WO2012058458A2 - Method for increasing the efficiency of double-strand break-induced mutagenesis - Google Patents
Method for increasing the efficiency of double-strand break-induced mutagenesis Download PDFInfo
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- WO2012058458A2 WO2012058458A2 PCT/US2011/058133 US2011058133W WO2012058458A2 WO 2012058458 A2 WO2012058458 A2 WO 2012058458A2 US 2011058133 W US2011058133 W US 2011058133W WO 2012058458 A2 WO2012058458 A2 WO 2012058458A2
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Definitions
- the present invention relates to a method for increasing double-strand break-induced mutagenesis at a genomic locus of interest in a cell, thereby providing new tools for genome engineering, including therapeutic applications and cell line engineering. More specifically, the present invention concerns a method for increasing double-strand break-induced mutagenesis at a genomic locus of interest, leading to a loss of genetic information and preventing any scarless re-ligation of said genomic locus of interest by NHEJ (nonhomologous end joining). The present invention also relates to engineered endonucleases, chimeric or not, vectors, compositions and kits used to implement this method.
- DSB double-strand breaks
- Haber 2000 Mammalian genomes constantly suffer from various types of damage of which double-strand breaks (DSB) are considered the most dangerous (Haber 2000).
- DSBs can arise when the replication fork encounters a nick or when ionizing radiation particles create clusters of reactive oxygen species along their path. These reactive oxygen species may in turn themselves cause DSBs.
- the need to repair DSBs arises commonly (Li, Vogel et al. 2007) and is critical for cell survival (Haber 2000). Failure to correct or incorrect repair can result in deleterious genomic rearrangements, cell cycle arrest, and/or cell death.
- NHEJ comprises at least two different processes (Feldmann, Schmiemann et al. 2000).
- the main and best characterized mechanism involves rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson 1998) or via the so-called microhomology-mediated end joining (MMEJ) (Ma, Kim et al. 2003).
- MMEJ microhomology-mediated end joining
- DNA repair processes there are three enzymatic activities required for repair of DSBs by the NHEJ pathway: ( ) nucleases to remove damaged DNA, ( ) polymerases to aid in the repair, and ( / ' ) a ligase to restore the phosphodiester backbone.
- DNA can be simply re-ligated or terminal nucleotides can be modified or removed by inherent enzymatic activities, such as phosphokinases and exo-nucleases. Missing nucleotides can also be added by polymerase ⁇ or ⁇ .
- NHEJ can be viewed as a flexible pathway for which the unique goal is to restore the chromosomal integrity, even at the cost of excision or insertion of nucleotide(s).
- DNA repair can be triggered by both physical and chemical means.
- Several chemicals are known to cause DNA lesions and are used routinely. Radiomimetic agents, for example, work through free-radical attack on the sugar moieties of DNA (Povirk 1996).
- a second group of drugs that induce DNA damage includes inhibitors of topoisomerase I (Topol) and II (TopoII) (Burden and N. 1998; Teicher 2008).
- Other classes of chemicals bind covalently to the DNA and form bulky adducts that are repaired by the nucleotide excision repair (NER) system (Nouspikel 2009). Chemicals inducing DNA damage have a diverse range of applications, however, although certain agents are more commonly applied in studying a particular repair pathway (e.g.
- GT gene targeting
- HR homologous recombination
- GT events occur in a fairly small population of treated mammalian cells and is extremely low in higher plant cells, ranging between 0.01-0.1 % of the total number of random integration events (Terada, Johzuka-Hisatomi et al. 2007).
- the low GT frequencies reported in various organisms are thought to result from competition between HR and NHEJ for repair of DSBs.
- the ends of a donor molecule are likely to be joined by NHEJ rather than participating in HR, thus reducing GT frequency.
- DSB repair by NHEJ is error-prone due to end-joining processes that generate insertions and/or deletions (Britt 1999).
- these NHEJ-based strategies might be more effective than HR-based strategies for targeted mutagenesis into cells.
- I-Scel a rare cutting endonuclease
- endonucleases is limited to rarely occurring natural recognition sites or to artificially introduced target sites.
- meganucleases with engineered specificity towards a chosen sequence have been developed. Meganucleases show high specificity to their DNA target. These proteins being able to cleave a unique chromosomal sequence and therefore do not affect global genome integrity.
- Natural meganucleases are essentially represented by homing endonucleases, a widespread class of proteins found in eukaryotes, bacteria and archae (Chevalier and Stoddard 2001 ). Early studies of the I-Scel and HO homing endonucleases illustrated how the cleavage activity of these proteins can be used to initiate HR events in living cells and demonstrated the recombinogenic properties of chromosomal DSBs (Dujon, Colleaux et al. 1986; Haber 1995). Since then, meganuclease- induced HR has been successfully used for genome engineering purposes in bacteria (Posfai, Kolisnychenko et al. 1999), mammalian cells (Sargent, Brenneman et al.
- ZFNs are chimeric proteins composed of a synthetic zinc-finger-based DNA binding domain fused to a DNA cleavage domain. By modification of the zinc-finger DNA binding domain, ZFNs can be specifically designed to cleave virtually any long stretch of dsDNA sequence (Kim, Cha et al. 1996; Cathomen and Joung 2008).
- a NHEJ-based targeted mutagenesis strategy was recently developed for several organisms by using synthetic ZFNs to generate DSBs at specific genomic sites (Lloyd, Plaisier et al. 2005; Beumer, Trautman et al. 2008; Doyon, McCammon et al. 2008; Meng, Noyes et al. 2008).
- the inventors have developed a new approach to increase the efficiency of targeted DSB-induced mutagenesis and have created a new type of meganucleases comprising several catalytic domains to implement this new approach. These novel enzymes allow a DNA cleavage that will lead to the loss of genetic information and any NHEJ pathway will produce targeted mutagenesis.
- the present invention relates to a method for increasing double-strand break-induced mutagenesis at a genomic locus of interest in a cell, thereby giving new tools for genome engineering, including therapeutic applications and cell line engineering. More specifically, in a first aspect, the present invention concerns a method for increasing double-strand break-induced mutagenesis at a genomic locus of interest, leading to a loss of genetic information and preventing any scarless re-ligation of said genomic locus of interest by NHEJ.
- the present invention relates to engineered enzymes and more particularly to chimeric rare-cutting endonucleases able to target a DNA sequence within a genomic locus of interest to generate at least one DNA double-strand break and a loss of genetic information around said DNA sequence thus preventing any scarless re-ligation of said genomic locus of interest by NHEJ.
- the present invention concerns a method for the generation of at least two-nearby DNA double-strand breaks at a genomic locus of interest to prevent any scarless re-ligation of said genomic locus of interest by NHEJ.
- the present invention relates to engineered enzymes and more particularly to engineered rare-cutting endonucleases, chimeric or not, able to target a DNA sequence within a genomic locus of interest to generate at said locus of interest at least two- nearby DNA double-strand breaks leading to at least the removal of a DNA fragment and thus preventing any scarless re-ligation of said genomic locus of interest by NHEJ.
- the present invention describes a method to identify at a genomic locus of interest a DNA target sequence cleavable at least twice by a fusion protein leading at least to a loss of genetic information and preventing any scarless re-ligation of said genomic locus of interest by NHEJ.
- the present invention relates to fusion proteins able to generate at least two nearby DNA double-strand breaks into a genomic locus of interest comprising one DNA target sequence cleavable by one rare-cutting endonuclease nearby one DNA target sequence cleavable by one frequent-cutting endonuclease.
- the present invention also relates to specific vectors, compositions and kits used to implement this method.
- Figure 1 Elimination of an intervening sequence enhances DSB-induced mutagenesis.
- the 22bp DNA sequences recognized by D21m (or D21 ) and Ri m (or R21 ), respectively, are introduced into a plasmid.
- a 10-bp intervening sequence is cloned between the two recognition sequences to avoid steric hindrance upon meganuclease binding.
- the intervening fragment comprising the 10-bp sequence surrounded by half of each target site is excised. Subsequent NHEJ, either via re-ligation of compatible or incompatible DNA ends, leads to mutagenic events since genetic information was lost.
- FIG. 2 Schematic representation of the analyses performed to detect DSB- induced mutations.
- HEK293 cells are simultaneously transfected with target plasmid and either one or two different meganuclease expressing plasmids. DNA is extracted two days post transfection and specific PCR is performed. PCR products are analyzed using deep sequencing technology (454, Roche). Alternatively, a mutation detection assay (Transgenomic, Inc. USA) is performed. PCR product from untreated cells is mixed (equimolar) with PCR products treated with the meganucleases. The melting/annealing step generates heteroduplex DNA, recognized and cleaved by the CEL-1 enzyme. After digestion, DNA bands are resolved on an analytic gel and each band is quantified by densitometry.
- FIG. 3 Sequence of the target DNA recognized by I-Crel.
- C I 221 represents a palindromic DNA sequence recognized and cleaved by the I-Crel meganuclease. Nucleotides are numbered outward (-/+) from the center of the target. Nucleotides at positions -2 to +2 do not directly contact the protein but rather interfere with the cleavage activity of the protein.
- the table represents a subset of the tested targets with nucleotide substitution at positions -2 to +2. The binding and cleavage activity of I-Crel on the target is indicated (++, strong, +, good, +/-, weak; -, no activity). Activities were determined in vitro.
- Figure 4 Strategies to enhance DSB-induced mutagenesis. Loss of genetic information can be obtained by one or any variations of the following described strategies as illustrating examples (slight vertical lanes indicate specific DNA recognition domains): - simultaneous DSBs generated by two different specific rare-cutting endonucleases (A); - chimeric rare-cutting endonucleases with two endonucleases catalytic domains (bi-functional) (B); - chimeric rare-cutting endonucleases with one DNA-binding domain and two endonucleases catalytic domains (bi-functional) (C); - fusion protein between a rare-cutting endonuclease, a endonuclease catalytic domain and a frequent-cutting endonuclease (multifunctional) (D); - chimeric rare-cutting endonucleases with one exonuclease catalytic domains capable to process DNA ends (bi-functional) (E
- Figure 5 Effect of Trex2 expression on SC_GS-induced mutagenic DSB repair.
- A Percentage of GFP+ cells induced on NHEJ model after transfection of SC_GS (SEQ ID NO: 153) with empty vector (SEQ ID NO: 175) or with increasing amount of Trex2 expression vector (SEQ ID NO: 154).
- B Percentage of mutagenesis (insertions and deletions) detected in the vicinity of the GS_CH01 target present on the NHEJ model induced by either SC_GS (SEQ ID NO: 153) with empty vector (SEQ ID NO: 175) or with two different doses of Trex2 encoding vector (SEQ ID NO: 154).
- Figure 6 Effect of Trex2 expression on the nature of deletions induced by different engineered meganucleases.
- SC RAGl SEQ ID NO: 58 encoded by plasmid pCLS2222, SEQ ID NO: 156
- SC XPC4 SEQ ID NO: 190 encoded by pCLS2510, SEQ ID NO: 157
- SC_CAPNS 1 SEQ ID NO: 192 encoded by pCLS6163, SEQ ID NO: 1548 only (grey histogram) or with Trex2 (SEQ ID NO: 194 encoded by pCLS7673, SEQ ID NO: 154) (black histogram).
- Figure 7 plasmid for SC GS and SC GS and Trex2 fusion expression
- CHO-K1 cells were co-transfected with the plasmid measuring SSA activity containing the GS CHOl . l target and an increasing amounts of SC GS (pCLS2690, SEQ ID NO: 153), SC_GS-5-Trex2 (pCLS8082, SEQ ID NO: 186), SC_GS-10-Trex2 (pCLS8052, SEQ ID NO: 187), Trex2-5-SC_GS (pCLS8053, SEQ ID NO: 188) or Trex2-10-SC_GS (pCLS8054, SEQ ID NO: 153).
- Beta-galactosidase activity was detected 72h after transfection using ONPG and 420 nm optical density detection. The entire process was performed on an automated Velocity 1 1 BioCel platform.
- A Percentage of GFP+ cells induced on NHEJ model 3 or 4 days after transfection with increasing dose of either SC_GS (pCLS2690, SEQ ID NO: 153), SC_GS-5-Trex2 (pCLS8082, SEQ ID NO: 186), SC_GS-10-Trex2 (pCLS8052, SEQ ID NO: 187), Trex2-5- SC GS (pCLS8053, SEQ ID NO: 188) or Trex2-10-SC_GS (pCLS8054, SEQ ID NO: 189).
- B Deep-sequencing analysis of deletion events induced by 1 or 6 ⁇ g of SC_GS (pCLS2690, SEQ ID NO: 153) or Trex2-10-SC_GS (pCLS8054, SEQ ID NO: 189).
- C Percentage of deletion events corresponding to a deletion of 2 (del2), 3 (del3) or 4 (del4) nucleotides at the end of double strand break generated by 1 or 6 ⁇ g of SC GS (pCLS2690, SEQ ID NO: 153) or Trex2-10-SC_GS (pCLS8054, SEQ ID NO: 189), other correspond to any other deletions events detected.
- Figure 10 Effect of Trex-SC_CAPNS1 (SEQ ID NO: 197) fusion on targeted mutagenesis in 293H cell line
- Panel A Percentage of Targeted Mutagenesis [TM] obtained in 293H cell line transfected with SC_CAPNS 1 (SEQ ID NO: 192) or Trex-SC CAPNS l (SEQ ID NO: 197).
- Panel B Nature of Targeted Mutagenesis obtained in 293H cell line transfected with
- Panel A Percentage of Targeted Mutagenesis obtained in Detroit551 cell line transfected with SC_CAPNS 1 (SEQ ID NO: 192) or Trex-SC_CAPNS 1 (SEQ ID NO: 197).
- Panel B Nature of Targeted Mutagenesis obtained in Detroit551 cell line transfected with SC_CAPNS1 (SEQ ID NO: 192) or Trex-SC_CAPNS1 (SEQ ID NO: 197).
- Del2, Del3 and Del4 correspond to 2, 3 and 4 base pairs deletion events at the cleavage site of CAPNS 1.
- "Other” represents all other TM events.
- Figure 12 Effect of Tdt expression on targeted mutagenesis in cell line monitoring NHEJ.
- Panel A Percentage of GFP+ cells induced on NHEJ model after co-transfection of 1 ⁇ g or 3 g of SC_GS expressing plasmid (SEQ ID NO: 153) and with either an increasing amount of Tdt expression vector (SEQ ID NO: 153) or with 2 ⁇ g of Tdt expressing plasmid (SEQ ID NO: 153), respectively.
- Panel B Percentage of targeted mutagenesis detected by deep sequencing in the vicinity of the GS_CH01 DNA target present on the NHEJ model, induced by either SC_GS with empty vector or with 2 ⁇ g of Tdt encoding vector.
- Panel C Percentage of insertion events within targeted mutagenesis events after co- transfection of the NHEJ model by 3 ⁇ g of SC GS expressing vector with 2 ⁇ g of an empty vector or with 2 ⁇ g of Tdt encoding plasmid.
- Panel D Percentage of insertion events in function of their size in presence (TDT) or absence (empty) of Tdt.
- Figure 13 Effect of Tdt expression on targeted mutagenesis induced by SC RAGl (SEQ ID NO: 58) at endogenous RAGl locus
- Panel A Percentage of targeted mutagenesis detected by deep sequencing in the vicinity of the SC RAGl target induced by co-transfection of 3 ⁇ g of SC RAGl encoding vector (SEQ ID NO: 156) with different amount of Tdt encoding vector (SEQ ID NO: 202) in 5 ⁇ g of total DNA (left part) or in 10 ⁇ g of total DNA (right part).
- Panel B Percentage of insertion events within targeted mutagenesis events after co- transfection of 3 ⁇ g of SC RAGl encoding vector (SEQ ID NO: 156) with different amount of Tdt encoding vector (SEQ ID NO: 202) in 5 ⁇ g of total DNA (left part) or in 10 ⁇ g of total DNA (right part).
- Panel C Percentage of insertion events in function of their size at endogenous RAGl locus after co-transfection of 3 ⁇ g of SC_RAG1 encoding vector (SEQ ID NO: 156) with different amounts of Tdt encoding vector (SEQ ID NO: 202) in 5 ⁇ g of total DNA (left part) or in 10 ⁇ g of total DNA (right part).
- Figure 14 Effect of Tdt expression on targeted mutagenesis induced by SC CAPNSl (SEQ ID NO: 192) at endogenous CAPNSl locus
- Panel A Percentage of targeted mutagenesis detected by deep sequencing in the vicinity of the SC CAPNS l target induced by co-transfection of 1 ⁇ g of SC CAPNS l expressing vector (SEQ ID NO: 158) with 2 ⁇ g of Tdt encoding plasmid (SEQ ID NO: 202).
- Panel B Percentage of insertion events within targeted mutagenesis events after co- transfection of 3 ⁇ g of SC_CAPNS 1 expressing vector (SEQ ID NO: 158) with 2 ⁇ g of Tdt encoding plasmid (SEQ ID NO: 202).
- Panel C Percentage of insertion events in function of their size at CAPNS 1 locus after co-transfection of 3 ⁇ g of SC_CAPNS1 expressing vector (SEQ ID NO: 158) with 2 ⁇ g of Tdt encoding plasmid (SEQ ID NO: 202).
- a first aspect of the present invention is a method for increasing double- strand break induced mutagenesis at a genomic locus of interest in a cell comprising the steps of:
- said rare-cutting endonuclease is able to generate one
- said another enzymatic activity is a nuclease activity.
- said another enzymatic activity is an exonuclease activity.
- said rare-cutting endonuclease is a chimeric rare-cutting endonuclease which generates one DNA double- strand break leading to DNA ends, thus processed by an exonuclease activity, allowing the loss of genetic information and preventing any scarless re-ligation of said genomic locus of interest.
- said rare-cutting endonuclease is a chimeric rare- cutting endonuclease which generates one DNA double-strand break leading to DNA ends, thus processed by an enzymatic activity (as illustrated in Figure 4E) other than a nuclease activity such as polymerase activity (TdT%), a dephosphatase activity, as non-limiting examples.
- said rare-cutting endonuclease of the present invention is a chimeric rare-cutting endonuclease comprising a catalytic domain given in Table 2 (SEQ ID NO: 38-57) and Table 3 (SEQ ID NO: 96-152), a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease of the present invention comprises a catalytic domain selected from the group consisting of Trex (SEQ ID NO: 145-149), and Tdt (SEQ ID NO: 201), functional mutants, variants or derivatives thereof.
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 194, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 194, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is a fusion protein comprising a single chain meganuclease and a protein of SEQ ID NO: 194, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 171 -174 and SEQ ID NO: 197.
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 201 , a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 201 , a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is a fusion protein comprising a single chain meganuclease and a protein of SEQ ID NO: 201 , a functional mutant, a variant or a derivative thereof.
- the present invention also relates to engineered enzymes and more particularly to chimeric rare-cutting endonucleases able to target a DNA sequence within a genomic locus of interest in order to generate at least one DNA double-strand break and a loss of genetic information by another enzymatic activity around said DNA sequence, thus preventing any scarless re-ligation of said genomic locus of interest by NHEJ.
- said chimeric rare-cutting endonuclease of the present invention is a fusion protein between a rare-cutting endonuclease which generates one DNA double-strand break at a targeted sequence within the genomic locus of interest, leading to DNA ends and an nuclease domain that is able to process said DNA ends in order to generate a loss of information at the genomic locus of interest.
- Said nuclease domain can be a exonuclease domain.
- said chimeric rare-cutting endonuclease of the present invention is a fusion protein between a rare-cutting endonuclease which generates one DNA double-strand break at a targeted sequence within the genomic locus of interest, leading to DNA ends and a polymerase activity, such as a template independent polymerase (TdT, 3) that is able to process said DNA ends and generate a loss of genetic information at the genomic locus of interest by adding at least one DNA fragment and preventing any scarless re-ligation.
- a polymerase activity such as a template independent polymerase (TdT, 3) that is able to process said DNA ends and generate a loss of genetic information at the genomic locus of interest by adding at least one DNA fragment and preventing any scarless re-ligation.
- said rare-cutting endonuclease of the present invention is a chimeric rare-cutting endonuclease comprising a catalytic domain given in Table 2 and Table 3, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease of the present invention comprises a catalytic domain selected from the group consisting of Trex (SEQ ID NO: 145-149), and Tdt (SEQ ID NO: 201 ), functional mutants, variants or derivatives thereof.
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 194, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 194, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is a fusion protein comprising a single chain meganuclease and a protein of SEQ ID NO: 194, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 171 -174 and SEQ ID NO: 197.
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 201 , a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 201 , a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is a fusion protein comprising a single chain meganuclease and a protein of SEQ ID NO: 201 , a functional mutant, a variant or a derivative thereof.
- the present invention concerns a method for the generation of at least two-nearby DNA double-strand breaks at a genomic locus of interest to prevent any scarless re-ligation of said genomic locus of interest by NHEJ.
- said method comprises the generation of two nearby DNA double-strand breaks into said genomic locus of interest by the introduction of at least one double-strand break creating agent able to generate at least two nearby double-strand breaks such that said at least two nearby DNA double-strand breaks allow the removal of an intervening sequence, as a non limiting example, to prevent any scarless re-ligation of said genomic locus of interest (as illustrated in Figure 4A to 4C).
- the present invention concerns a method comprising the steps of:
- said rare-cutting endonuclease of the method is engineered to provide one chimeric rare-cutting endonuclease that is able to generate two nearby DNA double-strand breaks in the genomic locus of interest (as illustrated in Figure 4B and 4C).
- said rare- cutting endonuclease of the method is engineered to provide one chimeric rare-cutting endonuclease that is able to generate more than two nearby DNA double-strand breaks in the genomic locus of interest; in this preferred embodiment, said one chimeric rare-cutting endonuclease is able to generate three nearby DNA double-strand breaks in the genomic locus of interest.
- said rare-cutting endonuclease of the present invention is a chimeric rare-cutting endonuclease comprising a catalytic domain given in Table 2 and Table 3, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease of the present invention comprises a catalytic domain selected from the group consisting of Colicin-E7 (SEQ ID NO: 97), I-Tevl (SEQ ID NO: 106 or SEQ ID NO: 60; SEQ ID NO: 107-108), NucA (SEQ ID NO: 41 and 1 12), NucM (SEQ ID NO: 43 and 1 13), SNase (SEQ ID NO: 45-47 and 1 16-1 18), BspD6I (SEQ ID NO: 124-125) a functional mutant, variant or derivative thereof.
- a catalytic domain selected from the group consisting of Colicin-E7 (SEQ ID NO: 97), I-Tevl (SEQ ID NO: 106 or SEQ ID NO: 60; SEQ ID NO: 107-108), NucA (SEQ ID NO: 41 and 1 12), NucM (SEQ ID NO: 43 and 1 13), SNase (SEQ ID NO: 45-47 and 1
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 84, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 84, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 54, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is a fusion protein comprising a meganuclease and a protein of SEQ ID NO: 54, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 85-87 and SEQ ID NO: 91 -93.
- said chimeric rare-cutting endonuclease comprises a catalytic domain selected from the group consisting of SEQ ID NO: 56 and 57, a functional mutant, a variant or a derivative thereof.
- said chimeric rare- cutting endonuclease comprises a catalytic domain of SEQ ID NO: 56, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 57, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 56, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is a fusion protein comprising a meganuclease and a protein of SEQ ID NO: 56, a functional mutant, a variant or a derivative thereof.
- said chimeric rare- cutting endonuclease is fused to a protein of SEQ ID NO: 57, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is a fusion protein comprising a meganuclease and a protein of SEQ ID NO: 57, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 61 - 66 and SEQ ID NO: 70-75.
- the present invention implies two engineered rare-cutting endonucleases and comprises the steps of:
- said two engineered rare-cutting endonucleases which respectively target a DNA sequence at a genomic locus of interest are not chimeric rare- cutting endonucleases (as illustrated in Figure 4A).
- said two engineered rare-cutting endonucleases which respectively target a DNA sequence at a genomic locus of interest are chimeric rare-cutting endonucleases.
- only one of said two engineered rare-cutting endonucleases, which respectively target a DNA sequence at a genomic locus of interest is a chimeric rare-cutting endonuclease.
- said at least two nearby DNA double-strand breaks induced into said genomic locus of interest are distant at least 12 bp. In another preferred embodiment, said at least two nearby DNA double-strand break-induced into said genomic locus of interest are distant at least 20 bp, 50bp, 100, 200, 500 or 1000 bp. In another preferred embodiment, the distance between said at least two nearby DNA double-strand breaks induced into said genomic locus of interest is between 12 bp and 1000 bp, more preferably between 12 bp and 500 bp, more preferably between 12 bp and 200 bp.
- the present invention relates to engineered rare-cutting endonucleases and more particularly to chimeric rare-cutting endonucleases, able to target a DNA sequence within a genomic locus of interest in order to generate at said locus of interest at least two-nearby DNA double-strand breaks leading to at least the removal of a DNA fragment and thus preventing any scarless re-ligation of said genomic locus of interest by NHEJ (as illustrated in Figure 4A, 4C and 4E).
- said chimeric rare- cutting endonucleases comprise at least two catalytic domains.
- said chimeric rare-cutting endonucleases comprise two nuclease domains.
- the present invention relates to a chimeric rare-cutting endonuclease to generate at least two nearby DNA double-strand breaks into a genomic locus of interest comprising: i) a rare-cutting endonuclease;
- said rare-cutting endonuclease part of said chimeric rare- cutting endonuclease is a meganuclease; in another preferred embodiment, said rare-cutting endonuclease part of said chimeric rare-cutting endonuclease is a I-Crel derived meganuclease. In another preferred embodiment, said rare-cutting endonuclease part of said chimeric rare-cutting endonuclease is a single chain meganuclease derived from I-Crel meganuclease.
- said chimeric rare-cutting endonuclease is a fusion protein between a meganuclease and at least one nuclease catalytic domain.
- said nuclease catalytic domain has an endonuclease activity; alternatively, said nuclease catalytic domain has an exonuclease activity.
- said rare-cutting endonuclease of the present invention is a chimeric rare-cutting endonuclease comprising a catalytic domain given in Table 2 and Table 3, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease of the present invention comprises a catalytic domain selected from the group consisting of Trex (SEQ ID NO: 145-149), Colicin E7 (SEQ ID NO: 97), I-Tevl (SEQ ID NO: 106 or SEQ ID NO: 60; SEQ ID NO: 107- 108), NucA (SEQ ID NO: 41 and 1 12), NucM (SEQ ID NO: 43 and 1 13), SNase (SEQ ID NO: 45-47 and 1 16- 1 18), BspD6I (SEQ ID NO: 124-125), a functional mutant, a variant or a derivative thereof.
- Trex SEQ ID NO: 145-149
- Colicin E7 SEQ ID NO: 97
- I-Tevl SEQ ID NO: 106 or SEQ ID NO: 60; SEQ ID NO: 107- 108
- NucA SEQ ID NO: 41 and 1 12
- NucM SEQ ID NO:
- said chimeric rare-cutting endonuclease is a fusion protein comprising a meganuclease and a protein of SEQ ID NO: 145- 149, SEQ ID NO: 97, SEQ ID NO: 106 or SEQ ID NO: 60, SEQ ID NO: 107-108, SEQ ID NO: 41 and 1 12, SEQ ID NO: 43 and 1 13, SEQ ID NO: 45-47 and 1 16-1 18, SEQ ID NO: 124-125, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 194, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 194, a functional mutant, a variant or a derivative thereof.
- said rare-cutting endonuclease is a fusion protein comprising a single-chain meganuclease and a protein of SEQ ID NO: 194.
- said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 171 - 174 and SEQ ID NO: 197.
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 84, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 84, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 54, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 85- 87 and SEQ ID NO: 91 -93.
- said chimeric rare-cutting endonuclease comprises a catalytic domain selected from the group consisting of SEQ ID NO: 56 and 57, functional mutants, variants or derivatives thereof.
- said chimeric rare- cutting endonuclease comprises a catalytic domain of SEQ ID NO: 56, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 57, a functional mutant, a variant or a derivative thereof.
- said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 56, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease is fused to a protein of SEQ ID NO: 57, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 61-66 and SEQ ID NO: 70-75.
- said chimeric rare-cutting endonuclease further comprises a second peptidic linker and a supplementary catalytic domain.
- the present invention relates to a chimeric rare-cutting endonuclease able to generate at least two nearby DNA double-strand breaks into a genomic locus of interest comprising:
- said supplementary catalytic domain is a nuclease domain; in this case, said chimeric rare-cutting endonuclease is a fusion protein between a rare-cutting endonuclease and two nuclease catalytic domains. In a more preferred embodiment, said chimeric rare-cutting endonuclease is a fusion protein between a meganuclease and two nuclease catalytic domains. In another more preferred embodiment, said chimeric rare-cutting endonuclease is a fusion protein between a meganuclease, one nuclease catalytic domain and one other catalytic domain.
- a chimeric rare-cutting endonuclease able to generate two-nearby double-strand breaks and composed of the DNA- binding domain of a rare-cutting endonuclease and two other nuclease catalytic domains.
- the present invention describes a method to identify at a genomic locus of interest a DNA target sequence cleavable at least twice by a fusion protein leading at least to a loss of genetic information and preventing any scarless re-ligation of said genomic locus of interest by NHEJ. More particularly, in this aspect is a method for increasing double- strand break induced mutagenesis at a genomic locus of interest in a cell comprising the steps of:
- the present invention relates to fusion proteins able to generate at least two nearby DNA double-strand breaks into a genomic locus of interest comprising one DNA target sequence cleavable by one rare-cutting endonuclease nearby one DNA target sequence cleavable by one frequent-cutting endonuclease.
- the present invention relates to a fusion protein comprising:
- said rare-cutting endonuclease part of said fusion protein is a meganuclease; in another preferred embodiment, said rare-cutting endonuclease part of said fusion protein is a I-Crel derived meganuclease. In another preferred embodiment, said rare-cutting endonuclease part of said fusion protein is a single chain meganuclease derived from I-Crel meganuclease.
- said further fusion protein comprises a second peptidic linker and a supplementary catalytic domain.
- the present invention relates to a fusion protein able to generate at least two nearby DNA double-strand breaks into a genomic locus of interest comprising one DNA target sequence cleavable by one rare- cutting endonuclease nearby one DNA target sequence cleavable by one frequent-cutting endonuclease, said fusion protein comprising:
- said supplementary catalytic domain is a nuclease domain
- said supplementary catalytic domain is a non-nuclease catalytic domain.
- the present invention also relates to polynucleotides encoding the endonuclease proteins of the invention, specific vectors (polynucleotidic or not) encoding and/or vectorizing them, compositions and/or kits comprising them, all of them being used or part of a whole to implement methods of the present invention for increasing double-strand break-induced mutagenesis at a genomic locus of interest in a cell.
- kits may contain instructions for use in increasing double-strand break-induced mutagenesis in a cell, packaging materials, one or more containers for the ingredients, and other components used for increasing double- strand break-induced mutagenesis
- - Amino acid residues in a polypeptide sequence are designated herein according to the one-letter code, in which, for example, Q means Gin or Glutamine residue, R means Arg or Arginine residue and D means Asp or Aspartic acid residue.
- - Amino acid substitution means the replacement of one amino acid residue with another, for instance the replacement of an Arginine residue with a Glutamine residue in a peptide sequence is an amino acid substitution.
- - Altered/enhanced/increased/improved cleavage activity refers to an increase in the detected level of meganuclease cleavage activity, see below, against a target DNA sequence by a second meganuclease in comparison to the activity of a first meganuclease against the target DNA sequence.
- the second meganuclease is a variant of the first and comprise one or more substituted amino acid residues in comparison to the first meganuclease.
- nucleosides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine.
- r represents g or a (purine nucleotides)
- k represents g or t
- s represents g or c
- w represents a or t
- m represents a or c
- y represents t or c (pyrimidine nucleotides)
- d represents g, a or t
- v represents g, a or c
- b represents g, t or c
- h represents a, t or c
- n represents g, a, t or c.
- meganuclease is intended an endonuclease having a double-stranded DNA target sequence of 12 to 45 bp.
- Said meganuclease is either a dimeric enzyme, wherein each domain is on a monomer or a monomeric enzyme comprising the two domains on a single polypeptide.
- “meganuclease domain” is intended the region which interacts with one half of the DNA target of a meganuclease and is able to associate with the other domain of the same meganuclease which interacts with the other half of the DNA target to form a functional meganuclease able to cleave said DNA target.
- meganuclease variant or “variant” it is intended a meganuclease obtained by replacement of at least one residue in the amino acid sequence of the parent meganuclease with a different amino acid.
- Variants include those with substitutions of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues.
- Such variants may have 75, 80, 85, 90, 95, 97.5, 98, 99, 99.5% or more homology or identity (or any intermediate value within this range) to a base or parental meganuclease sequence.
- peptide linker by "peptide linker", “peptidic linker” or “peptide spacer” it is intended to mean a peptide sequence which allows the connection of different monomers in a fusion protein and the adoption of the correct conformation for said fusion protein activity and which does not alter the specificity of either of the monomers for their targets.
- Peptide linkers can be of various sizes, from 3 amino acids to 50 amino acids as a non limiting indicative range. Non- limiting examples of such peptidic linkers are given in Table 1.
- one cell type related to the chosen cell type or organism is intended a cell type or an organism sharing characteristics with said chosen cell type or said chosen organism; this cell type or organism related to the chosen cell type or organism, can be derived from said chosen cell type or organism or not.
- subdomain it is intended the region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half- site.
- targeting DNA construct/minimal repair matrix/repair matrix it is intended to mean a DNA construct comprising a first and second portions which are homologous to regions 5' and 3 ' of the DNA target in situ.
- the DNA construct also comprises a third portion positioned between the first and second portion which comprise some homology with the corresponding DNA sequence in situ or alternatively comprise no homology with the regions 5' and 3' of the DNA target in situ.
- a homologous recombination event is stimulated between the genome containing the targeted gene comprised in the locus of interest and the repair matrix, wherein the genomic sequence containing the DNA target is replaced by the third portion of the repair matrix and a variable part of the first and second portions of the repair matrix.
- - by "functional variant” is intended a variant which is able to cleave a DNA target sequence, preferably said target is a new target which is not cleaved by the parent meganuclease.
- such variants have amino acid variation at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target.
- selection or selecting it is intended to mean the isolation of one or more meganuclease variants based upon an observed specified phenotype, for instance altered cleavage activity.
- This selection can be of the variant in a peptide form upon which the observation is made or alternatively the selection can be of a nucleotide coding for selected meganuclease variant.
- - by "screening” it is intended to mean the sequential or simultaneous selection of one or more meganuclease variant (s) which exhibits a specified phenotype such as altered cleavage activity.
- - by "derived from” it is intended to mean a meganuclease variant which is created from a parent meganuclease and hence the peptide sequence of the meganuclease variant is related to (primary sequence level) but derived from (mutations) the sequence peptide sequence of the parent meganuclease.
- I-Od is intended the wild-type I-Od having the sequence of pdb accession code l g9y, corresponding to the sequence SEQ ID NO: 1 in the sequence listing.
- I-Od variant with novel specificity is intended a variant having a pattern of cleaved targets different from that of the parent meganuclease.
- the terms “novel specificity”, “modified specificity”, “novel cleavage specificity”, “novel substrate specificity” which are equivalent and used indifferently, refer to the specificity of the variant towards the nucleotides of the DNA target sequence.
- all the I-Cre ⁇ variants described comprise an additional Alanine after the first Methionine of the wild type I-Cre ⁇ sequence as shown in SEQ ID NO: 195.
- These variants also comprise two additional Alanine residues and an Aspartic Acid residue after the final Proline of the wild type I-Cre ⁇ sequence.
- I-Od site is intended a 22 to 24 bp double-stranded DNA sequence which is cleaved by I-Od.
- I-Od sites include the wild-type non-palindromic I-Od homing site and the derived palindromic sequences such as the sequence 5'- ⁇ 2 ⁇ ⁇ & ⁇ - ⁇ -%&- ⁇ - ⁇ , ⁇ - ⁇ - ia + ic +2 g + 3a + 4C + 5g + 6t + 7t +8 t + 9t + iog + i ia + i 2 (SEQ ID NO: 2), also called C 1221 .
- LAGLIDADG homing endonuclease core domain which is the characteristic ⁇ fold of the homing endonucleases of the LAGLIDADG family, corresponding to a sequence of about one hundred amino acid residues.
- Said domain comprises four beta-strands ( ⁇ ⁇ ⁇ 2 ⁇ 3 ⁇ 4 ) folded in an anti-parallel beta- sheet which interacts with one half of the DNA target.
- This domain is able to associate with another LAGLIDADG homing endonuclease core domain which interacts with the other half of the DNA target to form a functional endonuclease able to cleave said DNA target.
- the LAGLIDADG homing endonuclease core domain corresponds to the residues 6 to 94.
- subdomain is intended the region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half- site.
- chimeric DNA target or “hybrid DNA target” it is intended the fusion of a different half of two parent meganuclease target sequences.
- at least one half of said target may comprise the combination of nucleotides which are bound by at least two separate subdomains (combined DNA target).
- a DNA target sequence comprising a rare-cutting endonuclease target sequence (20-24 bp) and a frequent-cutting endonuclease target sequence (4-8 bp), recognized by a chimeric rare-cutting endonuclease according to the present invention.
- beta-hairpin is intended two consecutive beta-strands of the antiparallel beta- sheet of a LAGLIDADG homing endonuclease core domain ( ⁇ ⁇ 2 0 ⁇ ⁇ 3 ⁇ 4 ) which are connected by a loop or a turn,
- single-chain meganuclease is intended a meganuclease comprising two LAGLIDADG homing endonuclease domains or core domains linked by a peptidic spacer as described in WO2009095793.
- the single-chain meganuclease is able to cleave a chimeric DNA target sequence comprising one different half of each parent meganuclease target sequence.
- cleavage site is intended a 20 to 24 bp double-stranded palindromic, partially palindromic (pseudo-palindromic) or non-palindromic polynucleotide sequence that is recognized and cleaved by a LAGLIDADG homing endonuclease such as I-Oel, or a variant, or a single-chain chimeric meganuclease derived from l-Crel.
- Said DNA target sequence is qualified of "cleavable" by an endonuclease, when recognized within a genomic sequence and known to correspond to the DNA target sequence of a given endonuclease or a variant of such endonuclease.
- These terms refer to a distinct DNA location, preferably a genomic location, at which a double stranded break (cleavage) is to be induced by the meganuclease.
- the DNA target is defined by the 5' to 3' sequence of one strand of the double-stranded polynucleotide, as indicate above for CI 221. Cleavage of the DNA target occurs at the nucleotides at positions +2 and -2, respectively for the sense and the antisense strand. Unless otherwise indicated, the position at which cleavage of the DNA target by an ⁇ -Cre I meganuclease variant occurs, corresponds to the cleavage site on the sense strand of the DNA target.
- DNA target half-site by "DNA target half-site", "half cleavage site” or half-site” is intended the portion of the DNA target which is bound by each LAGLIDADG homing endonuclease core domain.
- chimeric DNA target or “hybrid DNA target” is intended the fusion of different halves of two parent meganuclease target sequences.
- at least one half of said target may comprise the combination of nucleotides which are bound by at least two separate subdomains (combined DNA target).
- exonuclease refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within of a DNA or RNA molecule, preferably a DNA molecule.
- Endonucleases do not cleave the DNA or RNA molecule irrespective of its sequence, but recognize and cleave the DNA or RNA molecule at specific polynucleotide sequences, further referred to as "target sequences" or "target sites”.
- Endonucleases can be classified as rare-cutting endonucleases when having typically a polynucleotide recognition site of about 12-45 base pairs (bp) in length, more preferably of 14-45 bp.
- Rare-cutting endonucleases can for example be a homing endonuclease (Paques and Duchateau 2007), a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic domain of a restriction enzyme such as Fokl (Porteus and Carroll 2005) or a chemical endonuclease (Eisenschmidt, Lanio et al. 2005 ; Arimondo, Thomas et al. 2006; Simon, Cannata et al. 2008).
- a restriction enzyme such as Fokl (Porteus and Carroll 2005) or a chemical endonuclease (Eisenschmidt, Lanio et al. 2005 ; Arimondo, Thomas et al. 2006; Simon, Cannata et al. 2008).
- a chemical or peptidic cleaver is conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific target sequence, thereby targeting the cleavage activity to a specific sequence.
- Chemical endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences (Kalish and Glazer 2005). Such chemical endonucleases are comprised in the term "endonuclease" according to the present invention.
- Rare-cutting endonucleases can also be for example TALENs, a new class of chimeric nucleases using a Fokl catalytic domain and a DNA binding domain derived from Transcription Activator Like Effector (TALE), a family of proteins used in the infection process by plant pathogens of the Xanthomonas genus (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2010).
- TALE Transcription Activator Like Effector
- the functional layout of a Fokl-based TALE-nuclease (TALEN) is essentially that of a ZFN, with the Zinc- finger DNA binding domain being replaced by the TALE domain.
- DNA cleavage by a TALEN requires two DNA recognition regions flanking an unspecific central region.
- Rare- cutting endonucleases encompassed in the present invention can also be derived from TALEN
- Rare-cutting endonuclease can be a homing endonuclease, also known under the name of meganuclease. Such homing endonucleases are well-known to the art (Stoddard 2005). Homing endonucleases recognize a DNA target sequence and generate a single- or double- strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length.
- the homing endonuclease according to the invention may for example correspond to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease.
- An expression such as "double-strand break creating agent" can be used to qualify a rare-cutting endonuclease according to the present invention.
- meganucleases are essentially represented by homing endonucleases.
- Homing Endonucleases are a widespread family of natural meganucleases including hundreds of proteins families (Chevalier and Stoddard 2001 ). These proteins are encoded by mobile genetic elements which propagate by a process called "homing”: the endonuclease cleaves a cognate allele from which the mobile element is absent, thereby stimulating a homologous recombination event that duplicates the mobile DNA into the recipient locus. Given their exceptional cleavage properties in terms of efficacy and specificity, they could represent ideal scaffolds to derive novel, highly specific endonucleases.
- HEs belong to four major families.
- the LAGLIDADG family named after a conserved peptidic motif involved in the catalytic center, is the most widespread and the best characterized group. Seven structures are now available. Whereas most proteins from this family are monomeric and display two LAGLIDADG motifs, a few have only one motif, and thus dimerize to cleave palindromic or pseudo-palindromic target sequences. Although the LAGLIDADG peptide is the only conserved region among members of the family, these proteins share a very similar architecture.
- the catalytic core is flanked by two DNA-binding domains with a perfect two-fold symmetry for homodimers such as I-Oel (Chevalier, Monnat et al.
- DNA binding depends on two typical saddle-shaped ⁇ folds, sitting on the DNA major groove.
- Other domains can be found, for example in inteins such as V ⁇ -Pfu ⁇ (Ichiyanagi, Ishino et al. 2000) and PI- Scel (Moure, Gimble et al. 2002), whose protein splicing domain is also involved in DNA binding.
- residues 28 to 40 and 44 to 77 of I-Oel were shown to form two partially separable functional subdomains, able to bind distinct parts of a homing endonuclease target half-site (Smith, Grizot et al. 2006); International PCT Applications WO 2007/049095 and WO 2007/057781 (Cellectis)).
- the combination of the two former steps allows a larger combinatorial approach, involving four different subdomains.
- the different subdomains can be modified separately and combined to obtain an entirely redesigned meganuclease variant (heterodimer or single- chain molecule) with chosen specificity.
- couples of novel meganucleases are combined in new molecules ("half-meganucleases") cleaving palindromic targets derived from the target one wants to cleave. Then, the combination of such "half-meganucleases" can result in a heterodimeric species cleaving the target of interest.
- endonuclease examples include I-Sce I, l-Chu I, I-Cre I, I-Csm I, Pl-Sce I, PITH I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, Pi-Civ I, PI-Ctr I, PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI- Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, Pl-Pfu I, PI- Rm
- a homing endonuclease can be a LAGLIDADG endonuclease such as l-Scel, ⁇ -CreI, I-Ceul, ⁇ -MsoI, and ⁇ -DmoI.
- Said LAGLIDADG endonuclease can be I-Sce I, a member of the family that contains two LAGLIDADG motifs and functions as a monomer, its molecular mass being approximately twice the mass of other family members like I-Crel which contains only one LAGLIDADG motif and functions as homodimers.
- Endonucleases mentioned in the present application encompass both wild-type (naturally-occurring) and variant endonucleases.
- Endonucleases according to the invention can be a "variant" endonuclease, i.e. an endonuclease that does not naturally exist in nature and that is obtained by genetic engineering or by random mutagenesis, i.e. an engineered endonuclease.
- This variant endonuclease can for example be obtained by substitution of at least one residue in the amino acid sequence of a wild-type, naturally-occurring, endonuclease with a different amino acid. Said substitution(s) can for example be introduced by site- directed mutagenesis and/or by random mutagenesis.
- such variant endonucleases remain functional, i.e. they retain the capacity of recognizing and specifically cleaving a target sequence to initiate gene targeting process.
- the variant endonuclease according to the invention cleaves a target sequence that is different from the target sequence of the corresponding wild-type endonuclease.
- Methods for obtaining such variant endonucleases with novel specificities are well-known in the art.
- Endonucleases variants may be homodimers (meganuclease comprising two identical monomers) or heterodimers (meganuclease comprising two non-identical monomers).
- Endonucleases with novel specificities can be used in the method according to the present invention for gene targeting and thereby integrating a transgene of interest into a genome at a predetermined location.
- parent meganuclease it is intended to mean a wild type meganuclease or a variant of such a wild type meganuclease with identical properties or alternatively a meganuclease with some altered characteristic in comparison to a wild type version of the same meganuclease.
- the parent meganuclease can refer to the initial meganuclease from which the first series of variants are derived in step (a) or the meganuclease from which the second series of variants are derived in step (b), or the meganuclease from which the third series of variants are derived in step (k).
- delivery vector or “ delivery vectors” is intended any delivery vector which can be used in the present invention to put into cell contact or deliver inside cells or subcellular compartments agents/chemicals and molecules (proteins or nucleic acids) needed in the present invention. It includes, but is not limited to liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions or other appropriate transfer vectors. These delivery vectors allow delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as plasmids, peptides developed by Diatos. In these cases, delivery vectors are molecule carriers. By “delivery vector” or “delivery vectors” is also intended delivery methods to perform transfection
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- a “vector” in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semi-synthetic or synthetic nucleic acids.
- Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
- Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double- stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.
- orthomyxovirus e. g., influenza virus
- rhabdovirus e. g., rabies and vesicular stomatitis virus
- paramyxovirus e. g. measles and Sendai
- viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
- retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV- BLV group, lentivirus, spumavirus (Coffin, J. M, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott- Raven Publishers, Philadelphia, 1996).
- lentiviral vector HIV-Based lentiviral vectors that are very promising for gene delivery because of their relatively large packaging capacity, reduced immunogenicity and their ability to stably transduce with high efficiency a large range of different cell types.
- Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells.
- lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface.
- the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex.
- the product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration in the DNA of infected cells.
- integrated lentiviral vectors or LV
- integration lentiviral vectors or LV
- non integrative lentiviral vectors or NILV
- efficient gene delivery vectors that do not integrate the genome of a target cell through the action of the virus integrase.
- One type of preferred vector is an episome, i.e. , a nucleic acid capable of extra- chromosomal replication.
- Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
- Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors.
- a vector according to the present invention comprises, but is not limited to, a YAC (yeast artificial chromosome), a BAC (bacterial artificial), a baculovirus vector, a phage, a phagemid, a cosmid, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic DNA.
- expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
- Vectors can comprise selectable markers, for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine-guanine phosphoribosyl transferase for eukaryotic cell culture; TRP1 for 5. cerevisiae; tetracyclin, rifampicin or ampicillin resistance in E. coli.
- selectable markers for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine
- said vectors are expression vectors, wherein a sequence encoding a polypeptide of interest is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said polypeptide. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a replication origin, a promoter operatively linked to said encoding polynucleotide, a ribosome binding site, a RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer or silencer elements. Selection of the promoter will depend upon the cell in which the polypeptide is expressed. Suitable promoters include tissue specific and/or inducible promoters.
- inducible promoters examples include: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-D-D- thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature.
- tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), a-antitrypsin protease, human surfactant (SP) A and B proteins, ⁇ -casein and acidic whey protein genes.
- Inducible promoters may be induced by pathogens or stress, more preferably by stress like cold, heat, UV light, or high ionic concentrations (reviewed in Potenza C et al. 2004, In vitro Cell Dev Biol 40: 1-22).
- Inducible promoter may be induced by chemicals (reviewed in (Moore, Samalova et al. 2006); (Padidam 2003); (Wang, Zhou et al. 2003); (Zuo and Chua 2000).
- Delivery vectors and vectors can be associated or combined with any cellular permeabilization techniques such as sonoporation or electroporation or derivatives of these techniques.
- -By cell or cells is intended any prokaryotic or eukaryotic living cells, cell lines derived from these organisms for in vitro cultures, primary cells from animal or plant origin.
- primary cell or “primary cells” are intended cells taken directly from living tissue (i.e. biopsy material) and established for growth in vitro, that have undergone very few population doublings and are therefore more representative of the main functional components and characteristics of tissues from which they are derived from, in comparison to continuous tumorigenic or artificially immortalized cell lines. These cells thus represent a more valuable model to the in vivo state they refer to.
- eukaryotic cells refer to a fungal, plant or animal cell or a cell line derived from the organisms listed below and established for in vitro culture. More preferably, the fungus is of the genus Aspergillus, Penicillium, Acremonium, Trichoderma, Chrysoporium, Mortierella, Kluyveromyces or Pichia; More preferably, the fungus is of the species Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Penicillium chrysogenum, Penicillium citrinum, Acremonium Chrysogenum, Trichoderma reesei, Mortierella alpine, Chrysosporium lucknowense, Kluyveromyces lactis, Pichia pastoris or Pichia ciferrii.
- the plant is of the genus Arabidospis, Nicotiana, Solanum, lactuca, Brassica, Oryza, Asparagus, Pisum, Medicago, Zea, Hordeum, Secale, Triticum, Capsicum, Cucumis, Cucurbita, Citrullis, Citrus, Sorghum; More preferably, the plant is of the species Arabidospis thaliana, Nicotiana tabaccum, Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Solanum esculentum, Lactuca saliva, Brassica napus, Brassica oleracea, Brassica rapa, Oryza glaberrima, Oryza sativa, Asparagus officinalis, Pisum sativum, Medicago sativa, zea mays, Hordeum vulgare, Secale cereal, Triticum aestivum, Triticum durum, Capsicum sativus, Cu
- the animal cell is of the genus Homo, Rattus, Mus, Sus, Bos, Danio, Canis, Felis, Equus, Salmo, Oncorhynchus, Gallus, Meleagris, Drosophila, Caenorhabditis; more preferably, the animal cell is of the species Homo sapiens, Rattus norvegicus, Mus musculus, Sus scrofa, Bos taurus, Danio rerio, Canis lupus, Felis catus, Equus caballus, Salmo salar, Oncorhynchus mykiss, Gallus gallus, Meleagris gallopavo, Drosophila melanogaster, Caenorhabditis elegans.
- homologous is intended a sequence with enough identity to another one to lead to homologous recombination between sequences, more particularly having at least 95 % identity, preferably 97 % identity and more preferably 99
- identity refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences.
- Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting.
- mutants are intended the substitution, deletion, insertion of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide sequence.
- Said mutation can affect the coding sequence of a gene or its regulatory sequence. It may also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA.
- double-strand break-induced mutagenesis refers to a mutagenesis event consecutive to an NHEJ event following an endonuclease-induced DSB, leading to insertion/deletion at the cleavage site of an endonuclease.
- gene is meant the basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome, which codes for a specific protein or segment of protein.
- a gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, a 3' untranslated region.
- the gene may further comprise a terminator, enhancers and/or silencers.
- transgene refers to a sequence encoding a polypeptide.
- the polypeptide encoded by the transgene is either not expressed, or expressed but not biologically active, in the cell, tissue or individual in which the transgene is inserted.
- the transgene encodes a therapeutic polypeptide useful for the treatment of an individual.
- gene of interest refers to any nucleotide sequence encoding a known or putative gene product.
- locus is the specific physical location of a DNA sequence
- locus usually refers to the specific physical location of an endonuclease's target sequence on a chromosome. Such a locus, which comprises a target sequence that is recognized and cleaved by an endonuclease according to the invention, is referred to as “locus according to the invention”. Also, the expression “genomic locus of interest” is used to qualify a nucleic acid sequence in a genome that can be a putative target for a double-strand break according to the invention. By “endogenous genomic locus of interest” is intended a native nucleic acid sequence in a genome, i.e.
- genomic locus of interest of the present invention can be between two overlapping genes the considered endonuclease's target sequences are located in two different genes. It is understood that the considered genomic locus of interest of the present invention can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e. , in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria or chloroplasts as non-limiting examples.
- loss of genetic information is understood the elimination or addition of at least one given DNA fragment (at least one nucleotide) or sequence, bordering the recognition sites of the endonucleases of the present invention and leading to a change of the original sequence around said endonuclease-cutting sites, within the genomic locus of interest.
- This loss of genetic information can be, as a non-limiting example, the elimination of an intervening sequence between two endonuclease-cutting sites; it can also be, in another non-limiting example, the result of an exonuclease DNA-ends processing activity after a unique endonuclease DNA double-strand break.
- loss of genetic information within the genomic locus of interest is generated "around said DNA target sequence", i.e. around the endonuclease-cutting site (DSB), taken as reference.
- DLB endonuclease-cutting site
- It can also be, in other non- limiting examples, the result of DNA-ends processing activities by other enzymes, after a unique endonuclease DNA double-strand break, such as polymerase activity (TdT%), dephosphatase activity...
- two nearby DNA double strand breaks within the genomic locus of interest, is meant two endonucleases cutting sites distant at between 12 bp and 1000 bp.
- scarless re-ligation is intended the perfect re-ligation event, without loss of genetic information (no insertion/deletion events) of the DNA broken ends through NHEJ process after the creation of a double-strand break event.
- the present invention relates to a method to increase double-strand break mediated mutagenesis by avoiding any such "scarless re-ligation” process.
- fusion protein is intended the result of a well-known process in the art consisting in the joining of two or more genes which originally encode for separate proteins, the translation of said "fusion gene” resulting in a single polypeptide with functional properties derived from each of the original proteins.
- chimeric rare-cutting endonuclease any fusion protein comprising a rare-cutting endonuclease. Said rare-cutting endonuclease might be at the N- terminus part of said chimeric rare-cutting endonuclease; at the opposite, said rare-cutting endonuclease might be at the C- terminus part of said chimeric rare-cutting endonuclease.
- a “chimeric rare-cutting endonuclease” according to the present invention which comprises two catalytic domains can be described as "bi-functional” or as "bi-functional meganuclease".
- a "chimeric rare-cutting endonuclease" according to the present invention which comprises more than two catalytic domains can be described as “multi-functional” or as “multi-functional meganuclease”.
- chimeric rare-cutting endonucleases according to the present invention can be a fusion protein between a rare-cutting endonuclease and one catalytic domain; chimeric rare-cutting endonucleases according to the present invention can also be a fusion protein between a rare-cutting endonuclease and two catalytic domains.
- the rare-cutting endonuclease part of chimeric rare-cutting endonucleases according to the present invention can be a meganuclease comprising either two identical monomers, either two non identical monomers, or a single chain meganuclease.
- the rare- cutting endonuclease part of chimeric rare-cutting endonucleases according to the present invention can also be the DNA-binding domain of a rare-cutting endonuclease.
- chimeric rare-cutting endonucleases according to the present invention can be derived from a TALE-nuclease (TALEN), i. e., a fusion between a DNA-binding domain derived from a Transcription Activator Like Effector (TALE) and one or two catalytic domains.
- TALE Transcription Activator Like Effector
- frequent-cutting endonuclease an endonuclease typically having a polynucleotide recognition site of about 4-8 base pairs (bp) in length, more preferably of 4-6 bp.
- TALE-nuclease TALEN
- TALEN Transcription Activator Like Effector
- catalytic domain is intended the protein domain or module of an enzyme containing the active site of said enzyme; by active site is intended the part of said enzyme at which catalysis of the substrate occurs.
- Enzymes, but also their catalytic domains are classified and named according to the reaction they catalyze.
- the Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze (http://www.chem.qmul.ac.uk/iubmb/enzyme/).
- any catalytic domain can be fused to a rare-cutting endonuclease to generate a chimeric rare-cutting endonuclease.
- Non-limiting examples of such catalytic domains are given in table 2 and in table 3 with a GenBank or NCBI or UniProtKB/Swiss- Prot number as a reference.
- nuclease catalytic domain is intended the protein domain comprising the active site of an endonuclease or an exonuclease enzyme.
- Non-limiting examples of such catalytic domains are given in table 2 and in table 3 with a GenBank or NCBI or UniProtKB/Swiss- Prot number as a reference.
- Rl or Ri m Two engineered single-chain meganucleases called Rl or Ri m (SEQ ID NO: 58) and D21 or D21 m (SEQ ID NO: 59) are produced using the methods disclosed in International PCT Applications WO2003078619, WO2004/067736, WO2006/097784, WO2006/097853, WO2007/060495, WO 2007/049156, WO 2006/097854, WO2007/034262, WO 2007/049095, WO2007/057781 and WO2009095793 (Cellectis) and in (Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006).
- meganucleases derived from I-Crel, are designed to recognize two different DNA sequences, neither of which are recognized by wild-type I-Crel. (recognition sequences, respectively, tgttctcaggtacctcagccag SEQ ID NO: 3 and aaacctcaagtaccaaatgtaa SEQ ID NO: 4). Expression of these two meganucleases is driven by a CMV promoter and a polyA signal sequence. The two corresponding recognition sites are cloned in close proximity to generate the target plasmid. For this example, the recognition sites are separated by 10 bp ( Figure 1 ).
- Human HEK293 cells are transiently co-transfected with two plasmids carrying the expression cassette for Rl (SEQ ID NO: 58) and D21 (SEQ ID NO: 59), as well as the target plasmid.
- HEK293 cells are transiently co-transfected with the target plasmid and only one meganuclease-expressing plasmid.
- DNA is extracted 2 days post-transfection and targeted mutagenesis is assessed by a mutation detection assay as depicted in figure 2 (surveyor assay from Transgenomic, Inc. USA). High-fidelity PCR amplification of the DNA encompassing the two recognition sites is performed using appropriate specific primers.
- PCR amplification is performed on genomic DNA extracted from cells transfected with the target plasmid alone. After quantification and purification, equimolar amounts of PCR products are mixed in an annealing buffer and a fraction of this mixture is subjected to a melting/annealing step, resulting in the formation of distorted duplex DNA through random re-annealing of mutant and wild-type DNA.
- CEL- 1 enzyme surveyor assay from Transgenomic, Inc. USA
- the CEL- 1 cleaved samples are resolved on analytical gel, stained with ethidium bromide and the DNA bands are quantified using densitometry. The frequency of mutagenesis can then be calculated essentially as described in Miller et al (2007).
- PCR products from cells transfected with the target plasmid and (a) an empty plasmid; (b) one meganuclease expressing plasmid or; (c) two plasmids expressing respectively Rl (SEQ ID NO: 58) and D21 (SEQ ID NO: 59), are also analyzed by high-throughput sequencing ( Figure 2).
- PCR amplification is performed with appropriate primers to obtain a fragment flanked by specific adaptor sequences (adaptor A: 5 '- CCATCTCATCCCTGCGTGTCTCCGAC-NNNN-3', SEQ ID NO: 5 and adaptor B, 5'- CCTATCCCCTGTGTGCCTTGGC AGTCTC AG-3 ' , SEQ ID NO: 6) provided by the company (GATC Biotech AG, Germany) offering sequencing service on a 454 sequencing system (454 Life Sciences). Approximately 10,000 exploitable sequences are obtained per PCR pool and then analyzed for the presence of site-specific insertion or deletion events.
- the inventers are able to show that deletion of the intervening sequence or microsequence following the creation of two DNA DSBs greatly enhances the site-specific NHEJ-driven mutation rate. This deletion is observed only when both meganucleases are introduced into the cells.
- an engineered single-chain meganuclease derived from I-Crel (described in International PCT Applications WO2003078619, WO 2004/067736, WO 2006/097784, WO 2006/097853, WO 2007/060495, WO 2007/049156, WO 2006/097854, WO 2007/034262, WO 2007/049095, WO 2007/057781 , WO 2008/010093 and WO2009095793 (Cellectis) and in (Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al.
- each chimeric protein is assessed using our yeast assay previously described in International PCT Applications WO 2004/067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006).
- an I-Crel DNA target sequence is selected that can be bound but not cleaved by the wild-type meganuclease. This target contains 4-nucleotide substitutions at positions -2 to +2 ( Figure 3). Enzymes exhibiting cleavage activity are then tested following protocols described in example 1 , except that the target plasmid carried only one cleavage site for the I-Crel meganuclease.
- GenBank CAA45962. 1 NucA vrvtpsmysgsgydrghiapsadrtkttednaatflmtnmm
- Table 2 sequences of the catalytic domains fused to meganucleases.
- Vvn 1 14 >gi I6635279IgbIAAF19759.11AF063303_1 nuclease precursor 759. 1 Vvn [Vibrio vulnificus]
- Vvn_CLS >Vvn_CLS (variant of AAF19759.1)
- Nb.BsrDI 122 >gi
- KQIKDLLFWGKPDFIAYDP NDKILFAVEETGAVPTGNQALQRCERIYGSARKQIPF WYLLSEFGQHKDGGTRRDSIWPTIMGLKLTQLVKTPSIILHYSDIN PEDYNSGNGL KFLFKSLLQIIINYCTLKNPLKGMLELLSIQYENMLEFIKSQWKEQIDFLPGEEILN TKTKELARMYASLAIGQTVKIPEELFNWPRTDKVNFKSPQGLIKYDELCYQLEKAVG SKKAYCLSN AGAKPQKLESLKEWINSQKKLFDKAPKLTPPAEFNMKLDAFPVTSNN NYYVTTS ILYLFDYWKDLRIAIETAFPRLKG LPTDIDEKPALIYICNSV PGRL FGDPFTGQLSAFSTIFGKKNIDMPRIWAYYPHQIYSQALPKNNKSNKGITLKKELT DFLIFHGGVWKLNEGKAY
- I-Crel meganuclease (SEQ ID NO: 76) was chosen as the parent scaffold on which to fuse the catalytic domain of I-Tevl (SEQ ID NO: 60). Wild-type I-Tevl functions as a monomeric cleavase of the GIY-YIG family to generate a staggered double-strand break in its target DNA. Guided by biochemical and structural data, variable length constructs were designed from the N-terminal region of I-Tevl that encompass the entire catalytic domain and deletion-intolerant region of its linker (SEQ ID NOs: 61 to 66).
- each "tri-functional" meganuclease was assessed using yeast assay previously described in International PCT Applications WO 2004/067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006). All constructs were able to cleave the CI 221 target DNA with an activity comparable to that of wild-type I-Crel (Table 4).
- D20N point mutants were made to inactivate the I-Crel scaffold (SEQ ID NOs: 69 to 75). Tests in yeast assays showed no visible activity from the inactivated I-Crel (D20N) mutant protein alone (Table 4). However, cleavage activity could be observed for fusions having the I-Tevl catalytic domain (Table 4).
- Table 4 Activity in Yeast assay for I-Tevl/I-Crel fusions. The relative activity of wild-type and fusion proteins on the two parent protein targets (C I 221 for I-Crel and Tev for I-Tevl) is shown. Maximal activity (++++) is seen with each given protein on its native DNA target.
- I-CreI_N20 is an inactive variant of the wild-type I-Crel scaffold. In all other cases, activity is only detected on the C I 221 target since DNA recognition is driven by the I-Crel scaffold.
- the "N20" fusion variants illustrate cleavage activity due to the I-Tevl catalytic domain.
- Relative activity is scaled as: -, no activity detectable; +, ⁇ 25% activity; ++, 25% to ⁇ 50% activity; +++, 50% to ⁇ 75% activity; ++++, 75% to 100% activity.
- Protein-fusion scaffolds were designed based on a truncated form of I-Crel (SEQ ID NO: 76, I-CreI_X: SEQ ID NO: 77) and three different linker polypeptides (SEQ ID NOs: 78 to 80) fused to either the N- or C-terminus of the protein. Structure models were generated in all cases, with the goal of designing a "baseline" fusion linker that would traverse the I-Crel parent scaffold surface with little to no effect on its DNA binding or cleavage activities. For the two N-terminal fusion scaffolds, the polypeptide spanning residues 2 to 153 of I-Crel was used, with a K82A mutation to allow for linker placement.
- the C-terminal fusion scaffold contains residues 2 to 155 of wild-type I-Crel.
- the "free" end of the linker i.e. onto which a polypeptide can be linked
- the linker is designed to be proximal to the DNA, as determined from models built using the I-Crel/DNA complex structures as a starting point (PDB id: l g9z).
- Colicin E7 is a non-specific nuclease of the H H family able to process single- and double-stranded DNA. Guided by biochemical and structural data, the region of ColE7 that encompasses the entire catalytic domain (SEQ ID NO: 84) was selected. This ColE7 domain was fused to the N-terminus of either I-CreI_NFS l (SEQ ID NO: 81 ) or I-CreI_NFS2 (SEQ ID NO: 83) to create hColE7Cre_D0101 (SEQ ID: 85) or hColE7Cre_D0102 (SEQ ID NO:
- each "tri-functional" meganuclease was assessed using yeast assay as previously mentioned (see Example 3). All constructs were able to cleave the C I 221 target DNA with an activity comparable to that of wild-type I-Crel (Table 4).
- D20N point mutants were made to inactivate the I-Crel scaffold (SEQ ID NOs: 88-93). Tests in our yeast assays showed no visible activity from the inactivated I-Crel (D20N) mutant proteins alone (Table 5). However, cleavage activity could be observed for fusions having the ColE7 catalytic domain (Table 5).
- I-CreI_X represents a truncated version of I-Crel based on the crystal structure and was used as the foundation for the fusion scaffolds (I-CreI_NFS l , I-CreI_NFS2 and I-CreI_CFS l).
- "N20" constructs are inactive variants of the respective I-Crel-based scaffolds. Activity is detected in all cases wherein the I-Crel scaffold is active or when DNA catalysis is provided by the ColE7 domain.
- Relative activity is scaled as: -, no activity detectable; +, ⁇ 25% activity; ++, 25% to ⁇ 50% activity; +++, 50% to ⁇ 75% activity; ++++, 75% to 100% activity.
- Example 5 Effect of Trex2 or TREX2 (SEQ ID NO: 145) on meganuclease-induced mutagenesis
- Trex2 protein (SEQ ID NO: 145) is known to exhibit a 3' to 5' exonuclease activity (Mazur and Perrino, 2001 ).
- a 236 amino acid functional version of Trex2 (SEQ ID NO: 194) has been fused to single-chain meganucleases (SC-MN) for measuring improvements on meganuclease-induced targeted mutagenesis of such chimeric rare-cutting endonucleases.
- SC-MN single-chain meganucleases
- Levels of mutagenesis induced by SC-MN-Trex2 have been compared to levels of mutagenesis induced by co-transfecting vectors independently expressing SC-MN and Trex2 protein in a dedicated cellular model and at endogenous loci in 293H cells.
- Example 5A Co-transfection of Trex2 (SEQ ID NO: 145) with meganucleases
- a vector encoding meganuclease SC_GS (pCLS2690, SEQ ID NO: 153) was co- transfected into a cell line for monitoring mutagenic events in the presence or absence of a vector encoding Trex2 (pCLS7673, SEQID NO: 154).
- the SC GS meganuclease is a single chain protein (SEQ ID NO: 193) derived from the fusion of two I-Crel variants. It recognizes a 22bp DNA sequence (5'-TGCCCCAGGGTGAGAAAGTCCA-3' : GS CHO.
- SC_RAG1 pCLS2222, SEQID NO: 156 i.e. the expression vector encoding SC_RAG1 , SEQ ID NO: 58
- SC_XPC4 pCLS2510, SEQID NO: 157 i.e. the expression vector encoding SC XPC4, SEQ ID NO: 190
- SC CAPNS l pCLS6163, SEQID NO: 158 i.e.
- the expression vector encoding SC_CAPNS 1 , SEQ ID NO: 192) were co-transfected with or without a Trex2 expression vector (pCLS7673, SEQID NO: 154) to analyze the effect on meganuclease-induced mutagenesis at endogenous loci .
- the sequence used to measure SC_GS-induced mutagenesis is made of an ATG start codon followed by (i) 2 codons for alanine; (ii) an HA-tag sequence; (iii) the SC_GS recognition site; (iv) a stretch of glycine-serine di-residues; (v) an additional 2 codons for alanine as in (i) and finally; (vi) a GFP reporter gene lacking its ATG start codon.
- the GFP reporter gene is inactive due to a frame-shift introduced by the GS recognition site.
- the creation of a DNA double-strand break (DSB) by the SC_GS meganuclease followed by error-prone NHEJ events can lead to restoration of the GFP gene expression in frame with the ATG start codon.
- the final construct was introduced at the RAG1 locus in 293H cell line using the hsRAG l Integration Matrix CMV Neo from cGPS® Custom Human Full Kit DD (Cellectis Bioresearch) following the provider's instructions. Using this kit, a stable cell line containing a single copy of the transgene at the RAG1 locus was obtained.
- Genomic DNA was extracted from cell populations transfected with 1 ⁇ g of SC GS expressing plasmid and 0, 4 and 9 ⁇ g of Trex2 encoding plasmid. Locus specific PCR were performed using the following primers: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG (forward adaptor sequence)- 10N-(sequences needed for PCR product identification)- GCTCTCTGGCTAACTAGAGAACCC (transgenic locus specific forward sequence) -3' (SEQ ID NO: 160) and 5'-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-(reverse adaptor sequence)-TCGATCAGCACGGGCACGATGCC (transgenic locus specific reverse sequence) (SEQ ID NO: 161 ), and PCR products were sequenced by a 454 sequencing system (454 Life Sciences).
- Cells were co- transfected with 3 ⁇ g of plasmid expressing SC RAGl or SC_XPC4 or SC_CAPNS 1 (pCLS2222, SEQID NO: 156; pCLS2510, SEQID NO: 157 and pCLS6163, SEQID NO: 158 respectively) and with 0 or 2 ⁇ g of plasmid encoding Trex2 (pCSL7673 SEQID NO: 154) in 5 g of total DNA by complementation with a pUC vector (pCLS0002 SEQID NO: 191 ) using 25 ⁇ of lipofectamine (Invitrogen) according to the manufacturer's instructions.
- Locus specific PCR were performed using the following primers: 5'- CCATCTCATCCCTGCGTGTCTCCGACTCAG-(forward adaptor sequence)- 10N- (sequences needed for PCR product identification)-locus specific forward sequence for RAGl : GGC AAAG ATG AATC AAAGATTCTGTCC-3 ' (SEQ ID NO: 162), for XPC4: - AAG AGGC AAG AAA ATGTGCAGC-3 ' (SEQ ID NO: 163) and for CAPNS 1 - CG AGTC AGGGCGGG ATTAAG-3 ' (SEQ ID NO: 164) and the reverse primer 5'- CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-(reverse adaptor sequence)-(endogenous locus specific reverse sequence for RAG1 :- GATCTCACCCGGAACAGCTTAAATTTC-3 ' (SEQ ID NO: 165), for XPC4: -GCTGGGC ATATATAAGGTGCTC AA-3 ' (S
- the percentage of GFP+ cells monitoring mutagenesis events induced by SC_GS meganuclease in a dedicated cellular model, was analyzed 96h after a transfection with SC GS expressing plasmid (pCLS2690 SEQID NO: 153) alone or with an increasing dose of Trex2 encoding vector (pCLS7673 SEQID NO: 154).
- the percentage of GFP+ cells increased with the amount of Trex2 expressing plasmid transfected. In absence of Trex2, SC_GS expression led to 0.3% of GFP+ cells whereas 2, 4, 6 and 9 ⁇ g of Trex2 encoding plasmid led to 1.3, 2.8, 3.4 and 4.8% of GFP+ respectively (Figure 5A).
- Trex2 effect on mutagenesis induced by engineered meganucleases was measured at RAG1 , XPC4 and CAPNS 1 endogenous loci by co-transfecting plasmids expressing SC RAGl or SC XPC4 or SC_CAPNS 1 with or without Trex2 encoding plasmid.
- Transfections of 3 ⁇ g of meganuclease expressing vector with 2 ⁇ g of Trex2 (3/2 ratio) encoding plasmid were performed.
- the mutagenesis induced by the different meganucleases was quantified and analyzed three days post transfection.
- Trex2 stimulates mutagenesis at all loci studied with a stimulating factor varying from 1.4 up to 5 depending on the locus (Table 6).
- the nature of mutagenic events was also analyzed. It showed a modification of the pattern of the deletions induced by the meganucleases. As showed in Figure 6, particularly at RAG1 (panelA) and CAPNS 1 loci (panelC), the frequency of small deletions corresponding to degradation of 3' overhangs is significantly increased in the presence of Trex2.
- Example 5B Fusion of the human Trex2 protein to the N- or C-terminus of an engineered meganuclease. Expressing Trex2 within a cell can lead to exonuclease activity at loci not targeted by the meganuclease. Moreover, for obvious reasons, co-tranfection of two expressing vectors makes difficult to control the optimum expression of both proteins. In order to bypass those difficulties and to target Trex2 activity to the DSB induced by the meganuclease, the human Trex2 protein was fused to the N- or C-terminus of the SC_GS engineered meganuclease (SEQ ID NO: 153). Four SC_GS/Trex2 fusion proteins were made and tested for their ability to cleave their target (GS_CH0.1 target). The level of mutagenesis induced by each construct was measured using the cellular model described in example 5A.
- Both SC_GS and Trex2 were initially cloned into the Ascl / Xhol restriction sites of the pCLS1853 ( Figure 7, SEQ ID NO: 175), a derivative of the pcDNA3.1 (Invitrogen), which drives the expression of a gene of interest under the control of the CMV promoter.
- the four fusion protein constructs were obtained by amplifying separately the two ORFs using a specific primer and the primer CMVfor (5'- CGC AA ATGGGCGGTAGGCGT-3 ' ; SEQ ID NO: 176) or V5reverse (5'- CGTAGAATCGAGACCGAGGAGAGG-3 ' ; SEQ ID NO: 177), which are located on the plasmid backbone.
- CHO-K1 cells were transfected with the expression vector for the protein of interest and the reporter plasmid in the presence of Polyfect transfection reagent in accordance with the manufacturer's protocol (Qiagen). Culture medium was removed 72 hours after transfection and lysis/detection buffer was added for the ⁇ -galactosidase liquid assay.
- lysis/detection buffer contains: 100 ml of lysis buffer (10 mM Tris-HCl pH 7.5, 150 raM NaCl, 0.1% Triton XI 00, 0.1 mg/ml BSA, protease inhibitors), 10 ml of 100X Mg buffer ( 100 mM MgCl 2 , 35% 2-mercaptoethanol), 1 10ml of a 8 mg/ml solution of ONPG and 780 ml of 0.1 M sodium phosphate pH 7.5. The OD 20 is measured after incubation at 37°C for 2 hours. The entire process was performed using a 96-well plate format on an automated Velocity 1 1 BioCel platform (Grizot, Epinat et al. 2009). c) Meganuclease-induced mutafieneis
- TCGATCAGCACGGGCACGATGCC transgenic locus specific reverse sequence
- SEQ ID NO: 161 PCR products were sequenced by a 454 sequencing system (454 Life Sciences). Approximately 10,000 sequences were obtained per PCR product and then analyzed for the presence of site-specific insertion or deletion events. Results
- the cell line described in example 5A was transfected with plasmids expressing either SC GS or the 4 different fusion proteins. Quantification of the percentage of GFP+ cells was determined by flow cytometry 4 days post transfection. SC_GS induced 0.5 to 1 % of GFP+ cells whereas the all four fusion constructs enhance the percentage of GFP+ cells in dose dependent manner from 2 up to 9% (Figure 9A). This strategy appears to be more efficient than the co-transfection strategy as the highest frequency of 4.5% of GFP+ cells was obtained using 9 ⁇ g of Trex2 expressing vector ( Figure 5A) whereas this frequency can be obtained using only 3 ⁇ g of any fusion expressing vector ( Figure 9A).
- the targeted locus was analyzed by PCR amplification followed by sequencing, after cellular transfection of lug and 6 ⁇ g of SC GS or Trex2-10-SC_GS expressing plasmid.
- the deletions events were greatly enhanced with the fusion construct compared to the native meganuclease.
- 1 ⁇ g or 6 ⁇ g of Trex2-10- SC_GS expressing plasmid led to 24% and 31% of mutagenic events all corresponding to deletions.
- These NHEJ frequencies were higher than the ones obtained using 4 or 9 ⁇ g of Trex2 expressing vector in co-transfection experiments (9% and 13% respectively), ( Figures 5B and 9B).
- Trex2 fused to SC_GS was shown to stimulate Targeted Mutagenesis [TM] at a transgenic locus in immortalized cell line.
- Trex2 was fused to SC_CAPNS 1 and TM was monitored at an endogenous locus in immortalized cell line as well as in primary cell line.
- Trex2-SC CAPNS 1 fusion protein The Trex2 protein (SEQ ID NO: 194) was fused to the SC CAPNS l meganuclease
- Trex ORF was amplified using CMVfor primer 5'- CGC AAATGGGCGGTAGGCGT-3 ' (SEQ ID NO: 176) and Linkl OTrexRev primer
- PCR for deep-sequencing were performed using the following primers: 5'- CCATCTCATCCCTGCGTGTCTCCGAC-(forward adaptor sequence)- 10N-(sequences needed for PCR product identification)-CGAGTCAGGGCGGGATTAAG-3'-(locus specific forward sequence) (SEQ ID NO: 199) and the reverse primer 5'- CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-(reverse adaptor sequence)-
- PCR products were sequenced by a 454 sequencing system (454 Life Sciences). Approximately 10,000 sequences were obtained per PCR product and then analyzed for the presence of site-specific insertion or deletion events. Results
- Wild-type 293H cells were transfected by SC_CAPNS 1 or Trex-SC CAPNS l in order to determine if those constructs could stimulate engineered meganuclease-induced targeted mutagenesis at an endogenous locus.
- Transfection with SC_CAPNS1 led to 1.6% of targeted mutagenesis (TM) whereas transfection with the fusion Trex-SC CAPNSl stimulated TM up to 12.4% (Figure 10, Panel A).
- the analysis of the mutagenic sequences showed that the proportion of small deletions events of 2, 3 and 4 base pairs was increased from 2% of the TM events with SC_CAPNS1 to 67% with the fusion Trex-SC CAPNS 1 ( Figure 10, Panel B).
- Wild type Detroit551 cells were transfected by SC CAPNS l or Trex-SC_CAPNS 1 in order to determine if those constructs could also stimulate engineered meganuclease-induced targeted mutagenesis at an endogenous locus in primary cells.
- Transfection with SC_CAPNS 1 led to 1.1 % of TM whereas transfection with the fusion Trex-CAPNSl stimulated TM up to 12.5% ( Figure 1 1 , Panel A).
- the analysis of the mutagenic sequences showed that the proportion of small deletions events of 2, 3 and 4 base pairs was increased from 35% of the TM events with SC_CAPNS 1 to 90% with the fusion Trex-SC_CAPNS1 ( Figure 1 1 , Panel B).
- Tdt Terminal deoxynucleotidyl transferase
- Homing endonucleases from the LAGLIDADG family or meganucleases recognize long DNA sequences and cleave the two DNA strands, creating a four nucleotides 3' overhang.
- the cell can repair the double strand break (DSB) mainly through two mechanisms: by homologous recombination using an intact homologous template or by non homologous end joining (NHEJ). NHEJ is considered as an error prone mechanism that can induce mutations (insertion or deletion of DNA fragments) after DSB repair.
- the measurement of the mutagenesis frequency at the meganuclease locus is a way to assess the meganuclease activity.
- Meganucleases derived from the I-Crel protein have been shown to induce mutagenesis at the genomic site, for which they have been designed (Munoz et al., 201 1 ).
- the human Tdt protein (SEQ ID NO: 201 ) is a 508 amino acids protein that catalyzes the addition of deoxynucleotides to the 3'-hydroxyl terminus of DNA ends.
- the encoded protein is expressed in a restricted population of normal and malignant pre-B and pre-T lymphocytes during early differentiation. It generates antigen receptor diversity by synthesizing non-germ line elements at DSB site after RAG1 and RAG2 endonucleases cleavage. After a meganuclease DSB induced event, such an activity could add DNA sequences at the targeted site and would thus stimulate targeted mutagenesis induced by meganuclease.
- Example 6A Co-transfection of Tdt (SEQ ID NO: 201) with meganucleases
- SC_GS vector encoding meganuclease SC_GS (pCLS2690, SEQ ID NO: 153) was co-transfected on a cell line monitoring mutagenic NHEJ events in presence or absence of a vector encoding Tdt (pCLS3841 of SEQID NO: 202 encoding the protein of SEQ ID NO: 201 ).
- the SC_GS meganuclease (SEQ ID NO: 193) is a single chain protein where two I-Crel variants have been fused. It recognizes a 22bp DNA sequence (5'- TGCCCCAGGGTGAGAAAGTCCA-3 ' : GS CHO.
- the plasmid pCLS6810 (SEQID NO: 159) was designed to quantify NHEJ repair frequency induced by the SC_GS meganuclease (SEQ ID NO: 193).
- the sequence used to measure SC GS-induced mutagenesis is made of an ATG start codon followed by i) 2 codons for alanine, ii) the tag HA sequence, iii) the SC_GS recognition site, iv) a glycine serine stretch, v) the same 2 codons for alanine as in i) and finally vi) a GFP reporter gene lacking its ATG start codon.
- PCR products were sequenced by a 454 sequencing system (454 Life Sciences). Approximately 10,000 sequences were obtained per PCR product and then analyzed for the presence of site-specific insertion or deletion events. f) Transfection on 293H cells to monitor meganuclease-induced mutagenesis at endogenous loci One million of cells were seeded one day prior to transfection.
- Cells were co-transfected with 3 ⁇ g of SC RAGl encoding vector (pCLS2222, SEQ ID NO: 156) with 0.5, 1 and 2 ⁇ g or with 1 , 3 and 7 ⁇ g of plasmid encoding Tdt (pCLS3841 , SEQ ID NO: 202) in, respectively, 5 or 10 ⁇ g of total DNA by complementation with a pUC vector (pCLS0002, SEQID NO: 191 ) using 25 ⁇ of lipofectamine (Invitrogen) according to the manufacturer's instructions.
- SC_CAPNS 1 encoding vector pCLS6163 SEQ ID NO: 158
- 2 ⁇ g of empty vector plasmid pCLS0002, SEQ ID NO: 191
- Tdt encoding plasmid pCSL3841 , SEQ ID NO: 202
- lipofectamine Invitrogen
- PCR for deep-sequencing were performed using the following primers: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-(forward adaptor sequence)- 10N-(sequences needed for PCR product identification)(SEQ ID NO: 5) - locus specific forward sequence for RAG1 : GGCAAAGATGAATCAAAGATTCTGTCC-3 ' (SEQ ID NO: 162) and for CAPNS 1 : CGAGTCAGGGCGGGATTAAG-3 ' (SEQ ID NO: 164) and the reverse primer 5 ' -CCTATCCCCTGTGTGCCTTGGC AGTCTC AG-(reverse adaptor sequence)(SEQ ID NO: 6)-(endogenous locus specific reverse sequence for RAG1 :- GATCTC ACCCGGAAC AGCTTAAATTTC-3 ' (SEQ ID NO: 165) and for CAPNS 1 : - CG AG ACTTC ACGGTTTCGCC-3 ' (SEQ ID NO: 167).
- a cell line measuring mutagenic NHEJ repair induced by SC GS was created.
- the percentage of GFP+ cells, monitoring the mutagenic NHEJ repair, was analyzed 96h after a transfection with SC GS (pCLS2690, SEQ ID NO: 153) alone or with an increasing dose of Tdt encoding vector (pCLS3841 , SEQ ID NO: 202).
- SC_GS transfection led to 0.2 +/-0.1 % of GFP+ cells whereas all doses of Tdt encoding plasmid led to 1.0 +/- 0.4 % of GFP+ cells ( Figure 12, panel A).
- Wild type 293H cells were transfected by SC RAGl encoding vector (pCLS2222, SEQ ID NO: 156) with different doses of Tdt encoding plasmid (pCLS3841 , SEQ ID NO: 202) in order to determine if Tdt could stimulate engineered meganuclease-induced targeted mutagenesis at an endogenous locus.
- Table 8 Example of sequences with insertion at RAGl endogenous locus in presence of Tdt. 3- At endogenous CAPNS1 locus
- Wild type 293H cells were transfected with 3 ⁇ g of plasmid encoding SC CAPNSl meganuclease (pCLS6163, SEQ ID NO: 158) with 0 or 2 ⁇ of Tdt encoding plasmid (pCLS3841 , SEQ ID NO: 202) (in 5 ⁇ g of total DNA) in order to determine Tdt expression effect at another endogenous locus.
- the targeted mutagenesis was 7.4%.
- Tdt was present TM was stimulated up to 13.9% ( Figure 14, panel A).
- the nature of mutagenic DSB repair was analyzed and showed a modification of the pattern of the TM events induced by the meganuclease.
- insertion events represented 10% of total TM events in absence of Tdt whereas in presence of Tdt expressing vector insertion events represented 65% of the TM events.
- the sizes of insertions were also analyzed and in presence of Tdt a specific pattern of insertions appeared corresponding to small insertions ranging from 2 to 6 bp. Finally, the sequence analysis of these insertions seems to show that they are apparently random (Table 9).
- Table 9 Example of sequences with insertion at CAPNS 1 endogenous locus in presence of Tdt.
- Example 6B Fusion of the human Tdt to meganucleases: effect on targeted mutagenesis
- Tdt SEQ ID NO: 201
- meganuclease encoding plasmids Co-transfection of Tdt (SEQ ID NO: 201 ) with meganuclease encoding plasmids was shown to increase the rate of mutagenesis induced by meganucleases.
- this strategy implies the presence of two plasmids within the cell at the same time.
- a chimeric protein comprising TdT and Meganuclease proteins is engineered.
- the human Tdt protein (SEQ ID NO: 201 ) is fused to the N- or C-terminus of different Single chain engineered meganucleases SC MN such as SC_GS (SEQ ID NO: 193), SC_RAG (SEQ ID NO: 58) and SC_CAPNS 1 (SEQ ID NO: 192).
- SC MN Single chain engineered meganucleases
- SC_GS SEQ ID NO: 193
- SC_RAG SEQ ID NO: 58
- SC_CAPNS 1 SEQ ID NO: 192
- Two SC MN fused to Tdt protein are made: either at the N terminal domain or C terminal domain of the considered meganuclease. Those constructed are tested for their ability to increase mutagenic activity at the locus of interest.
- the Tdt protein is fused to the SC MN meganuclease either to its C-terminus or to its N- terminus using a ten amino acids linker (GGGGS) 2 (SEQ ID NO: 170).
- GGGGS ten amino acids linker
- All SC MN were initially cloned into the Ascl / Xhol restriction sites of the pCLS 1853 ( Figure 7, SEQ ID NO: 175), a derivative of the pcDNA3.1 (Invitrogen), which drives the expression of a gene of interest under the control of the CMV promoter.
- the two fusion proteins for each SC_MN/Tdt constructs are obtained by amplifying separately the two ORFs using specific primers.
- the following table 10 gives the oligonucleotidic sequences that are used to create the different SC GS/Tdt constructs.
- a PCR assembly is realized using the CMVfor (SEQ ID NO: 176) and TDTRev (SEQ ID NO: 204) oligonucleotides for Cter fusion of Tdt to SC MN or using TDTFor (SEQ ID NO: 205) and V5Rev (SEQ ID NO: 177) for Nter fusion of Tdt to SC MN.
- the final PCR product is cloned in a pTOPO vector then digested by Ascl and Xhol and ligated into the pCLS 1853 vector (SEQ ID NO: 175) pre- digested with these same enzymes.
- Example 7 Impact of co-transfection with two nucleases targeting two sequences separated by 173 base pairs (bp) on mutagenesis frequency.
- Nucleases consist of an engineered meganuclease (Nl) (SC RAG of SEQ ID NO: 216) encoded by pCLS2222 (SEQ ID NO: 156) cleaving the DNA sequence 5 ' -TTGTTCTC AGGTACCTCAGCC AGC-3 ' (Tl) (SEQ ID NO: 207) and a TALEN (N2) [SEQ ID NO: 209-210 respectively encoded by pCLS8964 (SEQ ID NO: 21 1 ) and pCLS8965 (SEQ ID NO: 212)] targeting DNA sequence 5'- TATATTTAAGC ACTTATATGTGTGTAAC AGGTATAAGTAACC ATAAAC A-3 ' (T2) (SEQ ID NO: 208). These two recognition sites are separated by 173 bp.
- Tl and T2 targets were amplified with specific primers flanked by specific adaptator needed for High Throughput Sequencing on the 454 sequencing system (454 Life Sciences)
- primers F_T2 At Tl and T2 loci, primers F_T2:
- the rate of mutations induced by the nucleases Nl and N2 at the targets Tl and T2 was measured by deep sequencing. Results are presented in Table 1 1. 0.63% of PCR fragments carried a mutation in samples corresponding to cells transfected with the Nl nuclease. Similarly, 1.46% of PCR fragments carried a mutation in sample corresponding to cells transfected with the N2 nuclease. The rate of induced mutagenesis increased up to 1.33% on Tl target and up to 2.48% on T2 target when the cells were transfected with plasmids expressing both N 1 and N2, showing that the presence of two nucleases targeting two nearby sequences stimulates up to about two folds the frequency of mutagenesis.
- TAL nucleases hybrid proteins composed of TAL effectors and Fokl DNA-cleavage domain. Nucleic Acids Res 39( 1 ): 359-72.
- thermostable archaeal intron- encoded endonuclease I-Dmol J Mol Biol 286(4): 1 123-36.
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CA2815512A1 (en) | 2012-05-03 |
WO2012058458A3 (en) | 2013-01-10 |
EP2633040A2 (en) | 2013-09-04 |
US20130337454A1 (en) | 2013-12-19 |
SG189482A1 (en) | 2013-05-31 |
AU2011319725A1 (en) | 2013-05-30 |
EP2633040B1 (en) | 2019-07-10 |
JP2013544082A (en) | 2013-12-12 |
IL225860A0 (en) | 2013-06-27 |
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