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Crispr-based genome modification and regulation

a genome modification and regulation technology, applied in the direction of peptides/protein ingredients, enzyme stabilisation, peptides, etc., can solve the problems of high cost and time-consuming preparation of custom-designed nucleases

Inactive Publication Date: 2016-10-13
SIGMA ALDRICH CO LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present patent provides an isolated RNA-guided endonuclease that can be used to modify chromosomal sequences in eukaryotic cells or embryos. The endonuclease has a nucleic acid sequence that includes a nuclear localization signal, a nuclease domain, and a domain that interacts with a guide RNA to target a specific nucleotide sequence for cleavage. The endonuclease can be introduced into cells or embryos along with a guide RNA and a donor polynucleotide, and the guide RNA can direct the endonuclease to a specific site in the chromosome to effect a double-stranded break. The endonuclease can also be fused with other proteins, such as a CRISPR / Cas-like protein, to create a fusion protein with additional functions. The technical effects of this patent include the ability to efficiently modify chromosomal sequences and regulate expression of those sequences in cells or embryos.

Problems solved by technology

Thus, these custom designed nucleases tend to be costly and time-consuming to prepare.

Method used

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  • Crispr-based genome modification and regulation

Examples

Experimental program
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Effect test

example 1

Modification of Cas9 Gene for Mammalian Expression

[0161]A Cas9 gene from Streptococcus pyogenes strain MGAS15252 (Accession number YP_005388840.1) was optimized with Homo sapiens codon preference to enhance its translation in mammalian cells. The Cas9 gene also was modified by adding a nuclear localization signal PKKKRKV (SEQ ID NO:1) at the C terminus for targeting the protein into the nuclei of mammalian cells. Table 1 presents the modified Cas9 amino acid sequence, with the nuclear localization sequence underlined. Table 2 presents the codon optimized, modified Cas9 DNA sequence.

TABLE 1Modified Cas9 Amino Acid Sequence(SEQ ID NO: 9)MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ...

example 2

Targeting Cas9

[0163]The adeno-associated virus integration site 1 (AAVS1) locus was used as a target for Cas9-mediated human genome modification. The human AAVS1 locus is located in intron 1 (4427 bp) of protein phosphatase 1, regulatory subunit 12C (PPP1R12C). Table 3 presents the first exon (shaded gray) and the first intron of PPP1R12C. The underlined sequence within the intron is the targeted modification site (i.e., AAVS1 locus).

TABLE 3First Exon and Intron of PPP1R12C (5′-3′)CAGGTCCACCCTCTGCTGCGCCACCTGGGGCATCCTCCTTCCCCGTTGCCAGTCTCGATCCGCCCCGTCGTTCCTGGCCCTGGGCTTTGCCACCCTATGCTGACACCCCGTCCCAGTCCCCCTTACCATTCCCCTTCGACCACCCCACTTCCGAATTGGAGCCGCTTCAACTGGCCCTGGGCTTAGCCACTCTGTGCTGACCACTCTGCCCCAGGCCTCCTTACCATTCCCCTTCGACCTACTCTCTTCCGCATTGGAGTCGCTTTAACTGGCCCTGGCTTTGGCAGCCTGTGCTGACCCATGCAGTCCTCCTTACCATCCCTCCCTCGACTTCCCCTCTTCCGATGTTGAGCCCCTCCAGCCGGTCCTGGACTTTGTCTCCTTCCCTGCCCTGCCCTCTCCTGAACCTGAGCCAGCTCCCATAGCTCAGTCTGGTCTATCTGCCTGGCCCTGGCCATTGTCACTTTGCGCTGCCCTCCTCTCGCCCCCGAGTGCCCTTGCTGTGCCGCCG...

example 3

Preparation of Donor Polynucleotide to Monitor Genome Modification

[0165]Targeted integration of a GFP protein into the N terminus of PPP1R12C was used to monitor Cas9-mediated genome modification. To mediate integration by homologous recombination a donor polynucleotide was prepared. The AAVS1-GFP DNA donor contained a 5′ (1185 bp) AAVS1 locus homologous arm, an RNA splicing receptor, a turbo GFP coding sequence, a 3′ transcription terminator, and a 3′ (1217 bp) AAVS1 locus homologous arm. Table 5 presents the sequences of the RNA splicing receptor and the GFP coding sequence followed by the 3′ transcription terminator. Plasmid DNA was prepared by using GenElute Endotoxin-Free Plasmid Maxiprep Kit (Sigma).

TABLE 5Sequences in the AAVS1-GFP DNA donor sequenceSEQ ID5′-3′ SequenceNO:RNA splicingCTGACCTCTTCTCTTCCTCCCACAG15receptorGFP codingGCCACCATGGACTACAAAGACGATG16sequence andACGACAAGGTCGACTCTAGAGCTGCtranscriptionAGAGAGCGACGAGAGCGGCCTGCCCterminatorGCCATGGAGATCGAGTGCCGCATCACCGGCACCCTGAA...

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Abstract

The present invention provides RNA-guided endonucleases, which are engineered for expression in eukaryotic cells or embryos, and methods of using the RNA-guided endonuclease for targeted genome modification in eukaryotic cells or embryos. Also provided are fusion proteins, wherein each fusion protein comprises a CRISPR / Cas-like protein or fragment thereof and an effector domain. The effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Also provided are methods for using the fusion proteins to modify a chromosomal sequence or regulate expression of a chromosomal sequence.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. patent application Ser. No. 14 / 649,777, filed Jun. 4, 2015, which is a U.S. National Stage application of PCT International Application No. PCT / US2013 / 073307, filed Dec. 5, 2013, which claims priority to U.S. Provisional Application Ser. No. 61 / 734,256, filed Dec. 6, 2012; U.S. Provisional Application Ser. No. 61 / 758,624, filed Jan. 30, 2013; U.S. Provisional Application Ser. No. 61 / 761,046, filed Feb. 5, 2013; and U.S. Provisional Application Ser. No. 61 / 794,422, filed Mar. 15, 2013, the disclosure of each is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]The present disclosure relates targeted genome modification. In particular, the disclosure relates to RNA-guided endonucleases or fusion proteins comprising CRISPR / Cas-like protein and methods of using said proteins to modify or regulate targeted chromosomal sequences.BACKGROUND OF THE INVENTION[0003]Targ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/90C12N15/67C12N7/00C12N15/11C07K7/06C12N15/86C12N9/22
CPCC12N15/907C12N15/86C12N15/67C12N9/22C12Y301/00C12N2750/14143C07K7/06C12N7/00C12N2310/3513C07K2319/09C12N15/11A61K38/00C12N15/102C12N15/63C12Y301/21004C12N9/96Y02A50/30C12N2800/22C12N15/85C12N2800/80C07K2319/81C07K2319/10C07K14/463C12N2310/20A61K9/0048A61P27/10
Inventor CHEN, FUQIANGDAVIS, GREGORY D.KANG, QIAOHUAKNIGHT, SCOTT W.
Owner SIGMA ALDRICH CO LLC
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