Programmable cas9-recombinase fusion proteins and uses thereof

A fusion protein and recombinase technology, applied in the fields of insertion, inversion, and deletion, which can solve problems such as limited applications

Pending Publication Date: 2019-05-24
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, such situations can lead to many random and unpredictable events, limiting potential applications

Method used

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  • Programmable cas9-recombinase fusion proteins and uses thereof
  • Programmable cas9-recombinase fusion proteins and uses thereof
  • Programmable cas9-recombinase fusion proteins and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0225] Example 1: Programmable Cas9-serine recombinase fusion protein that functions on DNA sequences in mammalian cells

[0226] Materials and methods

[0227] Oligonucleotides and PCR

[0228] All oligonucleotides were purchased from Integrated DNA Technologies (IDT, Coralville, CA) and are listed in Tables 1-5. Enzymes were purchased from New England Biolabs (Ipswich, MA) unless otherwise stated. Plasmid SafeATP-dependent DNase was purchased from Epicenter (Madison, WI). All assembled vectors were transformed into One Shot Mach1-T1 phage-resistant chemically competent cells (Fisher Scientific, Waltham, MA). Unless otherwise stated, all PCR reactions were performed using the Q5 Hot Start High-Fidelity 2X Master Mix. Phusion polymerase is used for circular polymerase extension clone (CPEC) assembly.

[0229] Table 1: Oligonucleotides used for gRNA construction

[0230]

[0231] Table 2: Oligonucleotides and gBlocks used for reporter construction

[0232]

[023...

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Abstract

Some aspects of this disclosure provide a fusion protein comprising a guide nucleotide sequence-programmable DNA binding protein domain (e.g., a nuclease-inactive variant of Cas9 such as dCas9), an optional linker, and a recombinase catalytic domain (e.g., a tyrosine recombinase catalytic domain or a serine recombinase catalytic domain such as a Gin recombinase catalytic domain). This fusion protein can recombine DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. The instant disclosure represents a step toward programmable, scarless genome editing inunmodified cells that is independent of endogenous cellular machinery or cell state.

Description

[0001] related application [0002] Pursuant to 35 U.S.C. § 119(e), this application claims priority to U.S. Provisional Patent Applications U.S.S.N. 62 / 372,755 filed on August 9, 2016 and U.S. Provisional Patent Application U.S.S.N. Each of which is incorporated herein by reference. [0003] Government funding [0004] This invention was made with government support under grant numbers R01EB022376 and R35GM118062 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the invention [0005] Efficient, programmable, and site-specific homologous recombination remains a long-standing goal of genetics and genome editing. Early attempts to direct recombination to a locus of interest relied on transfecting donor DNA with long flanking sequences homologous to the target locus. This strategy is hampered by very low efficiency and thus requires stringent selection to identify integrants. Recent efforts have exploited the ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/11A61K48/00
CPCA61K48/00C12N9/22C12N15/113C07K2319/09C07K2319/20C07K2319/43C12N2320/11C12N2310/20C07K4/00C07K2319/21C12N9/1241C12Y207/07
Inventor D.R.刘B.柴金德J.L.贝森
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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