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EP Patent status and claim 1 of SIGMA ALDRICH‘s CRISPR-Cas9 Patents.

執筆用メモ
 
SIGMA ALDRICHの欧州特許(2019/02/22)
SIGMAの技術の特徴は、HR修復を使った核酸の導入と、ニッカーゼ型で切る点。
 
EP3138910B:異議申立中(口頭審理未定)

1.  A method for integrating an exogenous sequence into a chromosomal sequence of a eukaryotic cell, the method comprising:

  a) introducing into the eukaryotic cell

      (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal,

         wherein the at least one RNA-guided endonuclease is a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) type II system protein and the CRISPR/Cas type II system protein is a Cas9 protein,

      (ii) at least one guide RNA or DNA encoding at least one guide RNA, and

      (iii) at least one donor polynucleotide comprising the exogenous sequence; and

  b) culturing the eukaryotic cell such that the guide RNA guides the RNA-guided endonuclease to a target site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break, and the double-stranded break is repaired by a DNA repair process such that the exogenous sequence is integrated into the chromosomal sequence,

   wherein the method does not comprise a process for modifying the germ line genetic identity of a human being and,

   wherein the method does not comprise a method for treatment of the human or animal body.

 

・HRの修復を使った核酸の導入

 

EP3138911B:異議申立中(始まったばっかり、口頭審理未定)

1.  A method for modifying a chromosomal sequence in a eukaryotic cell by integrating a donor sequence, the method comprising:

  a) introducing into the eukaryotic cell

      (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal,

       wherein the at least one RNA-guided endonuclease is a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) type II system protein and the CRISPR/Cas type II system protein is a Cas9 protein,

      (ii) at least one guide RNA or DNA encoding at least one guide RNA, and

      (iii) a donor polynucleotide comprising the donor sequence; and

  b) culturing the eukaryotic cell such that each guide RNA guides an RNA-guided endonuclease to a target site in the chromosomal sequence, the RNA-guided endonuclease introduces a double-stranded break at the target site, and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified by insertion or substitution of the donor sequence into the chromosomal sequence,

   wherein the target site in the chromosomal sequence is immediately followed by a protospacer adjacent motif (PAM),

   the method does not comprise a process for modifying the germ line genetic identity of a human being and,

   wherein the method does not comprise a method for treatment of the human or animal body by surgery or therapy.

 

・HRの修復を使った核酸の導入

 

EP3138912B:異議申立中(始まったばっかり、口頭審理未定)

1.  A method for modifying a chromosomal sequence in a eukaryotic cell, the method comprising:

  a) introducing into the eukaryotic cell two RNA-guided nickase systems or nucleic acid encoding said systems, and, optionally, a donor polynucleotide,

     wherein each RNA-guided nickase system comprises

      (i) a RNA-guided endonuclease that is modified to cleave one strand of a double-stranded sequence; and

      (ii) a guide RNA comprising a first region having complementarity to a target site in the chromosomal sequence and a second region that interacts with the RNA-guided endonuclease,

          wherein each target site is immediately followed by a protospacer adjacent motif (PAM), and the target sites of the two RNA-guided endonucleases are on opposite strands of the chromosomal sequence; and

  b) culturing the eukaryotic cell such that the two RNA-guided endonucleases cleave opposite strands of the chromosomal sequence in close enough proximity to introduce a double-stranded break in the chromosomal sequence, and repair of the double-stranded break by a DNA repair process leads to modification of the chromosomal sequence,

   wherein the method does not comprise a process for modifying the germ line genetic identity of a human being and,

   wherein the method does not comprise a method for treatment of the human or animal body by surgery or therapy.

 

・HRの修復を使った核酸の導入×ニッカーゼ型

・ニッカーゼは、Cas9に限定されず、RNAガイドエンドヌクレアーゼ(他のCasを含む)