Patent Application
20130337454
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 (non-homologous end joining). The present invention also relates to engineered endonucleases, chimeric or not, vectors, compositions and kits used to implement this method.
Mammalian genomes constantly suffer from various types of damage of which double-strand breaks (DSB) are considered the most dangerous (Haber 2000). For example, 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. For cultured mammalian cells that are dividing, 5-10% appear to have at least one chromosomal break (or chromatid gap) at any one time (Lieber and Karanjawala 2004). Hence, 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.
Repair of DSBs can occur through diverse mechanisms that can depend on cellular context. Repair via homologous recombination, the most accurate process, is able to restore the original sequence at the break. Because of its strict dependence on extensive sequence homology, this mechanism is suggested to be active mainly during the S and G2 phases of the cell cycle where the sister chromatids are in close proximity (Sonoda, Hochegger et al. 2006). Single-strand annealing is another homology-dependent process that can repair a DSB between direct repeats and thereby promotes deletions (Paques and Haber 1999). Finally, non-homologous end joining (NHEJ) of DNA is a major pathway for the repair of DSBs because it can function throughout the cell cycle and because it does not require a homologous chromosome (Moore and Haber 1996).
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). Although perfect re-ligation of the broken ends is probably the most frequent event, it could be accompanied by the loss or gain of several nucleotides.
Like most DNA repair processes, there are three enzymatic activities required for repair of DSBs by the NHEJ pathway: (i) nucleases to remove damaged DNA, (ii) polymerases to aid in the repair, and (iii) a ligase to restore the phosphodiester backbone. Depending on the nature of the DNA ends, 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 μ or λ. In addition, an alternative or so-called back-up pathway has been described that does not depend on ligase IV and Ku components and has been involved in class switching and V(D)J recombination (Ma, Kim et al. 2003). Overall, 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 (TopoI) 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., cross-linking agents are favored for NER studies), most drugs simultaneously provoke a variety of lesions (Nagy and Soutoglou 2009). Furthermore, the overall yield of induced mutations using these classical strategies is quite low, and the DNA damage leading to mutagenesis cannot be targeted to a precise genomic DNA sequence.
The most widely used site-directed mutagenesis strategy is gene targeting (GT) via homologous recombination (HR). Efficient GT procedures in yeast and mouse have been available for more than 20 years (Capecchi 1989; Rothstein 1991). Successful GT has also been achieved in Arabidopsis and rice plants (Hanin, Volrath et al. 2001) (Terada, Urawa et al. 2002; Endo, Osakabe et al. 2006; Endo, Osakabe et al. 2007). Typically, 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. As a consequence, the ends of a donor molecule are likely to be joined by NHEJ rather than participating in HR, thus reducing GT frequency. There are extensive data indicating that DSB repair by NHEJ is error-prone due to end-joining processes that generate insertions and/or deletions (Britt 1999). Thus, these NHEJ-based strategies might be more effective than HR-based strategies for targeted mutagenesis into cells.
Expression of I-SceI, a rare cutting endonuclease, has been shown to introduce mutations at I-SceI cleavage sites in Arabidopsis and tobacco (Kirik, Salomon et al. 2000). However, the use of endonucleases is limited to rarely occurring natural recognition sites or to artificially introduced target sites. To overcome this problem, 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-SceI 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. 1997; Cohen-Tannoudji, Robine et al. 1998; Donoho, Jasin et al. 1998), mice (Gouble, Smith et al. 2006) and plants (Puchta, Dujon et al. 1996; Siebert and Puchta 2002). Meganucleases have emerged as scaffolds of choice for deriving genome engineering tools cutting a desired target sequence (Paques and Duchateau 2007).
Combinatorial assembly processes allowing for the engineering of meganucleases with modified specificities have been described by Arnould et al. (Arnould, Chames et al. 2006; Arnould, Perez et al. 2007); Smith et al. (Smith, Grizot et al. 2006), Grizot et al. (Grizot, Smith et al. 2009). Briefly, these processes rely on the identification of locally engineered variants with a substrate specificity that differs from that of the wild-type meganuclease by only a few nucleotides. Another type of specific nucleases are the so-called Zinc-finger nucleases (ZFNs). 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). Subsequent repair of the DSBs by NHEJ frequently produces deletions and/or insertions at the joining site. For example, in zebrafish embryos the injection of mRNA coding for engineered ZFNs led to animals carrying the desired heritable mutations (Doyon, McCammon et al. 2008). In plants, similar NHEJ-based targeted mutagenesis has also been successfully applied (Lloyd, Plaisier et al. 2005). Although these powerful tools are available, there is still a need to further improve double-strand break-induced mutagenesis.
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.
In one of its embodiments, 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.
In a second aspect, 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.
In a third aspect, 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.
In a fourth aspect, 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. In a fifth aspect, 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.
In a sixth aspect, 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.
The above objects highlight certain aspects of the invention. Additional objects, aspects and embodiments of the invention are found in the following detailed description of the invention.
In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, as well as to the appended drawings. A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following Figures in conjunction with the detailed description below.
Size of deletion events were analyzed and the frequency of indicated deletion among all deletion events were calculated after treatment with meganucleases SC_RAG1 (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) and SC_CAPNS1 (SEQ ID NO: 192 encoded by pCLS6163, SEQ ID NO: 158) only (grey histogram) or with Trex2 (SEQ ID NO: 194 encoded by pCLS7673, SEQ ID NO: 154) (black histogram).
All fusion constructs were cloned in pCLS1853 (SEQ ID NO: 175), driving their expression by a CMV promoter.
CHO-K1 cells were co-transfected with the plasmid measuring SSA activity containing the GS_CHO1.1 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 72 h after transfection using ONPG and 420 nm optical density detection. The entire process was performed on an automated Velocity 11 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 μg 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 μg 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.
Panel A: Percentage of Targeted Mutagenesis [TM] obtained in 293H cell line transfected with SC_CAPNS1 (SEQ ID NO: 192) or Trex-SC_CAPNS1 (SEQ ID NO: 197).
Panel B: Nature of Targeted Mutagenesis obtained in 293H 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 CAPNS1. “Other” represents all other TM events.
Panel A: Percentage of Targeted Mutagenesis obtained in Detroit551 cell line transfected with SC_CAPNS1 (SEQ ID NO: 192) or Trex-SC_CAPNS1 (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 CAPNS1. “Other” represents all other TM events.
Panel A: Percentage of GFP+ cells induced on NHEJ model after co-transfection of 1 μg or 3 μg 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 μg 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_CHO1 DNA target present on the NHEJ model, induced by either SC_GS with empty vector or with 2 μg of Tdt encoding vector.
Panel C: Percentage of insertion events within targeted mutagenesis events after co-transfection of the NHEJ model by 3 μg of SC_GS expressing vector with 2 μg of an empty vector or with 2 μg of Tdt encoding plasmid.
Panel D: Percentage of insertion events in function of their size in presence (TDT) or absence (empty) of Tdt.
Panel A: Percentage of targeted mutagenesis detected by deep sequencing in the vicinity of the SC_RAG1 target induced by co-transfection of 3 μg of SC_RAG1 encoding vector (SEQ ID NO: 156) with different amount of Tdt encoding vector (SEQ ID NO: 202) in 5 μg of total DNA (left part) or in 10 μg of total DNA (right part).
Panel B: Percentage of insertion events within targeted mutagenesis events after co-transfection of 3 μg of SC_RAG1 encoding vector (SEQ ID NO: 156) with different amount of Tdt encoding vector (SEQ ID NO: 202) in 5 μg of total DNA (left part) or in 10 μg of total DNA (right part).
Panel C: Percentage of insertion events in function of their size at endogenous RAG1 locus after co-transfection of 3 μg of SC_RAG1 encoding vector (SEQ ID NO: 156) with different amounts of Tdt encoding vector (SEQ ID NO: 202) in 5 μg of total DNA (left part) or in 10 μg of total DNA (right part).
Panel A: Percentage of targeted mutagenesis detected by deep sequencing in the vicinity of the SC_CAPNS1 target induced by co-transfection of 1 μg of SC_CAPNS1 expressing vector (SEQ ID NO: 158) with 2 μg of Tdt encoding plasmid (SEQ ID NO: 202).
Panel B: Percentage of insertion events within targeted mutagenesis events after co-transfection of 3 μg of SC_CAPNS1 expressing vector (SEQ ID NO: 158) with 2 μg of Tdt encoding plasmid (SEQ ID NO: 202).
Panel C: Percentage of insertion events in function of their size at CAPNS1 locus after co-transfection of 3 μg of SC_CAPNS1 expressing vector (SEQ ID NO: 158) with 2 μg of Tdt encoding plasmid (SEQ ID NO: 202).
Unless specifically defined herein below, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology.
All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
According to 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:
In a preferred embodiment, said rare-cutting endonuclease is able to generate one DNA double-strand break in the genomic locus of interest and a loss of genetic information by another enzymatic activity. In a more preferred embodiment, said another enzymatic activity is a nuclease activity. In another more preferred embodiment, said another enzymatic activity is an exonuclease activity. In this preferred embodiment, 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.
In another preferred embodiment, 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
In a preferred embodiment, 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. In another preferred embodiment, 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.
In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 194, 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: 194, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, 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. In another preferred embodiment, said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 171-174 and SEQ ID NO: 197.
In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 201, 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: 201, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, 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.
In another aspect 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. For instance, as a non limiting example, 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. As another non limiting example, 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, . . . ) 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.
In a preferred embodiment, 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. In another preferred embodiment, 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.
In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 194, 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: 194, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, 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. In another preferred embodiment, said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 171-174 and SEQ ID NO: 197.
In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 201, 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: 201, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, 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.
In a third aspect, 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. In other words, 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
According to this third aspect, the present invention concerns a method comprising the steps of:
In a preferred embodiment of this third aspect, 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
In a preferred embodiment, 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. In another preferred embodiment, 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-TevI (SEQ ID NO: 106 or SEQ ID NO: 60; SEQ ID NO: 107-108), NucA (SEQ ID NO: 41 and 112), NucM (SEQ ID NO: 43 and 113), SNase (SEQ ID NO: 45-47 and 116-118), BspD6I (SEQ ID NO: 124-125) a functional mutant, variant or derivative thereof.
In another preferred embodiment, 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 a fusion protein comprising a meganuclease and 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.
In another preferred embodiment, 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. In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 56, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 57, 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: 56, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, 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. 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 a fusion protein comprising a meganuclease and 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.
In another embodiment of this third aspect, the present invention implies two engineered rare-cutting endonucleases and comprises the steps of:
In a preferred embodiment, 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
In a preferred embodiment, 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, 50 bp, 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.
In a fourth aspect, 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
In a preferred embodiment, 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-CreI 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-CreI meganuclease.
In a more preferred embodiment said chimeric rare-cutting endonuclease is a fusion protein between a meganuclease and at least one nuclease catalytic domain. In said more preferred embodiment, said nuclease catalytic domain has an endonuclease activity; alternatively, said nuclease catalytic domain has an exonuclease activity.
In a preferred embodiment, 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. In another preferred embodiment, 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), 1-TevI (SEQ ID NO: 106 or SEQ ID NO: 60; SEQ ID NO: 107-108), NucA (SEQ ID NO: 41 and 112), NucM (SEQ ID NO: 43 and 113), SNase (SEQ ID NO: 45-47 and 116-118), BspD6I (SEQ ID NO: 124-125), a functional mutant, a variant or a derivative thereof.
In another preferred embodiment, 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 112, SEQ ID NO: 43 and 113, SEQ ID NO: 45-47 and 116-118, SEQ ID NO: 124-125, a functional mutant, a variant or a derivative thereof.
In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 194, 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: 194, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said rare-cutting endonuclease is a fusion protein comprising a single-chain meganuclease and a protein of SEQ ID NO: 194. In another preferred embodiment, said chimeric rare-cutting endonuclease is selected from the group consisting of SEQ ID NO: 171-174 and SEQ ID NO: 197.
In another preferred embodiment, 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.
In another preferred embodiment, 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. In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 56, a functional mutant, a variant or a derivative thereof. In another preferred embodiment, said chimeric rare-cutting endonuclease comprises a catalytic domain of SEQ ID NO: 57, 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: 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.
In another preferred embodiment, said chimeric rare-cutting endonuclease further comprises a second peptidic linker and a supplementary catalytic domain. In other words, 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:
In a preferred embodiment, 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.
Also encompassed within the scope of the present invention is 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.
In a fifth aspect, 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:
In a sixth aspect, 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. In other words, the present invention relates to a fusion protein comprising:
In a preferred embodiment, 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-CreI derived meganuclease. In another preferred embodiment, said rare-cutting endonuclease part of said fusion protein is a single chain meganuclease derived from I-CreI meganuclease.
In another preferred embodiment, said further fusion protein comprises a second peptidic linker and a supplementary catalytic domain. In other words, 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:
In a preferred embodiment, said supplementary catalytic domain is a nuclease domain (as illustrated in
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. Such 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
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.
In the wild, meganucleases are essentially represented by homing endonucleases. Homing Endonucleases (HEs) 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-CreI (Chevalier, Monnat et al. 2001), I-MsoI (Chevalier, Turmel et al. 2003) and I-CeuI (Spiegel, Chevalier et al. 2006) and with a pseudo symmetry for monomers such as I-SceI (Moure, Gimble et al. 2003), I-DmoI (Silva, Dalgaard et al. 1999) or I-AniI (Bolduc, Spiegel et al. 2003). Both monomers and both domains (for monomeric proteins) contribute to the catalytic core, organized around divalent cations. Just above the catalytic core, the two LAGLIDADG peptides also play an essential role in the dimerization interface. DNA binding depends on two typical saddle-shaped αββαββα folds, sitting on the DNA major groove. Other domains can be found, for example in inteins such as PI-PfuI (Ichiyanagi, Ishino et al. 2000) and PI-SceI (Moure, Gimble et al. 2002), whose protein splicing domain is also involved in DNA binding.
The making of functional chimeric meganucleases, by fusing the N-terminal I-DmoI domain with an I-CreI monomer (Chevalier, Kortemme et al. 2002; Epinat, Arnould et al. 2003); International PCT Application WO 03/078619 (Cellectis) and WO 2004/031346 (Fred Hutchinson Cancer Research Center, Stoddard et al)) have demonstrated the plasticity of LAGLIDADG proteins.
Different groups have also used a semi-rational approach to locally alter the specificity of the I-CreI (Seligman, Stephens et al. 1997; Sussman, Chadsey et al. 2004); International PCT Applications WO 2006/097784, WO 2006/097853, WO 2007/060495 and WO 2007/049156 (Cellectis); (Arnould, Chames et al. 2006; Rosen, Morrison et al. 2006; Smith, Grizot et al. 2006), I-SceI (Doyon, Pattanayak et al. 2006), PI-SceI (Gimble, Moure et al. 2003) and I-MsoI (Ashworth, Havranek et al. 2006).
In addition, hundreds of I-CreI derivatives with locally altered specificity were engineered by combining the semi-rational approach and High Throughput Screening:
Two different variants were combined and assembled in a functional heterodimeric endonuclease able to cleave a chimeric target resulting from the fusion of two different halves of each variant DNA target sequence ((Arnould, Chames et al. 2006; Smith, Grizot et al. 2006); International PCT Applications WO 2006/097854 and WO 2007/034262).
Furthermore, residues 28 to 40 and 44 to 77 of I-CreI 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 mutations from the two subdomains of I-CreI within the same monomer allowed the design of novel chimeric molecules (homodimers) able to cleave a palindromic combined DNA target sequence comprising the nucleotides at positions ±3 to 5 and ±8 to 10 which are bound by each subdomain ((Smith, Grizot et al. 2006); International PCT Applications WO 2007/049095 and WO 2007/057781 (Cellectis)).
The method for producing meganuclease variants and the assays based on cleavage-induced recombination in mammal or yeast cells, which are used for screening variants with altered specificity are described in the International PCT Application WO 2004/067736; (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006). These assays result in a functional LacZ reporter gene which can be monitored by standard methods.
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. In a first step, 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. The assembly of four sets of mutations into heterodimeric endonucleases cleaving a model target sequence or a sequence from different genes has been described in the following Cellectis International patent applications: XPC gene (WO2007/093918), RAG gene (WO2008/010093), HPRT gene (WO2008/059382), beta-2 microglobulin gene (WO2008/102274), Rosa26 gene (WO2008/152523), Human hemoglobin beta gene (WO2009/13622) and Human interleukin-2 receptor gamma chain gene (WO2009019614).
These variants can be used to cleave genuine chromosomal sequences and have paved the way for novel perspectives in several fields, including gene therapy.
Examples of such endonuclease include I-Sce I, I-Chu I, I-Cre I, I-Csm I, PI-Sce I, PI-Tli 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, PI-Pfu I, PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko I, PI-Tsp I, I-MsoI.
A homing endonuclease can be a LAGLIDADG endonuclease such as I-SceI, I-CreI, I-CeuI, I-MsoI, and I-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-CreI 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. In the frame of the present invention, 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.
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.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of 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).
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. In general, 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. Large numbers of suitable vectors are known to those of skill in the art. 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 S. cerevisiae; tetracyclin, rifampicin or ampicillin resistance in E. coli. Preferably 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. Examples of inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-□-D-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature. Examples of tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), α-antitrypsin protease, human surfactant (SP) A and B proteins, β-casein and acidic whey protein genes.
Delivery vectors and vectors can be associated or combined with any cellular permeabilization techniques such as sonoporation or electroporation or derivatives of these techniques.
More preferably 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, Cucurbita pepo, Citrullus lanatus, Cucumis melo, Citrus aurantifolia, Citrus maxima, Citrus medica, Citrus reticulata.
More preferably 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.
The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.
As used above, the phrases “selected from the group consisting of,” “chosen from,” and the like include mixtures of the specified materials.
Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.
The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.
Two engineered single-chain meganucleases called R1 or R1m (SEQ ID NO: 58) and D21 or D21m (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). These meganucleases, derived from I-CreI, are designed to recognize two different DNA sequences, neither of which are recognized by wild-type I-CreI. (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 (
Human HEK293 cells are transiently co-transfected with two plasmids carrying the expression cassette for R1 (SEQ ID NO: 58) and D21 (SEQ ID NO: 59), as well as the target plasmid. For comparison, 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
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 R1 (SEQ ID NO: 58) and D21 (SEQ ID NO: 59), are also analyzed by high-throughput sequencing (
In this example, an engineered single-chain meganuclease derived from I-CreI (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. 2006)) is fused to various nuclease domains to create a bi-functional meganuclease. To obtain maximal activity, 31 different linkers are tested ranging in size from 3 to 26 amino acids (Table 1). Fusions are made using 18 different catalytic domains (Table 2) that are chosen based on their having essentially non-specific nuclease activity. Altogether, a library of 1116 different constructs are created via fusion to the N- or C-terminus of the engineered single-chain I-CreI-derived meganuclease, generating a collection of potential bi-functional meganucleases. Expression of these chimeric meganucleases are driven by a CMV promoter and a polyA signal sequence. The activity of 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). To monitor DNA cleavage activity resulting from the addition of the new catalytic domain, an I-CreI 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 (
To further validate the high rate of site-specific mutagenesis induced by a bi-functional chimeric endonuclease, the same strategy is applied to an engineered single-chain meganuclease designed to cleave the human RAG1 gene as described in International PCT Applications WO2003078619, WO 2008/010093 and WO2009095793. The bi-functional meganuclease is tested for its ability to induced NHEJ-driven mutation at its endogenous cognate recognition site. The mutagenesis activity is quantified by high-throughput sequencing of PCR products as described in example 1. PCR amplification is performed on genomic DNA extracted from meganuclease-transfected cells using appropriate primers.
The bi-functional meganucleases displayed an increased mutation rate since the intervening sequence is deleted, thereby preventing “scarless” re-ligation of DNA ends through NHEJ.
coli
pneumonia
Staphylococcus
aureus
Staphylococcus
hyicus
flexneri
Bacillus subtilis
varians]
stearothermophilus]
stearothermophilus]
lylae]
stearothermophilus]
stearothermophilus]
thermoglucosidasius]
thermoglucosidasius]
thermoglucosidasius]
E. coli ExoI
coli DH1]
norvegicus]
I-CreI meganuclease (SEQ ID NO: 76) was chosen as the parent scaffold on which to fuse the catalytic domain of I-TevI (SEQ ID NO: 60). Wild-type I-TevI 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-TevI that encompass the entire catalytic domain and deletion-intolerant region of its linker (SEQ ID NOs: 61 to 66). In all but one case, fragments were fused to the N-terminus of I-CreI with an intervening 5-residue polypeptide linker (-QGPSG-=SEQ ID NO: 67). The linker-less fusion construct naturally contained residues (-LGPDGRKA-=SEQ ID NO: 68) similar to those in the artificial linker. As I-CreI is a homodimer, all fusion constructs contain three catalytic centers (
The activity of 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 C1221 target DNA with an activity comparable to that of wild-type I-CreI (Table 4).
To validate the activity of the I-TevI catalytic domain independent of the I-CreI catalytic core, D20N point mutants were made to inactivate the I-CreI scaffold (SEQ ID NOs: 69 to 75). Tests in yeast assays showed no visible activity from the inactivated I-CreI (D20N) mutant protein alone (Table 4). However, cleavage activity could be observed for fusions having the I-TevI catalytic domain (Table 4).
Protein-fusion scaffolds were designed based on a truncated form of I-CreI (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-CreI 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-CreI 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-CreI. For both fusion scaffold types, the “free” end of the linker (i.e. onto which a polypeptide can be linked) is designed to be proximal to the DNA, as determined from models built using the I-CreI/DNA complex structures as a starting point (PDB id: 1g9z). The two I-CreI N-terminal fusion scaffolds (I-CreI_NFS1=SEQ ID NO: 81 and I-CreI_NFS2=SEQ ID NO: 82) and the single C-terminal fusion scaffold (I-CreI—CFS1: SEQ ID NO: 83) were tested in our yeast assay (see Example 3) and found to have activity similar to that of wild-type I-CreI (Table 5).
Colicin E7 is a non-specific nuclease of the HNH 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_NFS1 (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: 86), respectively. In addition, a C-terminal fusion construct, hCreColE7_D0101 (SEQ ID: 87), was generated using I-CreI_CFS1 (SEQ ID NO: 83). As I-CreI is a homodimer, all fusion constructs contain three catalytic centers (
The activity of each “tri-functional” meganuclease was assessed using yeast assay as previously mentioned (see Example 3). All constructs were able to cleave the C1221 target DNA with an activity comparable to that of wild-type I-CreI (Table 4). To validate the activity of the ColE7 catalytic domain independent of the I-CreI catalytic core, D20N point mutants were made to inactivate the I-CreI scaffold (SEQ ID NOs: 88-93). Tests in our yeast assays showed no visible activity from the inactivated I-CreI (D20N) mutant proteins alone (Table 5). However, cleavage activity could be observed for fusions having the ColE7 catalytic domain (Table 5).
Human 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. 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.
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, SEQ ID NO: 154). The SC_GS meganuclease is a single chain protein (SEQ ID NO: 193) derived from the fusion of two I-CreI variants. It recognizes a 22 bp DNA sequence (5′-TGCCCCAGGGTGAGAAAGTCCA-3′: GS_CHO.1 target, SEQ ID NO: 155) located in the first exon of the Cricetulus griseus glutamine synthetase gene. Different meganucleases such as SC_RAG1 (pCLS2222, SEQ ID NO: 156 i.e. the expression vector encoding SC_RAG1, SEQ ID NO: 58), SC_XPC4 (pCLS2510, SEQ ID NO: 157 i.e. the expression vector encoding SC_XPC4, SEQ ID NO: 190) and SC_CAPNS1 (pCLS6163, SEQ ID NO: 158 i.e. the expression vector encoding SC_CAPNS1, SEQ ID NO: 192) were co-transfected with or without a Trex2 expression vector (pCLS7673, SEQ ID NO: 154) to analyze the effect on meganuclease-induced mutagenesis at endogenous loci.
Material and Methods
a) Cellular Model to Monitor Meganuclease-Induced Mutagenesis
The plasmid pCLS6810 (SEQ ID NO: 159) was designed to quantify the NHEJ repair frequency induced by the SC_GS meganuclease (pCLS2690, SEQ ID NO: 153). 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 hsRAG1 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. Thus, after transfection of this cell line by SC_GS meganuclease expressing plasmid with or without a plasmid encoding Trex2 (pCLS7673, SEQ ID NO: 154), the percentage of GFP positive cells is directly correlated to the mutagenic NHEJ repair frequency induced by the transfected molecular entity/ies.
b) Transfection in a Cellular Model Monitoring Meganuclease-Induced Mutagenesis
One million of cells were seeded one day prior to transfection. Cells were co-transfected with 1 μg of SC_GS encoding vector (pCLS2690, SEQ ID NO: 153) and with 0, 2, 4, 6 or 9 μg of plasmid encoding Trex2 (pCLS7673 SEQ ID NO: 154) in 10 μg of total DNA by complementation with a pUC vector (pCLS0002, SEQ ID NO: 191) using 25 μl of lipofectamine (Invitrogen) according to the manufacturer's instructions. Four days following transfection, cells were harvested for flow cytometry analysis using Guava instrumentation. Genomic DNA was extracted from cell populations transfected with 1 μg of SC_GS expressing plasmid and 0, 4 and 9 μg 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). Approximately 10,000 sequences were obtained per PCR product and then analyzed for the presence of site-specific insertion or deletion events.
c) 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 μg of plasmid expressing SC_RAG1 or SC_XPC4 or SC_CAPNS1 (pCLS2222, SEQ ID NO: 156; pCLS2510, SEQ ID NO: 157 and pCLS6163, SEQ ID NO: 158 respectively) and with 0 or 2 μg of plasmid encoding Trex2 (pCSL7673 SEQ ID NO: 154) in 5 μg of total DNA by complementation with a pUC vector (pCLS0002 SEQ ID NO: 191) using 25 μl 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 RAG 1: GGCAAAGATGAATCAAAGATTCTGTCC-3′ (SEQ ID NO: 162), for XPC4:-AAGAGGCAAGAAAATGTGCAGC-3′ (SEQ ID NO: 163) and for CAPNS1-CGAGTCAGGGCGGGATTAAG-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: -GCTGGGCATATATAAGGTGCTCAA-3′ (SEQ ID NO: 166) and for CAPNS1: -CGAGACTTCACGGTTTCGCC-3′ (SEQ ID NO: 167). 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
1—On Cellular Model Measuring Meganuclease-Induced Mutagenesis
The percentage of GFP+ cells, monitoring mutagenesis events induced by SC_GS meganuclease in a dedicated cellular model, was analyzed 96 h after a transfection with SC_GS expressing plasmid (pCLS2690 SEQ ID NO: 153) alone or with an increasing dose of Trex2 encoding vector (pCLS7673 SEQ ID 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 μg of Trex2 encoding plasmid led to 1.3, 2.8, 3.4 and 4.8% of GFP+ respectively (
2—At Endogenous Loci
Trex2 effect on mutagenesis induced by engineered meganucleases was measured at RAG1, XPC4 and CAPNS1 endogenous loci by co-transfecting plasmids expressing SC_RAG1 or SC_XPC4 XPC4 or SC_CAPNS1 with or without Trex2 encoding plasmid. Transfections of 3 μg of meganuclease expressing vector with 2 μg of Trex2 (3/2 ratio) encoding plasmid were performed. The mutagenesis induced by the different meganucleases was quantified and analyzed three days post transfection. In these conditions, 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
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_CHO.1 target). The level of mutagenesis induced by each construct was measured using the cellular model described in example 5A.
a) Making of SC_GS/Trex2 Fusion Proteins
The Trex2 protein was fused to the SC_GS meganuclease either to its C-terminus or to its N-terminus using a five amino acids glycin stretch (sequence GGGGS) (SEQ ID NO: 169) or a ten amino acids glycin stretch (GGGGS)2 (SEQ ID NO: 170) as linkers. This yielded to four protein constructs named respectively SC_GS-5-Trex, SC_GS-10-Trex, Trex-5-SC_GS, Trex-10-SC_GS (SEQ ID NO: 171 to 174). Both SC_GS and Trex2 were initially cloned into the AscI/XhoI restriction sites of the pCLS1853 (
b) Extrachromosomal SSA Activity
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 β-galactosidase liquid assay. One liter of lysis/detection buffer contains: 100 ml of lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X100, 0.1 mg/ml BSA, protease inhibitors), 10 ml of 100× Mg buffer (100 mM MgCl2, 35% 2-mercaptoethanol), 110 ml of a 8 mg/ml solution of ONPG and 780 ml of 0.1M sodium phosphate pH 7.5. The OD420 is measured after incubation at 37° C. for 2 hours. The entire process was performed using a 96-well plate format on an automated Velocityll BioCel platform (Grizot, Epinat et al. 2009).
c) Meganuclease-Induced Mutageneis
One million of cells were seeded one day prior transfection. Cells were transfected with an increasing amount (from 1 μg up to 9 μg) of plasmid encoding SC_GS (pCLS2690, SEQ ID NO: 153) or SC_GS-5-Trex (pCLS8082 SEQ ID NO: 186), SC_GS-10-Trex (pCLS8052 SEQ ID NO: 187), Trex-5-SC_GS (pCLS8053 SEQ ID NO: 188) and Trex-10-SC_GS (pCLS8054 SEQ ID NO: 189) in 10 μg of total DNA by complementation with a pUC vector (SEQ ID NO: 191) using 25 μl of lipofectamine (Invitrogen) according to the manufacturer's instructions. Three to four days following transfection, cells were harvested for flow cytometry analysis using Guava instrumentation. Cells transfected with 1 μg and 6 μg of SC_GS or SC_GS-10-Trex2 expressing plasmid were harvested for genomic DNA extraction. 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). 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.
The activity of the four fusion proteins was first monitored using an extrachromosomal assay in CHO-K1 cells (Grizot, Epinat et al. 2009). The fusion of Trex2 to the SC_GS could indeed impair its folding and/or its activity.
1—On Cellular Model Measuring Meganuclease-Induced Mutagenesis
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% (
Trex2 fused to SC_GS was shown to stimulate Targeted Mutagenesis [TM] at a transgenic locus in immortalized cell line. In order to apply the fusion to other engineered meganucleases and to stimulate TM in primary cell line Trex2 was fused to SC_CAPNS1 and TM was monitored at an endogenous locus in immortalized cell line as well as in primary cell line.
d) Making of Trex2-SC_CAPNS1 Fusion Protein
The Trex2 protein (SEQ ID NO: 194) was fused to the SC_CAPNS1 meganuclease
(SEQ ID NO: 192) at its N-terminus using a (GGGGS)2 ten amino acids linker (SEQ ID NO: 170). Cloning strategy was the same as used for the fusion Trex-SC_GS. Both SC_CAPNS1 and Trex2 were initially cloned into the AscI/XhoI restriction sites of the pCLS1853 (
was used with V5 reverse primer 5′-CGTAGAATCGAGACCGAGGAGAGG-3′ (SEQ ID NO: 177); Trex ORF was amplified using CMVfor primer 5′-CGCAAATGGGCGGTAGGCGT-3′ (SEQ ID NO: 176) and Link10TrexRev primer
Then, after a gel purification of the two PCR fragments, a PCR assembly was realized using the CMVfor/V5reverse oligonucleotides. The final PCR product was then digested by AscI and XhoI and ligated into the pCLS 1853 plasmid digested with these same enzymes leading to Trex-SC_CAPNS1 encoding vector (pCLS8518 of SEQ ID NO: 196 encoding Trex-SC_CAPNS1 protein of SEQ ID NO: 197).
e) Transfection on 293H Cells to Monitor Trex2-Meganuclease Fusion on Mutagenesis at an Endogenous Locus
One million of cells were seeded one day prior to transfection. Cells were transfected with 100 ng of either SC_CAPNS1 or Trex-SC_CAPNS1 encoding vector (respectively, protein sequence of SEQ ID NO: 192 encoded by pCLS6163 of SEQ ID NO: 158 and protein sequence of SEQ ID NO: 197 encoded by pCLS8518 of SEQ ID NO: 196) in 5 μg of total DNA by complementation with a pUC vector (pCLS0002 SEQ ID NO: 191) using 25 μl of lipofectamine (Invitrogen) according to the manufacturer's instructions. Three days following transfection, cells were harvested for genomic DNA extraction.
f) Transfection on Detroit Cells to Monitor Trex2-Meganuclease Fusion on Mutagenesis at an Endogenous Locus
One million of cells were seeded one day prior to transfection. Cells were co-transfected with 6 μg of either SC_CAPNS1 or Trex-SC_CAPNS1 encoding vector (respectively pCLS6163 of SEQ ID NO: 158 and pCLS8518 of SEQ ID NO: 196) in 10 μg of total DNA by complementation with a pUC vector (pCLS0002, SEQ ID NO: 191) using Amaxa (LONZA) according to the manufacturer's instructions. Three days following transfection, cells were harvested for genomic DNA extraction.
g) Deep-Sequencing at CAPNS1 Locus
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)-CGAGACTTCACGGTTTCGCC-3′ (endogenous locus specific reverse sequence) (SEQ ID NO: 200). 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.
3—In Immortalized 293H Cell Line
Wild-type 293H cells were transfected by SC_CAPNS1 or Trex-SC_CAPNS1 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_CAPNS1 stimulated TM up to 12.4% (
4—In Primary Detroit Cell Line
Wild type Detroit551 cells were transfected by SC_CAPNS1 or Trex-SC_CAPNS1 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_CAPNS1 led to 1.1% of TM whereas transfection with the fusion Trex-CAPNS1 stimulated TM up to 12.5% (
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. Hence, after the transfection of a meganuclease into the cell, the measurement of the mutagenesis frequency at the meganuclease locus is a way to assess the meganuclease activity. Meganucleases derived from the I-CreI protein have been shown to induce mutagenesis at the genomic site, for which they have been designed (Munoz et al., 2011).
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.
To test this hypothesis, 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 SEQ ID 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-CreI variants have been fused. It recognizes a 22 bp DNA sequence (5′-TGCCCCAGGGTGAGAAAGTCCA-3′: GS_CHO.1 target, SEQ ID NO: 155) located in the first exon of Cricetulus griseus glutamine synthetase gene. Moreover, two different meganucleases SC_RAG1 (pCLS2222, SEQ ID NO: 156 encoding SC_RAG1 of SEQ ID NO: 58), and SC_CAPNS1 (pCLS6163, SEQ ID NO: 158 encoding SC_CAPNS1 of SEQ ID NO: 192) were co-transfected with or without Tdt expression plasmid (pCLS3841, SEQ ID NO: 202) and the effects on meganuclease-induced mutagenesis at the endogenous loci were analyzed by deep-sequencing.
d) Cellular Model to Monitor Meganuclease-Induced Mutagenesis
The plasmid pCLS6810 (SEQ ID 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. Since by itself GFP reporter gene is inactive due to a frame-shift introduced by GS recognition sites, creation of a DNA double strand break (DSB) by SC_GS meganuclease followed by a mutagenic DSB repair event by NHEJ can lead to restoration of GFP gene expression in frame with the ATG start codon. These sequences were placed in a plasmid used to target the final construct at the RAG1 locus in 293H cell line using the hsRAG1 Integration Matrix CMV Neo from cGPS® Custom Human Full Kit DD (Cellectis Bioresearch). Using this kit, a stable cell line containing a single copy of the transgene at the RAG1 locus was obtained. Thus, after transfection of this cell line by the SC_GS meganuclease and with or without a plasmid encoding Tdt (pCLS3841, SEQ ID NO: 202), the percentage of GFP positive cells is directly correlated to the mutagenesis frequency induced by the transfected specie.
e) Transfection on Cellular Model Monitoring Meganuclease-Induced Mutagenesis
One million of cells were seeded one day prior to transfection. Cells were co-transfected either with 1 μg of SC_GS encoding vector (pCLS2690, SEQ ID NO: 153) and with 0, 4, 6 or 9 μg of plasmid encoding Tdt (pCLS3841 SEQ ID NO: 202) or with 3 μg of SC_GS encoding plasmid with 0 or 2 μg of Tdt encoding vector in 5 or 10 μg of total DNA, respectively, by complementation with a pUC vector (pCLS0002, SEQ ID NO: 191) using 25 μl of lipofectamine (Invitrogen) according to the manufacturer's instructions. Three days following transfection, cells were harvested for flow cytometry analysis using Guava instrumentation. Conditions corresponding to 3 μg of SC_GS encoding vector with 0 or 2 μg of Tdt encoding plasmid were harvested for genomic DNA extraction. PCR for deep-sequencing 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)-3′ (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.
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 μg of SC_RAG1 encoding vector (pCLS2222, SEQ ID NO: 156) with 0.5, 1 and 2 μg or with 1, 3 and 7 μg of plasmid encoding Tdt (pCLS3841, SEQ ID NO: 202) in, respectively, 5 or 10 μg of total DNA by complementation with a pUC vector (pCLS0002, SEQ ID NO: 191) using 25 μl of lipofectamine (Invitrogen) according to the manufacturer's instructions. Three μg of SC_CAPNS1 encoding vector (pCLS6163 SEQ ID NO: 158) were co-transfected with 2 μg of empty vector plasmid (pCLS0002, SEQ ID NO: 191) or Tdt encoding plasmid (pCSL3841, SEQ ID NO: 202) using 25 μl of lipofectamine (Invitrogen) according to the manufacturer's instructions. Seven days following transfection, cells were harvested for genomic DNA extraction. 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 RAG 1: GGCAAAGATGAATCAAAGATTCTGTCC-3′ (SEQ ID NO: 162) and for CAPNS1: CGAGTCAGGGCGGGATTAAG-3′(SEQ ID NO: 164) and the reverse primer 5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-(reverse adaptor sequence) (SEQ ID NO: 6)-(endogenous locus specific reverse sequence for RAG1: -GATCTCACCCGGAACAGCTTAAATTTC-3′ (SEQ ID NO: 165) and for CAPNS1: -CGAGACTTCACGGTTTCGCC-3′ (SEQ ID NO: 167). 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.
1—On Cellular Model Measuring Meganuclease-Induced Mutagenic NHEJ Repair
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 96 h 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). Without the presence of Tdt, 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 (
2—At Endogenous RAG1 Locus
Wild type 293H cells were transfected by SC_RAG1 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. Two different transfections were performed with 3 μg of SC_RAG1 encoding vector (pCLS2222, SEQ ID NO: 156) with either 0.5, 1 and 2 μg or 1, 3 and 7 μg of plasmid expressing Tdt (pCLS3841, SEQ ID NO: 202) in 5 or 10 μg of total DNA by complementation with an empty vector (pCLS0002, SEQ ID NO: 191) respectively. In absence of Tdt expressing vector, the targeted mutagenesis (TM) varies between 0.5 and 0.8%. When Tdt was present TM was stimulated up to 1.6% (
attgttctcaggcgtacctcagccagc
attgttctcag
gtac
atctcagccagc
attgttctcag
gtac
ccctcagccagc
attgttctcag
gtac
gggctcagccagc
attgttctcagggcgtacctcagccagc
attgttctcag
gtac
agtctcagccagc
attgttctcag
gtac
ggggctcagccag
attgttctcagacccgtacctcagccagc
attgttctcagcctcgtacctcagccagc
attgttctcagcttcgtacctcagccagc
attgttctcag
gtac
tggactcagccagc
attgttctcag
gtac
agggctcagccagc
attgttctcag
gtac
gggaactcagccagc
attgttctcag
gtac
gaaggctcagccagc
attgttctcagttcctgtacctcagccagc
attgttctcag
gtac
gggtggctcagccagc
attgttctcag
gtac
tggttactcagccagc
attgttctcag
gtac
ccatacctcagccagc
attgttctcaggttacctgtacctcagccagc
attgttctcag
gtac
aagggggctcagccagc
attgttctcagggccgcccgtacctcagccagc
3—At Endogenous CAPNS1 Locus
Wild type 293H cells were transfected with 3 μg of plasmid encoding SC_CAPNS1 meganuclease (pCLS6163, SEQ ID NO: 158) with 0 or 2 μg of Tdt encoding plasmid (pCLS3841, SEQ ID NO: 202) (in 5 μg of total DNA) in order to determine Tdt expression effect at another endogenous locus. In absence of Tdt expressing vector, the targeted mutagenesis (TM) was 7.4%. When Tdt was present TM was stimulated up to 13.9% (
cagggccgcg
gtg
c
gcagtgtccgac
cagggccgcgccgtgcagtgtccgac
cagggccgcggcgtgcagtgtccgac
cagggccgcg
gtgc
acagtgtccgac
cagggccgcggccgtgcagtgtccgac
cagggccgcg
gtgc
tgcagtgtccgac
cagggccgcgcctgtgcagtgtccgac
cagggccgcgttctgtgcagtgtccgac
cagggccgcg
gtgc
gggcagtgtccgac
cagggccgcggtccgtgcagtgtccgac
cagggccgcg
gtgc
aggcagtgtccgac
cagggccgcg
gtgc
aaagcagtgtccgac
cagggccgcggtgcagtgcagtgtccgac
cagggccgcggtgcggtgcagtgtccgac
cagggccgcgtgtctgtgcagtgtccgac
cagggccgcg
gtgc
aaggtcagtgtccgac
cagggccgcggtgcccgtgcagtgtccgac
cagggccgcggtgcaagtgcagtgtccgac
cagggccgcg
gtgc
aagcagggagtgtccgac
Co-transfection of Tdt (SEQ ID NO: 201) with meganuclease encoding plasmids was shown to increase the rate of mutagenesis induced by meganucleases. However, this strategy implies the presence of two plasmids within the cell at the same time. Moreover it would be of benefit to target the Tdt activity at the newly created DSB upon Meganuclease's cleavage. Thus, 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_CAPNS1 (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.
h) Making of SC_MN/Tdt Fusion Proteins
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). This yields to two protein constructs named respectively SC_MN-Tdt or Tdt-SC_MN. All SC_MN were initially cloned into the AscI/XhoI restriction sites of the pCLS 1853 (
Then, after a gel purification of the two PCR fragments, 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 AscI and XhoI and ligated into the pCLS 1853 vector (SEQ ID NO: 175) pre-digested with these same enzymes.
To investigate the impact on mutagenesis frequency induced by two nucleases targeting two nearby sites, co-transfection with two engineered nucleases targeting DNA sequences within the RAG1 gene was performed. Nucleases consist of an engineered meganuclease (N1) (SC_RAG of SEQ ID NO: 216) encoded by pCLS2222 (SEQ ID NO: 156) cleaving the DNA sequence 5′-TTGTTCTCAGGTACCTCAGCCAGC-3′ (T1) (SEQ ID NO: 207) and a TALEN (N2) [SEQ ID NO: 209-210 respectively encoded by pCLS8964 (SEQ ID NO: 211) and pCLS8965 (SEQ ID NO: 212)] targeting DNA sequence 5′-TATATTTAAGCACTTATATGTGTGTAACAGGTATAAGTAACCATAAACA-3′ (T2) (SEQ ID NO: 208). These two recognition sites are separated by 173 bp.
Cells Transfection
The human 293H cells (ATCC) were plated at a density of 1.2×106 cells per 10 cm dish in complete medium [DMEM supplemented with 2 mM L-glutamine, penicillin (100 IU/ml), streptomycin (100 μg/ml), amphotericin B (Fongizone: 0.25 μg/ml, Invitrogen-Life Science) and 10% FBS]. The next day, cells were transfected with 10 μg of total DNA containing both nucleases expressing plasmids (3 μg of N1 and 0.25 μg of each monomer of N2), with Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. As control, each nuclease was expressed alone. For all conditions, samples were completed at 10 μg of total DNA with an empty vector pCLS0003 (SEQ ID NO: 213).
Two days after, cells were collected and genomic extraction was performed. The mutagenesis frequency was determined by Deep sequencing. The T1 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)
were used. 5,000 to 10,000 sequences per sample were analyzed.
The rate of mutations induced by the nucleases N1 and N2 at the targets T1 and T2 was measured by deep sequencing. Results are presented in Table 11. 0.63% of PCR fragments carried a mutation in samples corresponding to cells transfected with the N1 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 T1 target and up to 2.48% on T2 target when the cells were transfected with plasmids expressing both N1 and N2, showing that the presence of two nucleases targeting two nearby sequences stimulates up to about two folds the frequency of mutagenesis. Interestingly, within the samples transfected with only one nuclease plasmid, the majority of deletions observed are small deletions. In contrast, within the sample co-transfected with both nucleases expressing plasmids a large fraction of deletions are large deletions (>197 bp), corresponding to the intervening sequences between the two cleavage sites.
Thus, it was observed that co-transfection of two nucleases targeting two nearby sequences separated by 173 bp stimulates the mutagenesis frequency.
This application claims priority to U.S. Provisional Applications U.S. 61/407,339, filed Oct. 27, 2010, U.S. 61/472,072, filed Apr. 5, 2011 and U.S. 61/505,783, filed Jul. 8, 2011; each of which is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2011/058133 | 10/27/2011 | WO | 00 | 7/24/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61407339 | Oct 2010 | US | |
| 61472072 | Apr 2011 | US | |
| 61505783 | Jul 2011 | US |
