JPWO2021186163A5 - - Google Patents
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- JPWO2021186163A5 JPWO2021186163A5 JP2022555852A JP2022555852A JPWO2021186163A5 JP WO2021186163 A5 JPWO2021186163 A5 JP WO2021186163A5 JP 2022555852 A JP2022555852 A JP 2022555852A JP 2022555852 A JP2022555852 A JP 2022555852A JP WO2021186163 A5 JPWO2021186163 A5 JP WO2021186163A5
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- 108020005004 Guide RNA Proteins 0.000 claims description 78
- 238000000034 method Methods 0.000 claims description 74
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 24
- 150000007523 nucleic acids Chemical group 0.000 claims description 24
- 108010042407 Endonucleases Proteins 0.000 claims description 12
- 102000004533 Endonucleases Human genes 0.000 claims description 12
- 230000008685 targeting Effects 0.000 claims description 11
- 241001465754 Metazoa Species 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000005094 computer simulation Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- 108020004414 DNA Proteins 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 4
- 210000001161 mammalian embryo Anatomy 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 2
- 108091033409 CRISPR Proteins 0.000 claims description 2
- 241000283707 Capra Species 0.000 claims description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 2
- 241000283073 Equus caballus Species 0.000 claims description 2
- 241000206602 Eukaryota Species 0.000 claims description 2
- 108700024394 Exon Proteins 0.000 claims description 2
- 241000282326 Felis catus Species 0.000 claims description 2
- 241000287828 Gallus gallus Species 0.000 claims description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 2
- 241001494479 Pecora Species 0.000 claims description 2
- 241000009328 Perro Species 0.000 claims description 2
- 241000288906 Primates Species 0.000 claims description 2
- 241000283984 Rodentia Species 0.000 claims description 2
- 241000282898 Sus scrofa Species 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 claims description 2
- 230000002538 fungal effect Effects 0.000 claims description 2
- 238000002513 implantation Methods 0.000 claims description 2
- 230000035772 mutation Effects 0.000 claims description 2
- 230000009261 transgenic effect Effects 0.000 claims description 2
- 241000699670 Mus sp. Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
Description
加えて、発明者らは、均一な編集を創出する能力だけではなく、マイクロホモロジーの低い領域を標的化することによってマウス中に所望の大規模な欠失を創出するために本方法を使用する能力も実証した。それにより、遺伝子ノックアウトモデルを生成するための効率的な代替アプローチを提供する。
本発明の態様を以下の項にさらに記載する：
［項１］
核酸配列のＣＲＩＳＰＲ－Ｃａｓ編集における使用のための１つ以上のガイドＲＮＡ配列を選択する方法であって、
－ 前記核酸配列を標的化する複数のガイドＲＮＡ配列を特定すること、
－ 前記複数のガイドＲＮＡ配列のそれぞれについて編集結果の頻度を決定すること、および
－ 最も豊富な編集結果の頻度が、２番目に豊富な編集結果の頻度の少なくとも２倍を超えると決定される、１つ以上のガイドＲＮＡ配列を選択すること、
を含む、方法。
［項２］
前記複数のガイドＲＮＡ配列のそれぞれについての編集結果の頻度が、コンピュータモデルを使用して決定される、前記項１に記載の方法。
［項３］
前記核酸配列が遺伝子配列であり、前記方法が、前記複数のガイドＲＮＡ配列を特定する前に、遺伝子の１つまたは複数の一次転写物を特定することをさらに含む、前記項１または前記項２に記載の方法。
［項４］
遺伝子の最初の約５０％に位置する領域を標的化するガイドＲＮＡ配列を選択することをさらに含む、前記項のいずれか一項に記載の方法。
［項５］
遺伝子のすべての主要な転写物中に存在しないオーファンエクソンを標的化するあらゆるガイドＲＮＡ配列を除外することをさらに含む、前記項のいずれか一項に記載の方法。
［項６］
前記方法が、フレームシフティング変異をもたらすと予測されるガイドＲＮＡ配列を選択することをさらに含む、前記項のいずれか一項に記載の方法。
［項７］
各ガイドＲＮＡ配列にオフターゲットスコアを割り当て、スコアが予め定めた閾値未満のあらゆるガイドＲＮＡ配列を除外することをさらに含む、前記項のいずれか一項に記載の方法。
［項８］
各ガイドＲＮＡ配列にオンターゲット活性スコアを割り当て、スコアが予め定めた閾値未満のあらゆるガイドＲＮＡ配列を除外することをさらに含む、前記項のいずれか一項に記載の方法。
［項９］
前記項１から８のいずれか一項に記載の方法を使用して選択されたガイドＲＮＡ配列を含むガイドＲＮＡ分子を生成することをさらに含む、前記項のいずれか一項に記載の方法。
［項１０］
前記ガイドＲＮＡ分子がシングルガイドＲＮＡである、前記項９に記載の方法。
［項１１］
選択された１つ以上のガイドＲＮＡ配列を含む１つ以上のガイドＲＮＡ分子を使用して細胞の試験集団中の標的配列を編集し、前記細胞における各ガイドＲＮＡ配列と関連する編集結果を決定することをさらに含む、前記項のいずれか一項に記載の方法。
［項１２］
予測される最も豊富な結果を前記試験集団の細胞中に最も一貫して引き起こすガイドＲＮＡ分子を、前記１つ以上のガイドＲＮＡ分子から選択することをさらに含む、前記項１１に記載の方法。
［項１３］
核酸配列のＣＲＩＳＰＲ－Ｃａｓ編集における使用のための一対のガイドＲＮＡ配列を選択する方法であって、
－ 前記核酸配列を囲む５’フランクと３’フランクを標的化する複数のガイドＲＮＡ配列を特定すること、
－ 前記複数のガイドＲＮＡ配列のそれぞれについて編集結果の頻度を決定すること、および
－ 前記５’フランクを標的化する第１のガイドＲＮＡおよび前記３’フランクを標的化する第２のガイドＲＮＡを含む一対のガイドＲＮＡ配列であって、それぞれのガイドＲＮＡについて最も豊富な編集結果の頻度が２番目に豊富な編集結果の頻度の４倍未満であると決定される一対のガイドＲＮＡ配列、を選択すること、
を含む、方法。
［項１４］
前記項２から１２に記載のいずれかの特徴をさらに含む、前記項１３に記載の方法。
［項１５］
生物、細胞または細胞の集団あるいはセルフリー発現システムにおいて核酸配列を編集するための方法であって、前記方法が、前記核酸配列を含む二本鎖（ｄｓＤＮＡ）を、Ｃａｓエンドヌクレアーゼおよび前記Ｃａｓエンドヌクレアーゼを前記核酸配列内の標的配列に誘導することができるガイドＲＮＡ分子に曝露することを含み、ここで、前記ガイドＲＮＡ分子は、ＣＲＩＳＰＲ－Ｃａｓ編集において使用された場合に、２番目に豊富な編集結果の少なくとも２倍を超える頻度を有する主要な編集結果をもたらす（またはもたらすと予測される、例えばコンピュータモデルによってもたらすと予測される）ガイドＲＮＡ配列を含む、方法。
［項１６］
ガイドＲＮＡ分子が、前記項１から１２のいずれか一項に記載の方法に従って選択されたガイドＲＮＡ配列を含む、前記項１３に記載の方法。
［項１７］
ガイドＲＮＡ分子およびＤＮＡエンドヌクレアーゼを細胞または複数の細胞に導入することをさらに含む、前記項１３または前記項１４に記載の方法。
［項１８］
前記Ｃａｓエンドヌクレアーゼが、前記核酸配列内の標的配列を切断して二本鎖切断末端を生じさせる、前記項１から１５のいずれか一項に記載の方法。
［項１９］
Ｃａｓエンドヌクレアーゼが、Ｃａｓ９エンドヌクレアーゼである、前記項１６に記載の方法。
［項２０］
前記生物、細胞または細胞の集団が真核生物である、前記項１３から１７のいずれか一項に記載の方法。
［項２１］
生物、細胞または細胞の集団が動物、真菌または植物由来であり、好ましくは前記生物、細胞または細胞の集団が哺乳動物である、前記項１８に記載の方法。
［項２２］
細胞が接合体または接合体を形成する細胞の集団である、前記項１９に記載の方法。
［項２３］
前記方法が、胚をレシピエントの雌の動物に移植して妊娠させることをさらに含み、移植前に前記胚を発生のより後期にまで適宜培養する、前記項２０に記載の方法。
［項２４］
非モザイクのトランスジェニック動物を生成するためのものである、前記項１３から２１のいずれか一項に記載の方法。
［項２５］
動物が、げっ歯類、ウサギ、ヒツジ、ヤギ、ウマ、ウシ、ブタ、イヌ、ネコ、ニワトリまたは霊長類である、前記項２２に記載の方法。
［項２６］
生物、細胞または細胞の集団あるいはセルフリー発現システムにおいて核酸配列を編集するための方法であって、前記方法が、前記核酸配列を含む二本鎖（ｄｓＤＮＡ）を、Ｃａｓエンドヌクレアーゼおよび前記Ｃａｓエンドヌクレアーゼを前記核酸配列を囲む５’フランクと３’フランクを標的化させるように誘導することができる一対のガイドＲＮＡ分子に曝露することを含み、ここで、前記一対のガイドＲＮＡ分子は、ＣＲＩＳＰＲ－Ｃａｓ編集において使用された場合に、２番目に豊富な編集結果の頻度の４倍未満である頻度を有する主要な編集結果をもたらす（またはもたらすと予測される、例えばコンピュータモデルによってもたらすと予測される）第１のガイドＲＮＡおよび第２のガイドＲＮＡを含む、方法。
［項２７］
前記項１６から２５に記載のいずれかの特徴をさらに含む、前記項２６に記載の方法。
［項２８］
前記項１５から２７のいずれか一項に記載の方法によって取得される、細胞、細胞集団およびヒト以外の生物。
In addition, the inventors have demonstrated the ability to not only create uniform edits, but also to use the method to create desired large deletions in mice by targeting regions of low microhomology, thereby providing an efficient alternative approach for generating gene knockout models.
Aspects of the invention are further described in the following sections:
[Item 1]
1. A method for selecting one or more guide RNA sequences for use in CRISPR-Cas editing of a nucleic acid sequence, comprising:
- identifying a plurality of guide RNA sequences targeting said nucleic acid sequence;
- determining a frequency of editing outcomes for each of said plurality of guide RNA sequences; and
- selecting one or more guide RNA sequences for which the frequency of the most abundant editing outcome is determined to be at least twice as high as the frequency of the second most abundant editing outcome;
A method comprising:
[Item 2]
2. The method of claim 1, wherein the frequency of editing outcomes for each of the plurality of guide RNA sequences is determined using a computer model.
[Item 3]
3. The method of claim 1 or 2, wherein the nucleic acid sequence is a gene sequence, and the method further comprises identifying one or more primary transcripts of the gene prior to identifying the plurality of guide RNA sequences.
[Item 4]
Item 11. The method of any one of the preceding paragraphs, further comprising selecting a guide RNA sequence that targets a region located in about the first 50% of the gene.
[Item 5]
4. The method of any one of the preceding paragraphs, further comprising filtering out any guide RNA sequences targeting orphan exons that are not present in all primary transcripts of the gene.
[Item 6]
Item 11. The method of any one of the preceding paragraphs, wherein the method further comprises selecting a guide RNA sequence that is predicted to result in a frameshifting mutation.
[Item 7]
The method of any one of the preceding paragraphs, further comprising assigning an off-target score to each guide RNA sequence and filtering out any guide RNA sequences having a score below a predetermined threshold.
[Item 8]
The method of any one of the preceding paragraphs, further comprising assigning an on-target activity score to each guide RNA sequence and filtering out any guide RNA sequences with a score below a predetermined threshold.
[Item 9]
9. The method of any one of the preceding paragraphs, further comprising generating a guide RNA molecule comprising a guide RNA sequence selected using the method of any one of paragraphs 1 to 8.
[Item 10]
10. The method of claim 9, wherein the guide RNA molecule is a single guide RNA.
[Item 11]
The method of any one of the preceding paragraphs, further comprising editing target sequences in a test population of cells using one or more guide RNA molecules comprising the selected one or more guide RNA sequences, and determining an editing outcome associated with each guide RNA sequence in the cells.
[Item 12]
12. The method of claim 11, further comprising selecting from the one or more guide RNA molecules a guide RNA molecule that most consistently induces the most abundant predicted outcome in cells of the test population.
[Item 13]
1. A method for selecting a pair of guide RNA sequences for use in CRISPR-Cas editing of a nucleic acid sequence, comprising:
- identifying multiple guide RNA sequences targeting the 5' and 3' flanks surrounding said nucleic acid sequence;
- determining a frequency of editing outcomes for each of said plurality of guide RNA sequences; and
- selecting a pair of guide RNA sequences comprising a first guide RNA targeting the 5' flank and a second guide RNA targeting the 3' flank, wherein the frequency of the most abundant editing outcome for each guide RNA is determined to be less than four times the frequency of the second most abundant editing outcome;
A method comprising:
[Item 14]
14. The method according to claim 13, further comprising any one of the features according to claims 2 to 12.
[Item 15]
1. A method for editing a nucleic acid sequence in an organism, a cell or population of cells, or a cell-free expression system, the method comprising exposing a double-stranded (dsDNA) comprising the nucleic acid sequence to a Cas endonuclease and a guide RNA molecule capable of directing the Cas endonuclease to a target sequence within the nucleic acid sequence, wherein the guide RNA molecule comprises a guide RNA sequence that, when used in CRISPR-Cas editing, results (or is predicted, e.g., predicted by a computer model, to result) in a primary editing outcome having a frequency at least twice as high as a second most abundant editing outcome.
[Item 16]
14. The method according to claim 13, wherein the guide RNA molecule comprises a guide RNA sequence selected according to the method according to any one of claims 1 to 12.
[Item 17]
15. The method of claim 13 or 14, further comprising introducing a guide RNA molecule and a DNA endonuclease into the cell or cells.
[Item 18]
16. The method of any one of paragraphs 1 to 15, wherein the Cas endonuclease cleaves a target sequence within the nucleic acid sequence to generate double-stranded break ends.
[Item 19]
17. The method according to claim 16, wherein the Cas endonuclease is a Cas9 endonuclease.
[Item 20]
18. The method of any one of paragraphs 13 to 17, wherein the organism, cell or population of cells is a eukaryote.
[Item 21]
20. The method according to claim 18, wherein the organism, cell or population of cells is of animal, fungal or plant origin, preferably the organism, cell or population of cells is mammalian.
[Item 22]
20. The method according to claim 19, wherein the cell is a zygote or a population of cells that form a zygote.
[Item 23]
21. The method of claim 20, further comprising implanting the embryo into a recipient female animal to cause gestation, and suitably culturing the embryo to a later stage of development prior to implantation.
[Item 24]
22. The method according to any one of items 13 to 21, for producing a non-mosaic transgenic animal.
[Item 25]
23. The method according to claim 22, wherein the animal is a rodent, rabbit, sheep, goat, horse, cow, pig, dog, cat, chicken or primate.
[Item 26]
1. A method for editing a nucleic acid sequence in an organism, a cell or population of cells or a cell-free expression system, the method comprising exposing double-stranded (dsDNA) comprising the nucleic acid sequence to a Cas endonuclease and a pair of guide RNA molecules capable of directing the Cas endonuclease to target 5' and 3' flanks surrounding the nucleic acid sequence, wherein the pair of guide RNA molecules comprises a first guide RNA and a second guide RNA that, when used in CRISPR-Cas editing, results (or is predicted, e.g., predicted by a computer model, to result) in a predominant editing outcome having a frequency that is less than four times the frequency of a second most abundant editing outcome.
[Item 27]
27. The method of claim 26, further comprising any one of the features of claims 16 to 25.
[Item 28]
28. A cell, a cell population, or a non-human organism obtained by the method according to any one of items 15 to 27.
Claims (28)
－ 前記核酸配列を標的化する複数のガイドＲＮＡ配列を特定すること、
－ 前記複数のガイドＲＮＡ配列のそれぞれについて編集結果の頻度を決定すること、および
－ 最も豊富な編集結果の頻度が、２番目に豊富な編集結果の頻度の少なくとも２倍を超えると決定される、１つ以上のガイドＲＮＡ配列を選択すること、
を含む、方法。 1. A method for selecting one or more guide RNA sequences for use in CRISPR-Cas editing of a nucleic acid sequence, comprising:
- identifying a plurality of guide RNA sequences targeting said nucleic acid sequence;
- determining a frequency of editing outcomes for each of said plurality of guide RNA sequences; and - selecting one or more guide RNA sequences in which the frequency of the most abundant editing outcome is determined to be at least twice as high as the frequency of the second most abundant editing outcome.
A method comprising:
－ 前記核酸配列を囲む５’フランクと３’フランクを標的化する複数のガイドＲＮＡ配列を特定すること、
－ 前記複数のガイドＲＮＡ配列のそれぞれについて編集結果の頻度を決定すること、および
－ 前記５’フランクを標的化する第１のガイドＲＮＡおよび前記３’フランクを標的化する第２のガイドＲＮＡを含む一対のガイドＲＮＡ配列であって、それぞれのガイドＲＮＡについて最も豊富な編集結果の頻度が２番目に豊富な編集結果の頻度の４倍未満であると決定される一対のガイドＲＮＡ配列、を選択すること、
を含む、方法。 1. A method for selecting a pair of guide RNA sequences for use in CRISPR-Cas editing of a nucleic acid sequence, comprising:
- identifying multiple guide RNA sequences targeting the 5' and 3' flanks surrounding said nucleic acid sequence;
- determining a frequency of editing outcomes for each of said plurality of guide RNA sequences; and - selecting a pair of guide RNA sequences comprising a first guide RNA targeting said 5' flank and a second guide RNA targeting said 3' flank, wherein the frequency of the most abundant editing outcome for each guide RNA is determined to be less than four times the frequency of the second most abundant editing outcome.
A method comprising:
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB2003814.7A GB202003814D0 (en) | 2020-03-16 | 2020-03-16 | Optimised methods for cleavage of target sequences |
| GB2003814.7 | 2020-03-16 | ||
| PCT/GB2021/050650 WO2021186163A1 (en) | 2020-03-16 | 2021-03-16 | Optimised methods for cleavage of target sequences |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2023518379A JP2023518379A (en) | 2023-05-01 |
| JPWO2021186163A5 true JPWO2021186163A5 (en) | 2024-04-22 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022555852A Pending JP2023518379A (en) | 2020-03-16 | 2021-03-16 | Optimized methods for cleavage of target sequences |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US20230167443A1 (en) |
| EP (1) | EP4121524A1 (en) |
| JP (1) | JP2023518379A (en) |
| CN (1) | CN115279900A (en) |
| AU (1) | AU2021238926A1 (en) |
| CA (1) | CA3171406A1 (en) |
| GB (1) | GB202003814D0 (en) |
| WO (1) | WO2021186163A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023105000A1 (en) | 2021-12-09 | 2023-06-15 | Zygosity Limited | Vector |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK3420080T3 (en) * | 2016-02-22 | 2019-11-25 | Caribou Biosciences Inc | PROCEDURE FOR MODULATING DNA REPAIR RESULTS |
| WO2019232494A2 (en) * | 2018-06-01 | 2019-12-05 | Synthego Corporation | Methods and systems for determining editing outcomes from repair of targeted endonuclease mediated cuts |
-
2020
- 2020-03-16 GB GBGB2003814.7A patent/GB202003814D0/en not_active Ceased
-
2021
- 2021-03-16 EP EP21713109.3A patent/EP4121524A1/en active Pending
- 2021-03-16 JP JP2022555852A patent/JP2023518379A/en active Pending
- 2021-03-16 CA CA3171406A patent/CA3171406A1/en active Pending
- 2021-03-16 CN CN202180021728.0A patent/CN115279900A/en active Pending
- 2021-03-16 WO PCT/GB2021/050650 patent/WO2021186163A1/en unknown
- 2021-03-16 AU AU2021238926A patent/AU2021238926A1/en active Pending
- 2021-03-16 US US17/911,184 patent/US20230167443A1/en active Pending
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