Chimeric genome engineering molecules and methods
Inventors
Park, Jongjin • Yoon, Ji Young • Choi, Sunmee • Park, Mijin • PARK, Slki • PARK, Aiden Y. • Lee, Jung Hyuk • LIM, Junghak • Kim, Dong Wook • Choe, Sunghwa
Assignees
SNU R&DB Foundation • G and Flas Life Sciences Ltd • Gflas Life Sciences Inc
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Publication Number
US-11293019-B2
Publication Date
2022-04-05
Expiration Date
Abstract
The present disclosure provides compositions and methods for increasing mutation efficiency and homologous recombination rates of site-specific endonucleases. The compositions and methods comprise a chimeric polypeptide comprising a site-specific endonuclease or a domain thereof and a functional moiety. The current inventions relate to functional enhancement of the CRISPR-Cas enzymes. Disclosed herein include possible variants and their intended improvements.
Core Innovation
The invention relates to chimeric CRISPR genome-editing polypeptides in which a programmable, sequence-specific endonuclease is combined with DNA modifying functions. A chimeric polypeptide is formed by an in-frame fusion of a site-specific endonuclease selected from Cas9 and Cpf1 with DNA modifying enzymes and/or DNA-binding proteins and/or TdT to increase mutation efficiency, including indel/NHEJ and homologous recombination (HDR). The programmable endonuclease binds a locus to generate a modified locus in a genomic sample.
The DNA modifying enzymes include RecE, RecJ, RecBCD, mungbean nuclease, ExoI, ExoIII, and ExoVII. The DMEs are proposed to generate processed ends such as 3′-OH overhangs and resection products, thereby biasing repair toward error-prone NHEJ and toward HDR. The document also includes embodiments in which DNA-binding proteins, including SSB and DSB/DBP, are fused to provide DNA binding in the chimeric construct.
The chimeric designs also include fusions of TdT to the sequence-specific endonuclease, and nucleic acids encoding the fusions. The invention targets a single locus for mutagenesis by contacting a genomic sample comprising the locus with a chimeric polypeptide and a programmable endonuclease, generating a modified locus and modifying a unique segment of the genomic sample. The mutagenesis, insertion, or deletion is limited to at most 40 bases.
Claims Coverage
The partial content provides independent claims that cover a method of targeting a single locus for mutagenesis using a chimeric polypeptide with an exonuclease DME and a programmable CRISPR endonuclease, and classes of fusion polypeptides that are in-frame fusions of a sequence-specific endonuclease with either a DNA binding protein (DBP) or TdT.
Targeting a single genomic locus using an in-frame exonuclease-CRISPR fusion
Selecting a locus for mutagenesis; contacting a genomic sample comprising the locus to a chimeric polypeptide comprising an exonuclease selected from RecE, RecJ, RecBCD, Mungbean nuclease, ExoIII, ExoVII, and ExoI, and a programmable endonuclease that binds to the locus selected from Cas9 and Cpf1, thereby generating a modified locus that mutagenizes, inserts, or deletes at most 40 bases.
In-frame fusion of a sequence-specific endonuclease to a DNA binding protein
A polypeptide comprising a sequence-specific endonuclease fused in frame to a DNA binding protein, wherein the DNA binding protein binds single-stranded DNA or double-stranded DNA, and the sequence-specific endonuclease is selected from Cas9 and Cpf1.
In-frame fusion of a sequence-specific endonuclease to TdT
A polypeptide comprising a sequence-specific endonuclease fused in frame to a terminal deoxyribonucleotidyl transferase, wherein the sequence-specific endonuclease is selected from Cas9 and Cpf1.
The claim coverage centers on in-frame chimeric CRISPR polypeptides combining a Cas9 or Cpf1 endonuclease with additional DNA-handling functions—either exonuclease DMEs for locus-specific mutagenesis limited to at most 40 bases, a DNA binding protein that binds single- or double-stranded DNA, or TdT as a fused terminal deoxyribonucleotidyl transferase.
Stated Advantages
Increased on-target indel and HDR efficiencies.
Comparable or reduced off-target mutagenesis versus the unfused corresponding sequence-specific endonuclease.
Enhanced genome editing by improving deletion/indel profiles and editing efficiency.
Reduction of CRISPR off-target effects.
In some described comparisons, certain exonuclease fusions are reported to improve on-target mutagenesis while having the same or lower off-target mutagenesis when unfused.
Documented Applications
Editing of a single locus for mutagenesis in a genomic sample, including applications described in human cells and plants.
Characterization and assessment of off-target effects using whole genome sequencing/amplicon deep sequencing and off-target detection via TdT labeling with non-canonical bases such as BrdU.
Targeted genome modification at a plant genome modification context, including plant cell recovery and examples involving Arabidopsis/lettuce protoplast regeneration.
Off-target evaluation using targeted assays and deep sequencing frameworks, including reported evaluation of off-target loci such as CCR5 and PD-1.
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