Repeat variable diresidues for targeting nucleotides
Inventors
Duchateau, Philippe • Juillerat, Alexandre • Bertonati, Claudia
Assignees
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Publication Number
US-11778993-B2
Publication Date
2023-10-10
Expiration Date
Abstract
The present invention relates to polypeptides and more particularly to Transcription Activator-Like Effector derived proteins that allow to efficiently target and/or process nucleic acids. The present invention also concerns methods to use these proteins. The present invention also relates to vectors, compositions and kits in which RVD domains and Transcription Activator-Like Effector (TALE) proteins of the present invention are used.
Core Innovation
The disclosed invention relates to TALE-derived proteins engineered with Repeat Variable Diresidue (RVD) regions that bind to one specific nucleotide within a nucleic-acid target sequence. The TALE domain includes a plurality of TALE repeat sequences, each repeat comprising an RVD responsible for nucleotide-specific binding to a chromosome nucleic acid target sequence.
A key element of the invention is engineering the TALE domain together with an endonuclease domain to cleave genetic material within the nucleic-acid target sequence. Upon contacting the engineered protein with the nucleic-acid target sequence in a cell, the engineered protein binds and cleaves the chromosome to create a double strand break. The resulting double strand break is repaired by the cell through non-homologous end joining (NHEJ) to produce a genetic modification.
The invention further provides a framework for inducing homologous gene targeting using the same TALE-endonuclease binding and cleavage concept. A chimeric protein comprising a TALE domain and an endonuclease domain creates a double strand break at the chromosomal nucleic acid target sequence, and an exogenous nucleic acid homologous to at least a portion of the target sequence is introduced. Homologous recombination occurs between the exogenous nucleic acid and the nucleic-acid target sequence to process genetic material within or adjacent to the specific nucleic acid target sequence.
Claims Coverage
The independent claim set includes two independent methods, one covering NHEJ-mediated genetic modification and the other covering homologous recombination-mediated homologous gene targeting. Across these independent claims, the coverage centers on a TALE domain configured with specified RVD selections for nucleotide recognition, coupled to an endonuclease domain that creates a double strand break, followed by repair via NHEJ or recombination using an exogenous homologous nucleic acid.
TALE domain with specified RVDs for nucleotide-specific binding
A TALE domain comprising a plurality of TALE repeat sequences each comprising an RVD responsible for binding to one specific nucleotide in a nucleic acid target sequence present on a chromosome of a mammalian cell, wherein one or more RVD are selected from PI, DL, FL, GL, IL, KL, LL, YL, MM, WY, PV, SW, XF for recognizing A (X represents one amino acid residue selected from A, G, V, L, I, M, S, T, C, P, D, E, F, Y, W, Q, N, and K), RE for recognizing C, and ER, FR, GR, LR, QR, VR for recognizing G.
TALE-endonuclease cleavage to create a double strand break
An endonuclease domain to cleave genetic material within the nucleic acid target sequence, wherein contacting the engineered protein with the nucleic acid target sequence results in binding and cleavage of the chromosome within the nucleic acid target sequence to create a double strand break.
NHEJ repair to yield genetic modification
The double strand break is repaired by the cell through non-homologous end joining (NHEJ) resulting in a genetic modification in the chromosome.
Exogenous homologous nucleic acid for homologous gene targeting
Introducing into the cell an exogenous nucleic acid comprising a sequence homologous to at least a portion of the nucleic acid target sequence, wherein homologous recombination occurs between the exogenous nucleic acid and the nucleic acid target sequence to process genetic material within or adjacent to the specific nucleic acid target sequence.
Overall, the claims cover engineering a TALE domain with specified RVD selections for nucleotide-specific binding, fusing the TALE domain to an endonuclease domain to generate a double strand break, and then driving the genetic outcome either via NHEJ to produce a genetic modification or via homologous recombination using an exogenous homologous nucleic acid.
Stated Advantages
Documented Applications
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