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Nucleobase editors and uses thereof

A technology of amino acids and nucleotides, applied in nucleic acid vectors, genetic engineering, chemical instruments and methods, etc.

Pending Publication Date: 2018-09-07
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] A disadvantage of current techniques is that both NHEJ and HDR are stochastic processes, which often result in modest gene editing efficiencies as well as unwanted genetic changes that can compete with desired changes 8

Method used

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  • Nucleobase editors and uses thereof
  • Nucleobase editors and uses thereof
  • Nucleobase editors and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0456] Embodiment 1: Cas9 deaminase fusion protein

[0457] A number of Cas9:deaminase fusion proteins were generated and the resulting fusions were characterized for deaminase activity. The following deaminases were tested:

[0458] Human AID (hAID):

[0459] MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPYLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGLLD(SEQ ID NO:607)

[0460] Human AID-DC (hAID-DC, a truncated form of hAID with 7-fold increased activity):

[0461]MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLR:(60NOGLHENSVRLSRQLR)

[0462] Rat APOBEC1 (rAPOBEC1):

[0463] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYP...

Embodiment 2

[0499] Example 2: Deamination of DNA Target Sequences

[0500] Exemplary deamination targets. The dCas9:deaminase fusion proteins described herein can be delivered to cells in vitro or ex vivo or to a subject in vivo, and can be used to achieve C when the target nucleotide is in position 3-11 with respect to the PAM. to T or G to A conversion. Exemplary deamination targets include, but are not limited to, the following: CCR5 truncation: Any codon encoding Q93, Q102, Q186, R225, W86, or Q261 of CCR5 can be deaminated to generate a stop codon, which results in CCR5 The non-functional truncation of has application in HIV treatment. APOE4 mutation: The mutant codons encoding the C11R and C57R mutant APOE4 proteins can be deaminated to restore wild-type amino acids, which has applications in the treatment of Alzheimer's disease. eGFP truncation: Any codon encoding Q158, Q184, Q185 can be deaminated to generate a stop codon, or the codon encoding M1 can be deaminated to encode I,...

Embodiment 3

[0507] Example 3: Uracil Glycosylase Inhibitor Fusions Improve Deamination Efficiency

[0508] Directly programmable nucleobase editing efficiency in mammalian cells via dCas9:deaminase fusion proteins can be significantly improved by fusing uracil glycosylase inhibitor (UGI) to dCas9:deaminase fusion proteins.

[0509] Figure 9 In vitro C→T editing efficiency in human HEK293 cells using rAPOBEC1-XTEN-dCas9 is shown:

[0510]

[0511]

[0512] The sequence of the protospacer is as follows:

[0513]

[0514] *PAMs are boxed, C residues within the target window (positions 3-11) are numbered and bold.

[0515] Figure 10 demonstrated that the efficiency of C→T editing of the same protospacer sequence in HEK293T cells was greatly enhanced when the UGI domain was fused to the rAPOBEC1:dCas9 fusion protein.

[0516]

[0517]

[0518] From the sequencing of both strands of the target sequence it was shown that Figure 9 and Figure 10 percentage in . Since only...

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Abstract

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome ofa cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 andnucleic acid editing proteins or domains, are provided.

Description

[0001] governmental support [0002] This invention was made under grant number R01EB022376 (formerly R01GM065400), supported by the National Institutes of Health, under training grant numbers F32 GM 112366-2 and F32GM 106601-2, and supported by the National Institutes of Health This was done with government support under Harvard Biophysics NIH training grant T32 GM008313. The government has certain rights in this invention. [0003] related application [0004] This application is required under 35 U.S.C. §119 of U.S. Provisional Patent Applications U.S.S.N. 62 / 245,828 filed October 23, 2015, U.S.S.N. 311,763, U.S.S.N. 62 / 322,178 filed April 13, 2016, U.S.S.N. 62 / 357,352 filed June 30, 2016, U.S.S.N. 62 / 398,490, priority of U.S.S.N. 62 / 408,686 filed October 14, 2016, and U.S.S.N. 62 / 357,332 filed June 30, 2016; each of which is incorporated herein by reference. Background of the invention [0005] Targeted editing of nucleic acid sequences, such as targeted cleavage of ge...

Claims

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Application Information

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IPC IPC(8): C07K14/32C12N9/22C12N9/78C12N15/10C12N15/90
CPCC07K14/32C07K2319/00C07K2319/09C07K2319/80C12N9/22C12N9/78C12Y305/04005C12N9/2497A61P13/02A61P13/12A61P17/00A61P19/02A61P21/00A61P21/02A61P25/00A61P25/28A61P29/00A61P3/00A61P31/12A61P31/18A61P35/00A61P43/00A61P7/04A61P9/00C12N2800/80C12N2310/20C12N15/907C12N15/11
Inventor D.R.刘A.C.科摩H.A.里斯Y.金
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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