JPWO2020264016A5 - - Google Patents
Download PDFInfo
- Publication number
- JPWO2020264016A5 JPWO2020264016A5 JP2021576666A JP2021576666A JPWO2020264016A5 JP WO2020264016 A5 JPWO2020264016 A5 JP WO2020264016A5 JP 2021576666 A JP2021576666 A JP 2021576666A JP 2021576666 A JP2021576666 A JP 2021576666A JP WO2020264016 A5 JPWO2020264016 A5 JP WO2020264016A5
- Authority
- JP
- Japan
- Prior art keywords
- protein
- sequence
- stranded dna
- exonuclease
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Claims (49)
ゲノム編集分子とHDR促進剤とを真核細胞に提供することであって、ゲノム編集分子が、(i)標的編集部位中のDNA配列を切断する少なくとも1つの配列特異的エンドヌクレアーゼ又は配列特異的エンドヌクレアーゼをコードする少なくとも1つのポリヌクレオチドと;(ii)標的編集部位との相同性を有するドナー鋳型DNA分子とを含み;及びHDR促進剤が、一本鎖DNAアニーリングタンパク質(SSAP)と、二本鎖DNA基質を一本鎖DNA産物へと少なくとも部分的に変換することのできるエキソヌクレアーゼと、一本鎖DNA結合タンパク質(SSB)とを含み;
それによってゲノム編集分子及びHDR促進剤が、対照と比較して増加した頻度でHDRによるドナー鋳型ポリヌクレオチドを用いた真核細胞ゲノムの標的編集部位の修飾をもたらすことを含む、方法。 A method of increasing homologous recombination repair (HDR)-mediated genomic modification of a targeted editing site in a eukaryotic genome, comprising:
Providing a eukaryotic cell with a genome-editing molecule and an HDR-enhancing agent, wherein the genome- editing molecule comprises (i) at least one sequence-specific endonuclease or sequence that cleaves a DNA sequence in the target editing site; (ii) a donor template DNA molecule having homology to a target editing site; and an HDR facilitator comprising a single-stranded DNA annealing protein ( SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB);
A method, wherein the genome -editing molecule and the HDR-enhancing agent result in targeted editing site modification of a eukaryotic cell genome with a donor template polynucleotide by HDR at an increased frequency compared to a control.
(a)RNA誘導型ヌクレアーゼ又はRNA誘導型ヌクレアーゼをコードするポリヌクレオチドと、ガイドRNA又はガイドRNAをコードするポリヌクレオチドとを含む、
(b)ジンクフィンガーヌクレアーゼ(ZFN)、転写活性化因子様エフェクターヌクレアーゼ(TALエフェクターヌクレアーゼ)、アルゴノート、メガヌクレアーゼ、又は操作されたメガヌクレアーゼを含む、
(c)RNA誘導型ヌクレアーゼを含み、標的編集部位が、PAM配列と、ガイドRNAに相補的な、且つプロトスペーサー隣接モチーフ(PAM)配列に直接隣接した位置にある配列とを含む、且つ/又は
(d)切断後に標的編集部位に5’オーバーハングを提供する、
請求項1に記載の方法。 A sequence- specific endonuclease is
(a) comprising an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease and a guide RNA or a polynucleotide encoding the guide RNA;
(b) zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TAL effector nucleases), Argonautes, meganucleases, or engineered meganucleases;
(c) comprising an RNA-guided nuclease, wherein the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and located directly adjacent to the protospacer adjacent motif (PAM) sequence; and/or
(d) providing a 5' overhang to the target editing site after cleavage;
The method of claim 1.
(b)配列特異的エンドヌクレアーゼがCas9及び/又はCas12aを含み、ガイドRNA分子が、標的編集部位のDNA配列と少なくとも1塩基のミスマッチを有する、且つ/又は
(c)エキソヌクレアーゼが、T7ファージエキソヌクレアーゼ、大腸菌(E.coli)エキソヌクレアーゼIII、同等のエキソヌクレアーゼ活性を有する関連するタンパク質、又は配列番号143又は144と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、
請求項3に記載の方法。 (a) the sequence- specific endonuclease comprises at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or a combination thereof;
(b) the sequence-specific endonuclease comprises Cas9 and/or Cas12a and the guide RNA molecule has at least one base mismatch with the DNA sequence of the target editing site; and/or
(c) the exonuclease is at least 70%, 75%, 80% with T7 phage exonuclease, E. coli exonuclease III, a related protein with comparable exonuclease activity, or SEQ ID NO: 143 or 144; comprising proteins with 85%, 90%, 95%, or 99% sequence identity;
4. The method of claim 3 .
(a)環状DNAベクター、ジェミニウイルスレプリコン上に、又は線状DNA断片として提供される、且つ/又は
(b)エンドヌクレアーゼ認識配列のコピーに隣接される、
請求項1~4のいずれか一項に記載の方法。 a donor template DNA molecule
(a) provided on a circular DNA vector, a geminivirus replicon, or as a linear DNA fragment; and/or
(b) flanked by copies of the endonuclease recognition sequence;
The method according to any one of claims 1-4 .
(a)DNA鎖交換及び相同DNA分子の相補DNA鎖の塩基対合を提供する、且つ/又は
(b)RecT/Redβファミリータンパク質、ERFファミリータンパク質、又はRAD52ファミリータンパク質を含み、
(i)RecT/Redβファミリータンパク質が、Rac細菌プロファージRecTタンパク質、バクテリオファージλベータタンパク質、バクテリオファージSPP1 35タンパク質、同等のSSAP活性を有する関連するタンパク質、又は配列番号1、2、又は3と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、
(ii)ERFファミリータンパク質が、バクテリオファージP22 ERFタンパク質、機能的に関連性のあるタンパク質、又は配列番号4と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、又は
(iii)RAD52ファミリータンパク質が、サッカロミセス・セレビシエ(Saccharomyces cerevisiae)Rad52タンパク質、シゾサッカロミセス・ポンベ(Schizosaccharomyces pombe)Rad22タンパク質、クルイベロミセス・ラクチス(Kluyveromyces lactis)Rad52タンパク質、機能的に関連性のあるタンパク質、又は配列番号5、6、又は7と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、
請求項1~5のいずれか一項に記載の方法。 S SAP is
(a) provide for DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules; and/or
(b) a RecT/Redβ family protein, an ERF family protein, or a RAD52 family protein,
(i) the RecT/Redβ family protein is at least the Rac bacterial prophage RecT protein, the bacteriophage λbeta protein, the bacteriophage SPP1 35 protein, a related protein with comparable SSAP activity, or SEQ ID NO: 1, 2, or 3; including proteins with 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity;
(ii) the ERF family protein is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% the bacteriophage P22 ERF protein, a functionally related protein, or SEQ ID NO:4 contains a protein having the sequence identity of
(iii) the RAD52 family protein is Saccharomyces cerevisiae Rad52 protein, Schizosaccharomyces pombe Rad22 protein, Kluyveromyces lactis Rad52 protein , functionally related proteins or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 5, 6, or 7;
The method according to any one of claims 1-5.
(a)真核細胞;
(b)一本鎖DNAアニーリングタンパク質(SSAP)と、二本鎖DNA基質を一本鎖DNA産物へと少なくとも部分的に変換することのできるエキソヌクレアーゼと、一本鎖DNA結合タンパク質(SSB)とを含むHDR促進剤;及び
(c)標的編集部位中のDNA配列を切断する少なくとも1つの配列特異的エンドヌクレアーゼ又は配列特異的エンドヌクレアーゼをコードする少なくとも1つのポリヌクレオチドと、標的編集部位との相同性を有するドナー鋳型DNA分子とを含む1つ又は複数のゲノム編集分子
を含み;
真核細胞が有効量のHDR促進剤及び1つ又は複数のゲノム編集分子と会合し、それに接触し、及び/又はそれを含有する、システム。 A system for increasing homologous recombination repair (HDR)-mediated genomic modification of targeted editing sites in a eukaryotic genome, comprising:
(a) a eukaryotic cell;
(b) a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); and (c) at least one sequence-specific endonuclease that cleaves a DNA sequence in the target editing site or at least one polynucleotide encoding a sequence -specific endonuclease, and the target editing site comprising one or more genome editing molecules comprising a donor template DNA molecule having homology to
A system wherein eukaryotic cells are associated with, contacted with and/or contain an effective amount of an HDR facilitating agent and one or more genome editing molecules.
(a)RNA誘導型ヌクレアーゼ又はRNA誘導型ヌクレアーゼをコードするポリヌクレオチドと、ガイドRNA又はガイドRNAをコードするポリヌクレオチドとを含む、
(b)ジンクフィンガーヌクレアーゼ(ZFN)、転写活性化因子様エフェクターヌクレアーゼ(TALエフェクターヌクレアーゼ)、アルゴノート、メガヌクレアーゼ、又は操作されたメガヌクレアーゼを含む、
(c)標的編集部位にある2つの個別のDNA配列で単一のDNA鎖を切断する1つ以上の配列特異的エンドヌクレアーゼ又は配列特異的エンドヌクレアーゼ及びガイドRNAを含み、配列特異的エンドヌクレアーゼが、
(i)少なくとも1つのCas9ニッカーゼ、Cas12aニッカーゼ、Cas12i、ジンクフィンガーニッカーゼ、TALEニッカーゼ、又はこれらの組み合わせを含む、且つ/又は
(ii)Cas9及び/又はCas12aを含み、ガイドRNA分子が、標的編集部位のDNA配列と少なくとも1塩基のミスマッチを有する、
(d)RNA誘導型ヌクレアーゼを含み、標的編集部位が、PAM配列と、ガイドRNAに相補的な、且つPAM配列に直接隣接した位置にある配列とを含む、且つ/又は
(e)少なくとも1つの配列特異的エンドヌクレアーゼを含み、配列特異的エンドヌクレアーゼが、切断後に標的編集部位に5’オーバーハングを提供する、
請求項7に記載のシステム。 Genome -editing molecules
(a) comprising an RNA-guided nuclease or a polynucleotide encoding the RNA-guided nuclease and a guide RNA or a polynucleotide encoding the guide RNA;
(b) zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TAL effector nucleases), Argonautes, meganucleases, or engineered meganucleases;
(c) one or more sequence-specific endonucleases or a sequence-specific endonuclease and a guide RNA that cleave a single DNA strand at two separate DNA sequences at the target editing site, wherein the sequence-specific endonuclease is ,
(i) comprises at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or combinations thereof; and/or
(ii) comprising Cas9 and/or Cas12a, wherein the guide RNA molecule has at least one base mismatch with the DNA sequence of the target editing site;
(d) comprising an RNA-guided nuclease, wherein the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and located directly adjacent to the PAM sequence; and/or
(e) comprising at least one sequence-specific endonuclease, the sequence-specific endonuclease providing a 5' overhang to the target editing site after cleavage;
A system according to claim 7 .
(a)DNA鎖交換及び相同DNA分子の相補DNA鎖の塩基対合を提供する、
(b)RecT/Redβファミリータンパク質、ERFファミリータンパク質、又はRAD52ファミリータンパク質を含み、
(i)RecT/Redβファミリータンパク質が、Rac細菌プロファージRecTタンパク質、バクテリオファージλベータタンパク質、バクテリオファージSPP1 35タンパク質、又は同等のSSAP活性を有する関連するタンパク質を含む、
(ii)RecT/Redβファミリータンパク質が、バクテリオファージλベータタンパク質、バクテリオファージSPP1 35タンパク質、Rac細菌プロファージRecTタンパク質、又は同等のSSAP活性を有する関連するタンパク質を含む、
(iii)RecT/Redβファミリータンパク質が、配列番号1、2、又は3と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、
(iv)ERFファミリータンパク質が、バクテリオファージP22 ERFタンパク質、機能的に関連性のあるタンパク質、又は配列番号4と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、且つ/又は
(v)RAD52ファミリータンパク質が、サッカロミセス・セレビシエ(Saccharomyces cerevisiae)Rad52タンパク質、シゾサッカロミセス・ポンベ(Schizosaccharomyces pombe)Rad22タンパク質、クルイベロミセス・ラクチス(Kluyveromyces lactis)Rad52タンパク質、機能的に関連性のあるタンパク質、又は配列番号5、6、又は7と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、
請求項7~9のいずれか一項に記載のシステム。 S SAP is
(a) providing DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules;
(b) a RecT/Redβ family protein, an ERF family protein, or a RAD52 family protein,
(i) the RecT/Redβ family protein comprises the Rac bacterial prophage RecT protein, the bacteriophage λbeta protein, the bacteriophage SPP1 35 protein, or related proteins with comparable SSAP activity;
(ii) the RecT/Redβ family protein comprises a bacteriophage lambda beta protein, a bacteriophage SPP1 35 protein, a Rac bacterial prophage RecT protein, or a related protein with comparable SSAP activity;
(iii) RecT/Redβ family proteins include proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 1, 2, or 3 ,
(iv) the ERF family protein is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% a bacteriophage P22 ERF protein, a functionally related protein, or SEQ ID NO:4 and/or
(v) the RAD52 family protein is Saccharomyces cerevisiae Rad52 protein, Schizosaccharomyces pombe Rad22 protein, Kluyveromyces lactis Rad52 protein, function related proteins or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 5, 6, or 7;
A system according to any one of claims 7-9 .
(a)線状dsDNA分子を含む好ましい基質を有する、
(b)リン酸化した5’末端を含む線状dsDNA分子を含む好ましい基質を有する、
(c)5’→3’エキソヌクレアーゼ活性を有し、平滑末端型dsDNA基質、一方の鎖に内部切断を有するdsDNA基質、5’オーバーハングを有するdsDNA基質、及び/又は3’オーバーハングを有するdsDNA基質を認識することができる、
(d)3’→5’エキソヌクレアーゼ活性を有し、平滑末端型dsDNA基質、一方の鎖に内部切断を有するdsDNA基質、5’オーバーハングを有するdsDNA基質、及び/又は3’オーバーハングを有するdsDNA基質を認識することができる、且つ/又は
(e)バクテリオファージラムダエキソタンパク質、RacプロファージRecEエキソヌクレアーゼ、アルテミスタンパク質、アポロタンパク質、DNA2エキソヌクレアーゼ、Exo1エキソヌクレアーゼ、ヘルペスウイルスSOXタンパク質、UL12エキソヌクレアーゼ、腸内細菌エキソヌクレアーゼVIII、T7ファージエキソヌクレアーゼ、大腸菌(E.coli)エキソヌクレアーゼIII、哺乳類Trex2エキソヌクレアーゼ、同等のエキソヌクレアーゼ活性を有する関連するタンパク質、又は配列番号8、9、136、137、138、139、140、141、142、143、144、又は145と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、
請求項1~6のいずれか一項に記載の方法、又は請求項7~10のいずれか一項に記載のシステム。 exonuclease is
(a) having preferred substrates comprising linear dsDNA molecules;
(b) having a preferred substrate comprising a linear dsDNA molecule containing a phosphorylated 5'end;
(c) blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5′ overhangs, and/or with 3′ overhangs, which have 5′→3′ exonuclease activity; capable of recognizing a dsDNA substrate,
(d) blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5′ overhangs, and/or with 3′ overhangs, which have 3′→5′ exonuclease activity; capable of recognizing dsDNA substrates and/or
(e) Bacteriophage lambda exoprotein, Rac prophage RecE exonuclease, Artemis protein, Apollo protein, DNA2 exonuclease, Exo1 exonuclease, Herpes virus SOX protein, UL12 exonuclease, enterobacterial exonuclease VIII, T7 phage exonuclease , E. coli exonuclease III, mammalian Trex2 exonuclease, related proteins with equivalent exonuclease activity, or proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with 144 or 145;
A method according to any one of claims 1-6, or a system according to any one of claims 7-10 .
(a)SSAPと同じ宿主生物から入手される、
(b)細菌SSBである、
(c)腸内細菌科種(Enterobacteriaceae sp.)SSBである、
(d)大腸菌属種(Escherichia sp.)、赤痢菌属種(Shigella sp.)、エンテロバクター属種(Enterobacter sp.)、クレブシエラ属種(Klebsiella sp.)、セラチア属種(Serratia sp.)、パントエア属種(Pantoea sp.)、又はエルシニア属種(Yersinia sp.)であり、且つ/又は
(e)配列番号31又は34~132と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、
請求項1~6及び11のいずれか一項に記載の方法、又は請求項7~11のいずれか一項に記載のシステム。 single -stranded DNA binding protein (SSB)
(a) obtained from the same host organism as the S SAP,
(b) is bacterial SSB;
(c) is Enterobacteriaceae sp. SSB;
(d) Escherichia sp., Shigella sp., Enterobacter sp., Klebsiella sp., Serratia sp., Pantoea sp. or Yersinia sp., and/or
(e) proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs: 31 or 34-132;
A method according to any one of claims 1-6 and 11, or a system according to any one of claims 7-11 .
(b)対照真核細胞がゲノム編集分子の提供を受けるが、前記HDR促進剤の少なくとも1つに曝露されない対照方法又はシステムと比較して、非相同末端結合(NHEJ)の頻度が維持されるか、又は少なくとも2分の1に低下する、
請求項1~6及び11~12のいずれか一項に記載の方法、又は請求項7~12のいずれか一項に記載のシステム。 (a) said HDR frequency is increased at least 2-fold compared to a control system in which control eukaryotic cells are provided with a genome- editing molecule but not exposed to at least one HDR-enhancing agent; and/or
(b) the frequency of non-homologous end joining (NHEJ) is maintained compared to a control method or system in which control eukaryotic cells are provided with a genome-editing molecule but not exposed to at least one of said HDR-enhancing agents; or is at least reduced by a factor of two,
A method according to any one of claims 1-6 and 11-12, or a system according to any one of claims 7-12 .
(a)作動可能に連結された核局在化シグナル(NLS)及び/又は細胞透過性ペプチド(CPP)を更に含む、且つ/又は
(b)SSAP、エキソヌクレアーゼ、及び/又はSSBの間に挿入されたプロテアーゼ認識部位又は自己プロセシングタンパク質配列を含むポリタンパク質として細胞に提供される、
請求項1~6及び11~13のいずれか一項に記載の方法、又は請求項7~13のいずれか一項に記載のシステム。 S SAPs, exonucleases , and/or single -stranded DNA binding proteins (SSBs)
(a) further comprising an operably linked nuclear localization signal (NLS) and/or a cell penetrating peptide (CPP), and/or
(b) provided to the cell as a polyprotein comprising protease recognition sites or self-processing protein sequences inserted between SSAPs, exonucleases, and/or SSBs;
A method according to any one of claims 1-6 and 11-13, or a system according to any one of claims 7-13 .
(a)植物細胞が一倍体、二倍体、又は倍数体である、
(b)植物細胞が、培養培地中、植物中、又は植物組織中にある、
(c)SSAP、エキソヌクレアーゼ、及び/又は一本鎖DNA結合タンパク質が、配列番号10、配列番号11、配列番号12、配列番号13、配列番号14、配列番号15、及び配列番号16からなる群から選択される作動可能に連結された核局在化シグナル(NLS)を更に含む、且つ/又は
(d)システムが、ゲノム修飾を含む植物細胞、植物細胞から入手された珠芽、又は植物を単離すること及び/又は成長させることを提供し、及び植物細胞、珠芽、又は植物のゲノムがゲノム修飾を含む、
請求項15に記載のシステム。 the cells are plant cells,
(a) the plant cell is haploid, diploid, or polyploid;
(b) the plant cell is in a culture medium, in a plant, or in a plant tissue;
(c) the group in which the SSAP, exonuclease, and/or single-stranded DNA binding protein consists of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 and/or further comprising an operably linked nuclear localization signal (NLS) selected from
(d) the system provides isolating and/or growing a plant cell, propagule obtained from the plant cell, or plant comprising the genomic modification; including modifications,
16. The system of claim 15 .
i)少なくとも1つの配列特異的エンドヌクレアーゼ、
ii)真核細胞の標的編集部位と相同性を有するドナー鋳型DNA分子、
iii)一本鎖DNAアニーリングタンパク質(SSAP)、
iv)二本鎖DNA基質を一本鎖DNA産物へと少なくとも部分的に変換することのできるエキソヌクレアーゼ、及び
v)一本鎖DNA結合タンパク質(SSB)
を提供することを含み、細胞の標的編集部位がドナー鋳型DNA分子によって修飾される、方法。 A method of genetically engineering a eukaryotic cell, comprising:
i) at least one sequence-specific endonuclease ;
ii) a donor template DNA molecule with homology to a eukaryotic target editing site ;
iii) single-stranded DNA annealing protein (SSAP) ,
iv) an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and
v) single-stranded DNA binding protein (SSB )
wherein the target editing site of the cell is modified by the donor template DNA molecule.
(a)RNA誘導型ヌクレアーゼ又はRNA誘導型ヌクレアーゼをコードするポリヌクレオチドを含み、ここで、方法が更に、真核細胞に、ガイドRNA又はガイドRNAをコードするポリヌクレオチドを提供し、
(b)ジンクフィンガーヌクレアーゼ(ZFN)、転写活性化因子様エフェクターヌクレアーゼ(TALエフェクターヌクレアーゼ)、アルゴノート、メガヌクレアーゼ、又は操作されたメガヌクレアーゼを含む、且つ/又は
(c)ニッカーゼを含む、
請求項18に記載の方法。 at least one sequence-specific endonuclease
(a) comprising an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease , wherein the method further provides the eukaryotic cell with the guide RNA or a polynucleotide encoding the guide RNA;
(b) a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL effector nuclease), an Argonaute, a meganuclease, or an engineered meganuclease; and/or
(c) a nickase,
19. The method of claim 18 .
(a)少なくとも1つのCas9ニッカーゼ、Cas12aニッカーゼ、ジンクフィンガーニッカーゼ、TALEニッカーゼ、又はこれらの組み合わせを含み、配列特異的エンドヌクレアーゼが、標的編集部位のエンドヌクレアーゼ認識配列に特異的である、且つ/又は
(b)Cas9及び/又はCas12aを含み、ガイドRNA分子が、標的編集部位のDNA配列と少なくとも1塩基のミスマッチを有する、
請求項18~21のいずれか一項に記載の方法。 A sequence- specific endonuclease is
(a) the sequence-specific endonuclease comprises at least one Cas9 nickase, Cas12a nickase, zinc finger nickase, TALE nickase, or a combination thereof, wherein the sequence -specific endonuclease is specific for the endonuclease recognition sequence of the target editing site; and/or
(b) comprising Cas9 and/or Cas12a, wherein the guide RNA molecule has at least one base mismatch with the DNA sequence of the target editing site;
A method according to any one of claims 18-21 .
(a)プラスミド又はジェミニウイルスゲノムに提供される、且つ/又は
(b)エンドヌクレアーゼ認識配列に隣接されている、
請求項7~17のいずれか一項に記載のシステム、又は請求項18~22のいずれか一項に記載の方法。 a donor template DNA molecule
(a) provided in a plasmid or geminivirus genome , and/or
(b) flanked by endonuclease recognition sequences;
A system according to any one of claims 7-17 or a method according to any one of claims 18-22 .
(a)線状dsDNA分子を含むエキソヌクレアーゼの好ましい基質を有する、
(b)リン酸化した5’末端を含む線状dsDNA分子を含む好ましい基質を有する、
(c)5’→3’エキソヌクレアーゼ活性を有し、平滑末端型dsDNA基質、一方の鎖に内部切断を有するdsDNA基質、5’オーバーハングを有するdsDNA基質、及び/又は3’オーバーハングを有するdsDNA基質を認識することができる、
(d)3’→5’エキソヌクレアーゼ活性を有し、平滑末端型dsDNA基質、一方の鎖に内部切断を有するdsDNA基質、5’オーバーハングを有するdsDNA基質、及び/又は3’オーバーハングを有するdsDNA基質を認識することができる、
(e)、バクテリオファージラムダエキソタンパク質、RacプロファージRecEエキソヌクレアーゼ、アルテミスタンパク質、アポロタンパク質、DNA2エキソヌクレアーゼ、Exo1エキソヌクレアーゼ、ヘルペスウイルスSOXタンパク質、UL12エキソヌクレアーゼ、腸内細菌エキソヌクレアーゼVIII、T7ファージエキソヌクレアーゼ、大腸菌(E.coli)エキソヌクレアーゼIII、哺乳類Trex2エキソヌクレアーゼ、同等のエキソヌクレアーゼ活性を有する関連するタンパク質、又は配列番号8、9、136、137、138、139、140、141、142、143、144、又は145と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、且つ/又は
(f)T7ファージエキソヌクレアーゼ、大腸菌(E.coli)エキソヌクレアーゼIII、同等のエキソヌクレアーゼ活性を有する関連するタンパク質、又は配列番号143又は144と少なくとも70%、75%、80%、85%、90%、95%、又は99%の配列同一性を有するタンパク質を含む、
請求項18~25のいずれか一項に記載の方法。 exonuclease is
(a) having preferred substrates for exonucleases comprising linear dsDNA molecules;
(b) having a preferred substrate comprising a linear dsDNA molecule containing a phosphorylated 5'end;
(c) blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5′ overhangs, and/or with 3′ overhangs, which have 5′→3′ exonuclease activity; capable of recognizing a dsDNA substrate,
(d) blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5′ overhangs, and/or with 3′ overhangs, which have 3′→5′ exonuclease activity; capable of recognizing a dsDNA substrate,
(e), bacteriophage lambda exoprotein, Rac prophage RecE exonuclease, artemis protein, apollo protein, DNA2 exonuclease, Exo1 exonuclease, herpes virus SOX protein, UL12 exonuclease, enterobacterial exonuclease VIII, T7 phage exo nuclease, E. coli exonuclease III, mammalian Trex2 exonuclease, related proteins with equivalent exonuclease activity, or SEQ ID NOs:8, 9, 136, 137, 138, 139, 140, 141, 142, 143 , 144, or 145, and/or
(f) T7 phage exonuclease, E. coli exonuclease III, related proteins with equivalent exonuclease activity, or at least 70%, 75%, 80%, 85%, 90 with SEQ ID NO: 143 or 144 %, 95%, or 99% sequence identity,
A method according to any one of claims 18-25 .
(a)植物細胞が一倍体、二倍体、又は倍数体である、
(b)植物細胞が、培養培地中、植物中、又は植物組織中にある、且つ/又は
(c)方法が、ゲノム修飾を含む植物細胞、植物細胞から入手された珠芽、植物を単離する及び/又は成長させるステップを更に含み、植物細胞、珠芽、又は植物のゲノムがゲノム修飾を含む、
請求項28に記載の方法。 the cells are plant cells,
(a) the plant cell is haploid, diploid, or polyploid;
(b) the plant cell is in a culture medium, in a plant, or in a plant tissue; and/or
(c) the method further comprises isolating and/or growing the plant cell, propagule obtained from the plant cell, plant comprising the genomic modification, wherein the genome of the plant cell, propagule, or plant comprises the genomic modification; ,
29. The method of claim 28 .
(a)1つ以上のベクターに提供される、
(b)染色体において提供される。
(c)ポリペプチド、DNA、mRNAを導入すること、及び/又は性的交雑によって提供される、且つ/又は
(d)i)~v)の1つ以上を含む前駆細胞によって提供され、ここで、前駆細胞が、i)~v)のうちの少なくとも1つを含まず、前駆細胞に含まれないi)~v)のうちの少なくとも1つが、ポリペプチド、DNA、又はmRNAの前駆細胞への送達によって且つ/又は前駆細胞の性的交雑によって、前駆細胞にその後提供される、
請求項18~29のいずれか一項に記載の方法。 i ) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to a eukaryotic target editing site, iii) a single-stranded DNA annealing protein (SSAP), iv) a double-stranded DNA substrate. into a single- stranded DNA product, and v) a single-stranded DNA binding protein (SSB),
(a) provided on one or more vectors,
(b) provided on a chromosome;
(c) introduced polypeptides, DNA, mRNA and/or provided by sexual mating; and/or
(d) provided by a progenitor cell comprising one or more of i)-v), wherein the progenitor cell does not comprise at least one of i)-v) and is not included in the progenitor cell i) at least one of -v) is subsequently provided to the progenitor cell by delivery of the polypeptide, DNA or mRNA to the progenitor cell and/or by sexual mating of the progenitor cell;
A method according to any one of claims 18-29 .
(b)標的編集部位が、タンパク質コード配列中又はプロモーター中にある、
(c)標的編集部位の修飾が、挿入、欠失、又は置換である、
(d)標的編集部位が、作物栽培学的に重要な形質をコードする遺伝子又は哺乳類疾患に関わる遺伝子である、
(e)方法が、修飾された標的編集部位を含む真核細胞を単離し又は増殖させることを更に含む、
(f)相同組換え修復(HDR)頻度が対照システムと比較して少なくとも2倍に増加し、ここで、対照真核細胞が少なくとも、少なくとも1つの配列特異的エンドヌクレアーゼ及びドナー鋳型DNA分子の提供を受けるが、SSAP、エキソヌクレアーゼ、及びSSBからなる群の少なくとも1つに対し曝露されない、且つ/又は
(g)HDR促進剤の少なくとも1つに曝露されない対照方法又はシステムと比較して、非相同末端結合(NHEJ)の頻度が維持されるか、又は少なくとも2分の1に低下し、ここで、対照真核細胞が少なくとも、少なくとも1つの配列特異的エンドヌクレアーゼ及びドナー鋳型DNA分子の提供を受けるが、SSAP、エキソヌクレアーゼ、及びSSBからなる群の少なくとも1つに対し曝露されない、
請求項18~31のいずれか一項に記載の方法。 (a) further comprising detecting the modification ;
(b) the target editing site is in the protein coding sequence or in the promoter;
(c) the modification of the target editing site is an insertion, deletion, or substitution;
(d) the target editing site is a gene encoding an agronomically important trait or a gene involved in a mammalian disease;
(e) the method further comprises isolating or growing eukaryotic cells comprising the modified target editing site;
(f) homologous recombination repair (HDR) frequency is increased by at least 2-fold compared to the control system, wherein the control eukaryotic cell provides at least at least one sequence-specific endonuclease and a donor template DNA molecule; but not exposed to at least one of the group consisting of SSAP, exonuclease, and SSB;
(g) the frequency of non-homologous end joining (NHEJ) is maintained or reduced by at least 2-fold compared to a control method or system not exposed to at least one of the HDR-enhancing agents, wherein control eukaryotic cells are provided with at least one sequence-specific endonuclease and a donor template DNA molecule, but are not exposed to at least one of the group consisting of SSAP, exonuclease, and SSB;
A method according to any one of claims 18-31 .
(a)前記標的編集部位中の少なくとも1つのエンドヌクレアーゼ認識配列DNA配列を切断する少なくとも1つの配列特異的エンドヌクレアーゼ又は前記少なくとも1つの配列特異的エンドヌクレアーゼをコードする少なくとも1つのポリヌクレオチドを提供すること、及び
(b)少なくとも1つの二本鎖DNA配列を含む少なくとも1つのドナー分子を提供することであって、(i)前記DNA配列が、標的編集部位に隣接する配列と少なくとも50ヌクレオチドの長さにわたって少なくとも90%の相同性を有し、及び(ii)前記ドナー配列が、前記標的編集部位と比較して少なくとも1つの修飾を含むこと;及び
(c)少なくとも1つの相同組換え修復(HDR)促進剤を提供することであって、HDR促進剤が、
(i)少なくとも1つの一本鎖DNAアニーリングタンパク質(SSAP)、及び
(ii)二本鎖DNA基質を一本鎖DNA産物へと少なくとも部分的に変換することのできる少なくとも1つのエキソヌクレアーゼ、及び
(iii)少なくとも1つの一本鎖DNA結合タンパク質(SSB)
を含み;
及びそれによって少なくとも1つの配列特異的エンドヌクレアーゼ、少なくとも1つのドナー分子、及び少なくとも1つのHDR促進剤が前記真核細胞の前記標的編集部位に前記修飾を導入すること;及び
(d)前記標的編集部位に修飾を含む真核細胞を単離すること又は増殖させること
を含む方法。 A method for producing a eukaryotic cell having a genetically modified target editing site, comprising:
(a) providing at least one sequence-specific endonuclease that cleaves at least one endonuclease recognition sequence DNA sequence in said target editing site or at least one polynucleotide encoding said at least one sequence-specific endonuclease; and (b) providing at least one donor molecule comprising at least one double-stranded DNA sequence, wherein (i) said DNA sequence comprises sequences flanking the target editing site and at least 50 nucleotides of has at least 90% homology over its length, and (ii) said donor sequence contains at least one modification relative to said target editing site; and (c) at least one homologous recombination repair ( HDR) enhancer, wherein the HDR enhancer comprises
(i) at least one single-stranded DNA annealing protein (SSAP); and (ii) at least one exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product; iii) at least one single-stranded DNA binding protein (SSB)
includes;
and whereby at least one sequence-specific endonuclease, at least one donor molecule, and at least one HDR facilitator introduce said modification into said target editing site of said eukaryotic cell; and (d) isolating or growing a eukaryotic cell containing a modification at said target editing site;
method including.
(b)標的編集部位の一部分が、前記標的編集部位における2つの配列特異的切断を用いることによって欠失され、ドナー分子によって提供される配列に置き換えられる、
請求項34に記載の方法。 (a) the modification is selected from the group consisting of insertion of one or more nucleotides, deletion of one or more nucleotides, or substitution of one or more nucleotides; and/or
(b) a portion of the target editing site is deleted by using two sequence-specific cleavages at said target editing site and replaced with a sequence provided by a donor molecule;
35. The method of claim 34 .
(b)修飾を含む真核細胞を増殖させることを更に含む、
請求項34又は35に記載の方法。 (a) the donor molecule is flanked by endonuclease recognition sequences in the vector, and/or
(b) further comprising growing the eukaryotic cell comprising the modification;
36. A method according to claim 34 or 35 .
(i)請求項34~36のいずれか一項に記載の遺伝子修飾された真核細胞を作製するステップ、及び
(ii)前記細胞を生物へと再生するステップ
を含む方法。 A method of making a genetically modified organism, comprising:
37. A method comprising the steps of (i) producing a genetically modified eukaryotic cell according to any one of claims 34-36 , and (ii) regenerating said cell into an organism.
(a)ドナー鋳型DNAと、配列特異的エンドヌクレアーゼと、ガイドRNAをコードするポリヌクレオチドとを含む、
(b)一本鎖DNAアニーリングタンパク質(SSAP)と、二本鎖DNA基質を一本鎖DNA産物へと少なくとも部分的に変換することのできるエキソヌクレアーゼと、一本鎖DNA結合タンパク質(SSB)とを含む、
(c)i)少なくとも1つの配列特異的エンドヌクレアーゼ、ii)真核細胞の標的編集部位と相同性を有するドナー鋳型DNA分子、iii)一本鎖DNAアニーリングタンパク質(SSAP)、iv)二本鎖DNA基質を一本鎖DNA産物へと少なくとも部分的に変換することのできるエキソヌクレアーゼ、及びv)一本鎖DNA結合タンパク質(SSB)をコードする核酸を含む、且つ/又は
(d)誘導性プロモーターに作動可能に連結されている配列特異的ヌクレアーゼを含む、
請求項41に記載のベクター。 the vector is
(a) comprising a donor template DNA, a sequence- specific endonuclease, and a polynucleotide encoding a guide RNA;
(b) a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); including,
(c) i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to a eukaryotic target editing site, iii) a single-stranded DNA annealing protein (SSAP), iv) double-stranded an exonuclease capable of at least partially converting a DNA substrate into a single-stranded DNA product; and v) a nucleic acid encoding a single-stranded DNA binding protein (SSB); and/or
(d) a sequence-specific nuclease operably linked to an inducible promoter;
42. The vector of claim 41.
(a)第1のベクターと第2のベクターとを含み、ここで
i)第1のベクターが、ドナー鋳型DNAと配列特異的エンドヌクレアーゼとを含む核酸を含み、配列特異的エンドヌクレアーゼが、RNA誘導型ヌクレアーゼをコードするポリヌクレオチドと、ガイドRNAをコードするポリヌクレオチドとを含み;及び
ii)第2のベクターが、一本鎖DNAアニーリングタンパク質(SSAP)と、二本鎖DNA基質を一本鎖DNA産物へと少なくとも部分的に変換することのできるエキソヌクレアーゼと、一本鎖DNA結合タンパク質(SSB)とを含む、且つ/又は
(b)遺伝子操作された細胞の検出用薬剤を更に含む、
請求項43に記載のキット。 the kit is
(a) a first vector and a second vector, wherein i) the first vector comprises a nucleic acid comprising donor template DNA and a sequence -specific endonuclease, wherein the sequence- specific endonuclease comprises a polynucleotide encoding an RNA-guided nuclease and a polynucleotide encoding a guide RNA; and ii) a second vector comprises a single-stranded DNA annealing protein (SSAP) and a double-stranded DNA substrate. an exonuclease capable of at least partially converting into a single-stranded DNA product and a single-stranded DNA binding protein (SSB); and/or
(b) further comprising an agent for detection of genetically engineered cells;
44. The kit of claim 43 .
(a)植物又は哺乳類細胞である、且つ/又は
(b)宿主細胞である、
請求項45に記載の細胞。 the cells
(a) is a plant or mammalian cell, and/or
(b) is a host cell;
46. The cell of claim 45 .
(b)細胞が生殖系列細胞である、
(c)細胞が前駆植物細胞であり、前駆植物細胞又は植物に含まれるi)~v)のうちの少なくとも1つが形質転換によって供給される、
(d)生物が植物であり、前駆植物に含まれないi)~v)のうちの少なくとも1つが、前駆植物に含まれないi)~v)のうちの少なくとも1つを含む第2の植物との性的交雑によって供給される、
(e)前駆真核細胞が動物細胞であり、前駆細胞に含まれないi)~v)のうちの少なくとも1つがトランスフェクションによって供給される、且つ/又は
(f)前駆生物が非ヒト動物であり、非ヒト動物に含まれないi)~v)のうちの少なくとも1つが、非ヒト動物に含まれないi)~v)のうちの少なくとも1つを含む非ヒト動物との性的交雑によって供給される、
請求項48に記載の前駆真核細胞又は生物。 (a) the donor template is a double-stranded DNA molecule;
(b) the cell is a germline cell;
(c) the cell is a progenitor plant cell and at least one of i) to v) contained in the progenitor plant cell or plant is provided by transformation;
(d) a second plant wherein the organism is a plant and at least one of i) to v) not included in the progenitor plant includes at least one of i) to v) not included in the progenitor plant supplied by sexual interbreeding with
(e) the eukaryotic progenitor cell is an animal cell and at least one of i) to v) not contained in progenitor cells is provided by transfection, and/or
(f) the precursor is a non-human animal, and at least one of i) to v) not included in non-human animals includes at least one of i) to v) not included in non-human animals supplied by sexual crosses with non-human animals, including
49. A progenitor eukaryotic cell or organism according to claim 48 .
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201962866317P | 2019-06-25 | 2019-06-25 | |
US62/866,317 | 2019-06-25 | ||
PCT/US2020/039410 WO2020264016A1 (en) | 2019-06-25 | 2020-06-24 | Improved homology dependent repair genome editing |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2022539338A JP2022539338A (en) | 2022-09-08 |
JPWO2020264016A5 true JPWO2020264016A5 (en) | 2023-07-04 |
Family
ID=71579688
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2021576666A Pending JP2022539338A (en) | 2019-06-25 | 2020-06-24 | Improved homology-dependent repair genome editing |
Country Status (13)
Country | Link |
---|---|
US (2) | US11041172B2 (en) |
EP (1) | EP3990633A1 (en) |
JP (1) | JP2022539338A (en) |
KR (1) | KR20220052905A (en) |
CN (1) | CN114630910A (en) |
AR (1) | AR123323A1 (en) |
AU (1) | AU2020301236A1 (en) |
BR (1) | BR112021026220A2 (en) |
CA (1) | CA3138663A1 (en) |
IL (1) | IL289106A (en) |
MX (1) | MX2021014861A (en) |
SG (1) | SG11202112092TA (en) |
WO (1) | WO2020264016A1 (en) |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190323019A1 (en) * | 2016-12-21 | 2019-10-24 | Yi-Hua Hsu | Composition for editing a nucleic acid sequence and method using the same |
CN109666693B (en) * | 2018-12-29 | 2022-08-16 | 北京市农林科学院 | Application of MG132 in editing receptor genome by base editing system |
US11242534B1 (en) | 2020-07-31 | 2022-02-08 | Inari Agriculture Technology, Inc. | INHT31 transgenic soybean |
US20240011042A1 (en) | 2020-07-31 | 2024-01-11 | Inari Agriculture Technology, Inc. | Excisable plant transgenic loci with signature protospacer adjacent motifs or signature guide rna recognition sites |
WO2023122625A2 (en) * | 2021-12-20 | 2023-06-29 | Intergalactic Therapeutics, Inc. | Production of gene therapy vector in engineered bacteria |
WO2023156585A1 (en) * | 2022-02-18 | 2023-08-24 | Pulmobiotics, S.L. | Systems, tools, and methods for engineering bacteria |
WO2023165613A1 (en) * | 2022-03-03 | 2023-09-07 | 清华大学 | Use of 5'→3' exonuclease in gene editing system, and gene editing system and gene editing method |
CN115960733B (en) * | 2022-09-19 | 2023-11-24 | 苏州泓迅生物科技股份有限公司 | Genetically engineered saccharomycete for assembling large fragment DNA, construction method and application thereof |
CN115976086B (en) * | 2023-01-19 | 2023-08-08 | 河南农业大学 | Method for editing bacteria CRISPR-Cas9 gene and application thereof |
WO2024168253A1 (en) * | 2023-02-10 | 2024-08-15 | Possible Medicines Llc | Delivery of an rna guided recombination system |
WO2024168265A1 (en) * | 2023-02-10 | 2024-08-15 | Possible Medicines Llc | Aav delivery of rna guided recombination system |
CN116873928A (en) * | 2023-06-28 | 2023-10-13 | 浙江工业大学 | Preparation method and application of tungsten carbide nanorod-carbon nanosheet composite material and iron-carbon microelectrode material |
CN116555346B (en) * | 2023-07-05 | 2023-09-01 | 中山大学 | Method for promoting growth of grass carp by adopting carbon nanotube gene carrier delivery system |
CN117487959B (en) * | 2024-01-02 | 2024-03-22 | 东北农业大学 | Molecular marker linked with gene related to quantity of Indian pumpkin lateral branches and application thereof |
Family Cites Families (83)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US39247A (en) | 1863-07-14 | Improvement in skates | ||
US5352605A (en) | 1983-01-17 | 1994-10-04 | Monsanto Company | Chimeric genes for transforming plant cells using viral promoters |
US5428147A (en) | 1983-04-15 | 1995-06-27 | Mycogen Plant Science, Inc. | Octopine T-DNA promoters |
US5322938A (en) | 1987-01-13 | 1994-06-21 | Monsanto Company | DNA sequence for enhancing the efficiency of transcription |
US5310667A (en) | 1989-07-17 | 1994-05-10 | Monsanto Company | Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases |
US5641876A (en) | 1990-01-05 | 1997-06-24 | Cornell Research Foundation, Inc. | Rice actin gene and promoter |
DE69132913T2 (en) | 1990-04-26 | 2002-08-29 | Aventis Cropscience N.V., Gent | New Bacillus thuringia strain and its gene coding for insect toxin |
US5633435A (en) | 1990-08-31 | 1997-05-27 | Monsanto Company | Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases |
US5866775A (en) | 1990-09-28 | 1999-02-02 | Monsanto Company | Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases |
US5277905A (en) | 1991-01-16 | 1994-01-11 | Mycogen Corporation | Coleopteran-active bacillus thuringiensis isolate |
ES2124738T3 (en) | 1991-08-02 | 1999-02-16 | Mycogen Corp | NEW MICROORGANISM AND INSECTICIDE. |
EP0625006A4 (en) | 1992-11-20 | 1996-04-24 | Agracetus | Transgenic cotton plants producing heterologous bioplastic. |
DE69428290T2 (en) | 1993-01-13 | 2002-04-18 | Pioneer Hi-Bred International, Inc. | DERIVATIVES OF ALPHA-HORDOTHIONIN WITH HIGHER LEVELS OF LYSINE |
EP0945431A3 (en) | 1994-02-04 | 2004-08-18 | Basf Aktiengesellschaft | Process and intermediates for the preparation of phenylacetic derivatives |
US5593881A (en) | 1994-05-06 | 1997-01-14 | Mycogen Corporation | Bacillus thuringiensis delta-endotoxin |
US5792931A (en) | 1994-08-12 | 1998-08-11 | Pioneer Hi-Bred International, Inc. | Fumonisin detoxification compositions and methods |
US5736514A (en) | 1994-10-14 | 1998-04-07 | Nissan Chemical Industries, Ltd. | Bacillus strain and harmful organism controlling agents |
BR9609200A (en) | 1995-06-02 | 1999-05-11 | Pioneer Hi Bred Int | High threonine derivatives of alpha-hordothionine |
MX9709352A (en) | 1995-06-02 | 1998-02-28 | Pioneer Hi Bred Int | HIGH METHIONINE DERIVATIVES OF 'alpha'-HORDOTHIONIN. |
US5703049A (en) | 1996-02-29 | 1997-12-30 | Pioneer Hi-Bred Int'l, Inc. | High methionine derivatives of α-hordothionin for pathogen-control |
US5850016A (en) | 1996-03-20 | 1998-12-15 | Pioneer Hi-Bred International, Inc. | Alteration of amino acid compositions in seeds |
AU728086B2 (en) | 1996-11-01 | 2001-01-04 | Pioneer Hi-Bred International, Inc. | Proteins with enhanced levels of essential amino acids |
AU746454B2 (en) | 1998-03-02 | 2002-05-02 | Massachusetts Institute Of Technology | Poly zinc finger proteins with improved linkers |
US6534261B1 (en) | 1999-01-12 | 2003-03-18 | Sangamo Biosciences, Inc. | Regulation of endogenous gene expression in cells using zinc finger proteins |
US6453242B1 (en) | 1999-01-12 | 2002-09-17 | Sangamo Biosciences, Inc. | Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites |
US6794136B1 (en) | 2000-11-20 | 2004-09-21 | Sangamo Biosciences, Inc. | Iterative optimization in the design of binding proteins |
ES2262514T3 (en) | 1999-04-29 | 2006-12-01 | Syngenta Limited | HERBICID RESISTANT PLANTS. |
US20020192813A1 (en) | 1999-08-18 | 2002-12-19 | Timothy W. Conner | Plant expression vectors |
US6683231B2 (en) | 2000-06-02 | 2004-01-27 | E. I. Du Pont De Nemours And Company | High level production of p-hydroxybenzoic acid in green plants |
US7169970B2 (en) | 2000-09-29 | 2007-01-30 | Syngenta Limited | Herbicide resistant plants |
US7151204B2 (en) | 2001-01-09 | 2006-12-19 | Monsanto Technology Llc | Maize chloroplast aldolase promoter compositions and methods for use thereof |
US7045684B1 (en) | 2002-08-19 | 2006-05-16 | Mertec, Llc | Glyphosate-resistant plants |
AU2007286176B2 (en) | 2006-08-11 | 2012-10-11 | Monsanto Technology Llc | Production of high tryptophan maize by chloroplast targeted expression of anthranilate synthase |
WO2010009353A1 (en) | 2008-07-16 | 2010-01-21 | Monsanto Technology Llc | Methods and vectors for producing transgenic plants |
DK2417262T3 (en) | 2009-04-07 | 2015-06-22 | Dow Agrosciences Llc | NANOPARTICALLY MEDICATED SUPPLY OF SEQUENCE-SPECIFIC NUCLEASES |
US8618354B2 (en) | 2009-10-06 | 2013-12-31 | The Regents Of The University Of California | Generation of haploid plants and improved plant breeding |
RU2571928C2 (en) | 2009-10-16 | 2015-12-27 | ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи | Use of dendrimer nanotechnology for delivery of biomolecules into plant cells |
AU2011238826B2 (en) | 2010-03-31 | 2014-11-13 | Corteva Agriscience Llc | Plant peptide gamma-zein for delivery of biomolecules into plant cells |
GB201009732D0 (en) | 2010-06-10 | 2010-07-21 | Gene Bridges Gmbh | Direct cloning |
UA113046C2 (en) | 2010-07-07 | 2016-12-12 | A METHOD OF INTRODUCING A LINEAR NUCLEIC ACID MOLECULES INTO A PLANT CELL, WHICH HAS A CELL WALL, WITH THE APPLICATION OF PEGILE NAPHID | |
JP6067671B2 (en) | 2011-03-23 | 2017-01-25 | ダウ アグロサイエンシィズ エルエルシー | Quantum dot carrier peptide conjugates suitable for imaging and delivery applications in plants |
US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
PT2768942T (en) | 2011-10-17 | 2020-01-21 | Massachusetts Inst Technology | Intracellular delivery |
US20130145488A1 (en) | 2011-12-06 | 2013-06-06 | Iowa State University Research Foundation, Inc. | Mesoporous silica nanoparticles suitable for co-delivery |
US20130185823A1 (en) | 2012-01-16 | 2013-07-18 | Academia Sinica | Mesoporous silica nanoparticle-mediated delivery of dna into arabidopsis root |
RS64622B1 (en) | 2012-05-25 | 2023-10-31 | Univ California | Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription |
US20140096284A1 (en) | 2012-10-01 | 2014-04-03 | Iowa State University Research Foundation, Inc. | Method for the delivery of molecules lyophilized onto microparticles to plant tissues |
BR112015008708A2 (en) | 2012-10-23 | 2017-09-26 | Toolgen Inc | a target DNA cleavage composition comprising a target DNA specific guide and nucleic acid encoding cas protein or cas protein and use thereof |
DK2898075T3 (en) | 2012-12-12 | 2016-06-27 | Broad Inst Inc | CONSTRUCTION AND OPTIMIZATION OF IMPROVED SYSTEMS, PROCEDURES AND ENZYME COMPOSITIONS FOR SEQUENCE MANIPULATION |
BR122021008308B1 (en) | 2012-12-12 | 2022-12-27 | The Broad Institute, Inc. | CRISPR-CAS COMPONENT SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION |
PL2931898T3 (en) | 2012-12-12 | 2016-09-30 | Le Cong | Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains |
PT2771468E (en) | 2012-12-12 | 2015-06-02 | Harvard College | Engineering of systems, methods and optimized guide compositions for sequence manipulation |
WO2014093694A1 (en) | 2012-12-12 | 2014-06-19 | The Broad Institute, Inc. | Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes |
US8697359B1 (en) | 2012-12-12 | 2014-04-15 | The Broad Institute, Inc. | CRISPR-Cas systems and methods for altering expression of gene products |
WO2014118719A1 (en) | 2013-02-01 | 2014-08-07 | Cellectis | Tevl chimeric endonuclease and their preferential cleavage sites |
US9677082B2 (en) | 2013-03-15 | 2017-06-13 | Syngenta Participations Ag | Haploid induction compositions and methods for use therefor |
US9902973B2 (en) | 2013-04-11 | 2018-02-27 | Caribou Biosciences, Inc. | Methods of modifying a target nucleic acid with an argonaute |
US20150040268A1 (en) | 2013-04-25 | 2015-02-05 | Morflora Israel Ltd | Methods and compositions for the delivery of nucleic acids to seeds |
AU2014275045A1 (en) | 2013-06-03 | 2015-12-17 | University Of Southern California | Targeted crosslinked multilamellar liposomes |
WO2014199358A1 (en) | 2013-06-14 | 2014-12-18 | Cellectis | Methods for non-transgenic genome editing in plants |
CA2920253A1 (en) | 2013-08-02 | 2015-02-05 | Enevolv, Inc. | Processes and host cells for genome, pathway, and biomolecular engineering |
WO2015021266A1 (en) | 2013-08-09 | 2015-02-12 | Massachusetts Institute Of Technology | Nanobionic engineering of organelles and photosynthetic organisms |
EP3036327B1 (en) | 2013-08-22 | 2019-05-08 | Pioneer Hi-Bred International, Inc. | Genome modification using guide polynucleotide/cas endonuclease systems and methods of use |
US9675078B2 (en) | 2013-10-15 | 2017-06-13 | Board Of Trustees Of The University Of Arkansas | Method of delivery of bio-active agents to plant cells by using nano-sized materials as carriers |
CA3194412A1 (en) | 2014-02-27 | 2015-09-03 | Monsanto Technology Llc | Compositions and methods for site directed genomic modification |
MX2021000857A (en) | 2014-06-23 | 2021-07-28 | Regeneron Pharma | Nuclease-mediated dna assembly. |
CA2954626A1 (en) | 2014-07-11 | 2016-01-14 | E. I. Du Pont De Nemours And Company | Compositions and methods for producing plants resistant to glyphosate herbicide |
CN107429241A (en) | 2014-08-14 | 2017-12-01 | 北京百奥赛图基因生物技术有限公司 | DNA knocks in system |
EP3845655A1 (en) | 2014-10-01 | 2021-07-07 | The General Hospital Corporation | Methods for increasing efficiency of nuclease-induced homology-directed repair |
CA2963820A1 (en) * | 2014-11-07 | 2016-05-12 | Editas Medicine, Inc. | Methods for improving crispr/cas-mediated genome-editing |
EP3234200B1 (en) | 2014-12-20 | 2021-07-07 | Arc Bio, LLC | Method for targeted depletion of nucleic acids using crispr/cas system proteins |
US10155938B2 (en) | 2015-04-14 | 2018-12-18 | City Of Hope | Coexpression of CAS9 and TREX2 for targeted mutagenesis |
US9790490B2 (en) | 2015-06-18 | 2017-10-17 | The Broad Institute Inc. | CRISPR enzymes and systems |
AU2016326711B2 (en) | 2015-09-24 | 2022-11-03 | Editas Medicine, Inc. | Use of exonucleases to improve CRISPR/Cas-mediated genome editing |
JP6990653B2 (en) | 2015-10-30 | 2022-02-03 | パイオニア ハイ-ブレッド インターナショナル, インコーポレイテッド | Methods and compositions for rapid plant transformation |
US20170175140A1 (en) * | 2015-12-16 | 2017-06-22 | Regents Of The University Of Minnesota | Methods for using a 5'-exonuclease to increase homologous recombination in eukaryotic cells |
WO2017184227A2 (en) * | 2016-02-04 | 2017-10-26 | President And Fellows Of Harvard College | Recombinase genome editing |
WO2018067846A1 (en) | 2016-10-05 | 2018-04-12 | President And Fellows Of Harvard College | Methods of crispr mediated genome modulation in v. natriegens |
WO2018085693A1 (en) | 2016-11-04 | 2018-05-11 | Inari Agriculture, Inc. | Novel plant cells, plants, and seeds |
CN108085328B (en) | 2016-11-21 | 2021-06-22 | 中国科学院分子植物科学卓越创新中心 | Method for editing DNA sequence |
KR102098915B1 (en) | 2017-12-22 | 2020-04-09 | (주)지플러스 생명과학 | Chimeric genome engineering molecules and methods |
WO2020003311A1 (en) | 2018-06-25 | 2020-01-02 | Yeda Research And Development Co. Ltd. | Systems and methods for increasing efficiency of genome editing |
WO2020041172A1 (en) | 2018-08-21 | 2020-02-27 | The Jackson Laboratory | Methods and compositions for recruiting dna repair proteins |
-
2020
- 2020-06-24 BR BR112021026220A patent/BR112021026220A2/en unknown
- 2020-06-24 CA CA3138663A patent/CA3138663A1/en active Pending
- 2020-06-24 US US16/911,156 patent/US11041172B2/en active Active
- 2020-06-24 JP JP2021576666A patent/JP2022539338A/en active Pending
- 2020-06-24 AU AU2020301236A patent/AU2020301236A1/en active Pending
- 2020-06-24 EP EP20739829.8A patent/EP3990633A1/en active Pending
- 2020-06-24 KR KR1020227002110A patent/KR20220052905A/en unknown
- 2020-06-24 SG SG11202112092TA patent/SG11202112092TA/en unknown
- 2020-06-24 CN CN202080044833.1A patent/CN114630910A/en active Pending
- 2020-06-24 MX MX2021014861A patent/MX2021014861A/en unknown
- 2020-06-24 WO PCT/US2020/039410 patent/WO2020264016A1/en unknown
- 2020-06-25 AR ARP200101810A patent/AR123323A1/en unknown
-
2021
- 2021-05-21 US US17/326,891 patent/US20210277422A1/en active Pending
- 2021-12-19 IL IL289106A patent/IL289106A/en unknown
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11639511B2 (en) | CRISPR enabled multiplexed genome engineering | |
JP6737974B1 (en) | Nuclease-mediated DNA assembly | |
US20230383290A1 (en) | High-throughput precision genome editing | |
EP3152312B1 (en) | Methods and compositions for modifying a targeted locus | |
JP2023073245A5 (en) | ||
CN109136248B (en) | Multi-target editing vector and construction method and application thereof | |
JPWO2020264016A5 (en) | ||
JP2019500036A (en) | Reconstruction of DNA end repair pathways in prokaryotes | |
JP2024125308A (en) | Genome editing in Bacteroides | |
US20190309283A1 (en) | Method for preparing long-chain single-stranded dna | |
KR20180082981A (en) | Composition and method for inserting specific nucleic acid sequence into target nucleic acid using overlapping guide nucleic acid | |
WO2024055664A1 (en) | Optimized guide rna, crispr/acc2c9 gene editing system, and gene editing method | |
WO2024125221A1 (en) | Base editing system and base editing method | |
JP2024509194A (en) | In vivo DNA assembly and analysis |