) inhibitor fused to the effector protein, or combinations thereof.

In a preferred embodiment, the HDR activator is selected from the group comprising: RAD51, RAD52, DMC1, CtIP, or any combination thereof. In a preferred embodiment, the NHEJ inhibitors is selected from the group comprising: E1B55K, E4orf6, 53BP1(DM), Rif, p53, or any combination thereof.

In a preferred embodiment, the one or more HDR activator and/or one or more NHEJ inhibitor proteins is fused to the effector protein. The second embodiment of the present invention refers to a method for gene edition which comprises the use of the CRISPR-Cas system defined above.

For the purpose of the present invention the following terms are defined:

• The term "comprising" means including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

• The expression "consisting of’ means including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. Description of the figures

Figure 1. Cas9 PDMR9 fusion. A) Scheme of cas9-PDRM9 fusion and split GFP reporter system. H5 stands for 5’ homologous recombination sequence, H3 for 3’ homologous recombination sequence, SA splicing acceptor, SD splicing donor B) HDR activity measured using the split-GFP reporter when acceptor split GFP construct is provided as an episomal plasmid; or integrated in the genome (C).

Figure 2. Traffic light reporter system for HDR and NHEJ. A) Scheme of the traffic light reporter system to measure HDR and NHEJ efficiencies. B) HDR and NHEJ measured by number of cells expressing GFP or RFP, and relative ratio of HDR versus NHEJ, after a double stranded break in the reporter DNA construct mediated by Cas9 or Cas9-PRDM9 fusion C) Synergistic HDR Enhancer technology: Cas9 (in blue) is fused to PRDM9 (green). Cas9 is associated with different HDR enhacers via sgRNA loops that include MS2 phage aptamer sequences bound by fusion proteins of HDR enhancers and the MS2 coat protein. D) HDR measurement of HDR comparing hCas9 alone or fused with PRDM9, as well as co delivery. E) Performance of different ssODN templates. Figure 3 HDR enhancement by Cas9 fusion proteins. A) HDR/NHEJ ratio of the Traffic Light Reporter, upon transfection with Cas9, Cas9-PRDM9 and Cas9-PRDM9 together with a different HDR enhancing factors. B) HDR/NHEJ ratio of the Traffic Light Reporter, upon transfection with Cas9, Cas9-PRDM9 and Cas9-PRDM9 together with a combination of different HDR enhancing factors. C) HDR/NHEJ ratio of the Traffic Light Reporter, upon transfection with Cas9-PRDM9 and Cas9-PRDM9 together with a the best HDR enhancing factors.

Figure 4. HDR enhancement in Cas9 fusion proteins by additional factors. A) Library of synergistic factors tested. B) SHE performance for HDR in iPSC cells for HDR and NHEJ comparison.

Detailed description of the invention

The present invention is illustrated by means of the Examples set below, without the intention of limiting its scope of protection.

Example 1. Material and methods

Example 1.1. Cloning and plasmids

PRDM9 catalytic domain together with a flexible linker (GGGGS) was purchased from Twist Bioscience. PRDM9 was cloned into a Cas9-expression vector Addgene #41815 using isothermal assembly following standard protocols. Dead (D10A and H840A) and nickase (D10 A) Cas9 mutations as well as PRDM9 catalytic mutation (G172A) were introduced by site directed mutagenesis (New England Biolabs). The collection of HDR enhancing factors fused to MS2 protein were constructed based on the pcDNA™3.1 vector backbone (Thermo Fisher). The following expression vectors were amplified by PCR: RAD51 (Addgene #41815), CtIP (Addgene #109403), Ad4orf2B (Addgene #64221), Ad4E4orf6 (Addgene #64221), 53BP1-DN (Addgene #131045), D-sup (Addgene #90019) and DMC1 (CRG CDS collection) were amplified by PCR. Similarly, MS2 expressing vector (Addgene #61423) was also amplified by PCR. HDR enhancing factors and MS2 tag were cloning between Esp3I sites by Golden Gate Assembly. In the case of UL12, a gBlock of MS2-UL12 was ordered to Twist Bioscience and cloned into pcDNA™3.1 vector backbone by Golden Gate Assembly. MS2-RAD52 was ordered as a plasmid vector to Twist Bioscience. gRNAs were cloned into sg-RNA(MS2) cloning vector (Addgene #61424) between Bbsl sites using standard cloning methods. Example 1.2. Cell culture, transfection and electroporation

Hek293T cells were infected with TLR lentivirus at a 0,2 multiplicity of infection (MOI) to generate the TLR cell line. After infection cells were selected with puromycin following standard protocols for 2 weeks before being used for subsequent assays. Hek293T cell line containing pT4 SMN1 2/2 emGFP was generated by PEI mediated transfection of SB100X and pT4 SMN1 2/2 emGFP DNA constructs, followed by single clone expansion and PCR genotyping. A positive clone was selected and expanded and used for subsequent assays.

TLR cell line and C2C12 cell line (ATCC) were cultured at 37°C in a 5% CO 2 incubator with Dulbecco’s modified eagle medium (DMEM), supplemented with high glucose (Gibco, Therm Fisher), 10% Fetal Bovine Serum (FBS), 2 mM glutamine and 100 U penicillin/0.1 mg/mL streptomycin.

FiPSC-Ctrll-Ep6F-5 cell line was purchased from the Biobank of the Barcelona Centre for Regenerative Medicine (CMRB). Cell’s transfection experiments were performed with Polyethyleneimine (PEI, Thermo Fisher Scientific) at 1:3 DNA-PEI ratio in OptiMem. Cells were seeded the day before to achieve 70% confluence on transfection day (usually 120.000 cells in adherent p24 well plate). Plasmid molar ratio was 1 of Cas9 or Cas9-PRDM9: 3 gRNA: 3 HDR template using 0,089 pmols of Cas9 or Cas9-PRDM9 for a p24 well plate. For the experiments with the HDR enhancers plasmid molar ratio was 1 Cas9-PRDM9: 2 HDR enhancer: 3 gRNA: 3 HDR template using 0,089 pmols of Cas9-PRDM9 for a p24 well plate.

Electroporation of C2C12 and iPS cells was performed according to the manufacturer’s instructions with Lonza 4D-Nucleofector System with CD-137 program (C2C12). In the case of the iPS the parameters P3 Primary Cell 4D-Nucleofector were followed and the program CM-113 was applied.

Example 1.3. Analysis of HDR efficiency by FACS

When targeting HDR efficiency in TLR cells, targeted integration results in cells becoming GFP-positive which can be easily monitored by FACS analysis. Cells were analysed by flow cytometry using BD LSR Fortessa (BD Bioscience. Blue 488nm laser with 530/30 filter and Yellow Green 561nm laser with 610/20 filter) 3-4 days after transfection. The relative HDR frequency was obtained by normalizing HDR by NHEJ frequency (RFP-positive cells).

Example 1.4. NGS Library Preparation

To quantify precise HDR genomic DNA was extracted using DNeasy Blood and tissue kit (Qiagen) and it was amplified using two-steps PCR and sequenced using Illumina sequencing platform (Miseq). DNA contains wild-type and edited DNA molecules, which were amplified together using the same pairs of primers. The two PCR reactions were performed with KAPA HiFi DNA Polymerase following manufacturer protocol: lOOng DNA, 0.3 mM of forward and reverse primers in a final reaction volume of 25 pi. Primers included sequencing adapters to their 3’ -ends, adding them to both termini of PCR products that amplified genomic DNA. For the second PCR the primers used appended dual sample indexes and flow cell adapters. PCR products were purified with a PCR purification kit (Qiagen) and quantified using the QuBIT dsDNA High Sensitivity Assay kit and the QuBIT 2.0 fluorometer according to the manufacturer’s instructions (Life Technologies) before preparing the sequencing reaction.

Example 2. Results

Example 2.1. Cas9-PDRM9 fusion.

The inventors of the present invention first constructed a C-terminal fusion of S. pyogenes cas9 and PDRM9 and tested recombination efficiency with one split GFP reporter, both as episomal DNA and in cells containing the split GFP reporter in the genome (Figure 1A). The inventors observed an increase of HDR of 40% both in episomal plasmid system and chromosome reporter system (Figure IB and Figure 1C respectively, Pval=0.04 and 0,03).

An alternative traffic light reporter system was used to estimate the increase in HDR compare with NHEJ (Non-homologous DNA end joining) (Figure 2A). The inventors generated stable cells lines with a copy of the TLR system for Hek293T. Although HDR absolute efficiency did not significantly changed in the conditions tested, a decrease in the NHEJ was observed when the catalytic domain of PRDM9 was fused to Cas9 (Figure 2B). There was a significant increase of HDR/NHEJ ration for the fusion with the HDR enhancer PRDM9. In addition, this enhancement was lost when the fusion was made to a mutated version of PRDM9 (Figure 2B)

Nickase Cas9 was also tested in the chimeric protein as it could outperforms Cas9 HDR activity when fused with hRad51 avoiding double-strand break (DSB) repair; however, lack of effective HDR activity was observed when using deadCas9 or nickaseCas9 version of the nuclease (Figure 2B right panel). Additionally, the inventors have created a library of chimeric cas9 proteins with PRDM9 at different topologies within the Cas9 peptide to increase HDR activity. Moreover, a library of flexible linkers was also screened to increase the HDR efficiency. In order to further increase the HDR efficiency we characterized the impact of different HDR templates by employing a library of templates (Figure 2E).

Example 2.2. Synergic HDR Enhancer System (SHE).

In order to explore additional factors that combined with PRDM9 could further increase the HDR efficiency we generated a Synergic HDR enhancer System or SHE (Figure 3A) by employing a modified gRNA with a tetra-loop of phage sequence MS2 described elsewhere for synthetic transcriptional activation (Konermann et al. 2015). This library (Figure 4A) consist of multiple selected rad51 orthologues and rad52 which are key in HDR, in addition to other HDR associated proteins such as CtIP and MREll, important in the resection of DNA after a DSB that have HDR enhancer activity when fussed to Cas9, as well as other proteins such as adenovirus 4 proteins E1B55K and E4orf6 which inhibit the competing pathway NHEJ. Expression of i53 also inhibit NHEJ, by suppressing 53BP1, a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR. Additionally, we tested the protein Dsup from radiation resistant organism Tardigrade.

Members of this library are summarized in Figure 4A.

The pooled library and chimeric Cas9 protein and additional factors was transfected a population of cells containing the HDR reporter. After transfection, FACS analysis was done. Many of the factors tested individually showed an increase in the HDR activity in the traffic light reporter (Figure 3A and C); in addition, the combinations of the best factors were also tested HDR activity (Figure 3B). We also validated the effect of our enhanced HDR machinery of Cas9-PRDM9 in addition to Ctpfl and 53BP1 in iPSC cells and editing outcome was measured by NGS technology by Illumina (Figure 4B).

We included factors important in recombination during meiosis such DMC1, a meiosis specific protein implicated in the repair of DSB using the homologous chromosome as the template for repairing the break.

The pooled library and chimeric Cas9 protein will be cloned into a lentiviral vector and transduced into a population of cells containing the HDR reporter. After transfection with gRNA and template, FACS sorting will be used to enrich for cells that underwent HDR. This population will be analyzed to find overrepresented library members which could be potential activators of HDR. Genotyping will be performed using a specific DNA barcode that will have been added previously to each library member.