Name: generation of genomic deletions in mammalian cell lines via crispr/cas9
Uploaded: 2015-01-03T14:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 at JoVE.com

Thanks to Jason Wright for suggesting the Golden Gate Assembly cloning strategy and Katherine Helming and members of Orkin lab, particularly Jian Xu, Guoji Guo, Elenoe Smith, and Partha Das for helpful discussions. This work was supported by NIH R01HL032259 and P30DK049216 (Center of Excellence in Molecular Hematology) to S.H.O. and NIDDK K08DK093705 to D.E.B.

<ol>
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We have no conflicts of interest related to this report.

CRISPR / Cas9システムは、サイズの範囲のゲノム欠失を生成するために使用され得る。私たちは、削除の周波数が意図された削除サイズに関して反比例して変化することを観察したが、私たちは日常的に複数の二対立削除されたクローンを得100キロバイトまで最大1 Mbの欠失、および削除を回復することができました。我々は、細胞株に順次導入欠失の効率の損失が観察されなかった。この戦略は、多数の遺伝子および要素の組み合わせの欠失を作成するために使用することができる。対立遺伝子欠失クローンを取得するプロセスは、二対立遺伝子欠失12クローンの所望の数を得るために、欠失サイズに基づいてスクリーニングするために必要なクローンの最小数を推定することにより促進することができる。 確率分布における二対立遺伝子欠失の不在とアレル削除を得る能力は、関数の完全な喪失に関連する細胞致死性を示す可能性があります。低FRequencyまたは不在欠失は、（削除をスクリーニングするためのPCRプライマーを検証するために、正対照の不足）が悪いトランスフェクション、非効率的なsgRNAsまたは非効率的なPCRスクリーニングプライマーを含む、多くのシナリオを反映している可能性があります。 GFP +細胞は、トランスフェクション効率（ ステップ5.2参照）の代わりとして使用することができるので、GFP +細胞の減少は、おそらく悪いトランスフェクションを反射し、結果として生じる、削除効率を低下させた。非効率的なsgRNAの制御を助けることができる独立したスクリーニングプライマーを有する2つの異なるsgRNA対を用いてPCRプライマーをスクリーニングし、対立遺伝子の欠失クローンを得る機会を最大化。 GFP +細胞のための細胞選別は、欠失対立遺伝子のために豊かにします。この工程を省略することができるが、省略可能性が高いアレルまたは対立遺伝子欠失を有するものを同定するためのより多くのクローンをスクリーニングする必要になる。トランスフェクション効率を最適化することができる程度に、我々は、ゲノム編集効率を向上させることが予想される。 図2B）の部位で欠失-主な成果は、小さな〜1である。多くの場合、これらの対立遺伝子は、マイクロホモロジーベースの修理34,35の結果であると思われる。それは我々が記述するPCRに基づく検出戦略は、より大きな又はより複雑な挿入、欠失、逆位、または再配列を識別しないことに留意すべきである。これらのイベントはあまり一般的ですが、私たちはどちらも削除も非削除アンプリコンを検出することができているクローンを観察し、さらなる調査の際に、これらのより複雑な成果を反映している。 我々は、大規模なCRISPR /アレルと非欠失クローンからの非欠失対立遺伝子のCas9媒介「瘢痕」（ 図2B参照） を観察した。これらの「傷は &quot;から構成され意図された欠失なしsgRNA切断部位で産生された小インデル（ すなわち、sgRNAs AとBの間に介在するセグメントの欠失）。これらの傷は、多くの場合、sgRNAによって標的認識を中断。そこで我々は、以前に同じsgRNAsを使用してsgRNAsに曝露した細胞における対立遺伝子をターゲット変更に注意を促すだろう。より成功した再標的化戦略は、認識部位の「瘢痕」以前とは異なる独特のsgRNAシーケンスを利用するであろう。 sgRNAs一対のエキソン配列（ 図1B、下部）を認識したときのケースでは、フレームシフト対立さえ欠失の不存在下で製造することができる。したがって、アレル欠失クローンは、機能喪失により非削除されたアレル12上のフレームシフト変異の高頻度にのために濃縮することができる。 38 - CRISPR / Cas9システムの一つの懸念は、オフターゲット効果、 すなわち、意図しないサイト36におけるゲノム修飾である。 R19ヌクレオチドCRISPR / Cas9ベースのオフターゲット効果39の頻度を減らすことができます- ecentレポートは17と短いガイドRNAがあることを示唆している。さらに、ニッカーゼで標的部位ごとに2つのガイドを使用して二重ニッキング戦略は、オフターゲット効果を最小限にしながら7 DSBを作成するために使用することができる。あるいは、RNAiに使用戦略と類似の、我々は、非重複protospacer配列とsgRNAsの異なる対がオフターゲット潜在的とは対照的に観察された表現型は、オンターゲットCRISPR / Cas9修飾の結果であることを実証するために使用されることを示唆している効果。便利な方法は、スクリーニングプライマー（ ステップ2を参照）の単一のセットは、複数のsgRNA対（ 図1A）のために使用することができるように、少なくとも二つの隣接するが重なり合わないsgRNA対を設計することであろう。また、不足している配列および/または破壊された遺伝子を再導入することによって削除細胞株を補完することとの因果関係を立証することができ与えられたゲノム欠失と表現型。 セルラーモデル系での作業生物学者のために、RNAiは機能ゲノミクスのための強力なツールを表現している。 42 -しかし、このアプローチの限界は、標的mRNA転写レベルの不完全な還元、同じ遺伝子を標的とする独立した試薬の効果の不均一性、及び種子ベースおよび非種子効果40を含む既知のオフターゲット効果が含まれている。ゲノム編集戦略は、これらの懸念の多くに対処することを約束し、将来の遺伝的摂動8,36,37のための刺激的な、補完的なアプローチを表している。従来の標的とすることによって、RNAiによる可能性と挑戦的ではない方法で、非コード遺伝的要素の研究は、25に近づくためにさらに、ゲノムの編集ができます。私たちが生産し、機能喪失型対立遺伝子を特徴づけるために、堅牢で具体的な方法として、CRISPR / Cas9によるゲノムの欠失の生成を促進する。

Recent advances in genome engineering technology have allowed for unprecedented opportunities for site-specific modification of the genome. This technology may be utilized to investigate the function of genes and regulatory elements via prospective genetic perturbation. Zinc finger nucleases (ZFNs), transcription-activator like (TAL) effector nucleases (TALENs), and CRISPR/Cas9 RNA-guided nucleases each leverage customizable DNA specificity to localize a nuclease for the introduction of DSBs1&#8211;3. The resulting DSBs can be repaired by indel-forming NHEJ or by homology-directed repair (HDR) using a donor template4.
The CRISPR/Cas9 nuclease pathway, an adaptive immune system in prokaryotic cells5, has been recently adapted for mammalian genome engineering2,3. This tool has been demonstrated to be an inexpensive, efficient, and reliable genome engineering technique6. Briefly, a complex of Streptococcus pyogenes-derived Cas9 nuclease and a sgRNA achieve target recognition via Watson-Crick base-pairing with cognate genomic DNA sequences. sgRNAs include 20-mer sequences complementary to genomic sequences adjacent to an obligate protospacer adjacent motif (PAM) NGG. Cas9 induces a DSB at a predictable position within the target site. Additionally, variants of Cas9 with single-strand cleavage capacity or catalytic inactivity may be used to facilitate &#8220;nicking&#8221; or transcriptional regulation respectively7&#8211;9. CRISPR/Cas9 has been used for a wide range of applications including both knock-in and knockout10,11, large-scale genomic deletions12&#8211;14, pooled library screening for gene discovery15,16, genetic engineering of numerous model organisms10,11,17&#8211;21, as well as gene therapy22,23.
Here we describe a protocol for efficient deletion of desired genomic regions. The protocol includes CRISPR design, cloning, and delivery, as well as deletion, identification, and characterization. Genomic deletions can be generated by the introduction of two CRISPR sgRNAs with Cas9 to induce repair of the resultant two DSBs by NHEJ with deletion of the intervening segment. This strategy has been used to create deletions ranging from one kilobase to over one megabase12. Deletions can be informative for the study of genes and other genetic elements, either in isolation or in combination. There are several potential advantages of genomic deletions as compared to HDR or single-site small indel production. First, this method capitalizes on the high efficiency of NHEJ in many cellular contexts7. The high frequency of deletion limits the number of clones needed to be screened to identify informative clones. Deletion frequency is inversely related to deletion size. Biallelic deletion clones may be retrieved at frequencies at least as great as of probabilistic expectation12. Second, both monoallelic and biallelic deletions may be easily identified and distinguished by conventional PCR, simplifying the screening process. Strategies relying on small indels or point mutations may require RFLP, allele-specific PCR, T7EN1 cleavage assay, Sanger sequencing, RT-qPCR, or immunoblotting, which may be more laborious. Third, by removing a substantial portion of a gene or element of interest, a reliable loss-of-function allele may be obtained. In contrast, frameshift mutations in protein-coding sequences may not always induce nonsense-mediated decay, may produce a hypomorphic or neomorphic allele, or target an exon excluded from an alternate isoform24. Finally, deletions may be particularly revealing for the study of non-coding DNA such as regulatory elements since frameshift mutations as produced by single-site indels would not be relevant25.

<table><tbody><tr><td>T4 polynucleotide kinase</td><td>New England Biolabs</td><td>M0201S</td><td></td></tr><tr><td>T4 DNA ligase (with associated ligation buffer)</td><td>New England Biolabs</td><td>M0202T</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate (ATP)</td><td>New England Biolabs</td><td>P0756S</td><td></td></tr><tr><td>BSA, molecular biology grade</td><td>New England Biolabs</td><td>B9000S</td><td></td></tr><tr><td>BbsI restriction enzyme (with associated NEB Buffer 2.1)</td><td>New England Biolabs</td><td>R0539S</td><td></td></tr><tr><td>pSpCas9(BB) (pX330)</td><td>Addgene</td><td>42230</td><td></td></tr><tr><td>S.O.C. medium</td><td>Life Technologies</td><td>15544-034</td><td></td></tr><tr><td>BTX ECM 830</td><td>Harvard Apparatus</td><td>45-0052</td><td></td></tr><tr><td>BTX solution and 2 mm cuvettes</td><td>Harvard Apparatus</td><td>45-0803</td><td></td></tr><tr><td>pmaxGFP plasmid</td><td>Lonza</td><td>VPA-1003</td><td></td></tr><tr><td>QuickExtract DNA Extraction Solution</td><td>Epicentre</td><td>QE09050</td><td></td></tr><tr><td>HotStarTaq PCR Master Mix Kit</td><td>Qiagen</td><td>203443</td><td></td></tr><tr><td>Zero Blunt PCR Cloning Kit</td><td>Life Technologies</td><td>K2700-20</td><td></td></tr><tr><td>Plasmid</td><td><a href="http://www.addgene.org/42230/" target="_blank"><img src="/img/new_window.png" /> Addgene</a></td><td>42230</td><td></td></tr></tbody></table>

generation of genomic deletions in mammalian cell lines via crispr/cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

この実験の目的は、MEL細胞におけるPIM1の削除だった。複数の非重複sgRNA対（ すなわち、独立protospacer配列）の使用は、オフターゲット効果（ 図1A）をコントロールするのに役立ち得る。複数の独立protospacer配列により共有される一般的なオフターゲット効果とは対照的に一貫性のある表現型は、オンターゲット効果に起因する可能性が高いであろう。各ペアは固有の削除ブレークポイントの生成につながる。 （ すなわち、n〜150塩基対未満で）近接している場合、同一のスクリーニングプライマーはsgRNAsの各セットによって生成される欠失を検出するために使用することができる。ゲノム欠失は、遺伝子（ 図1B）に関して、様々な場所にsgRNAのペアを使用して、遺伝子を破壊することができる。例えば、sgRNAペアが全遺伝子本体の削除のための遺伝子に隣接すること。ペアは、フレームシフトインデルを作成する可能性を有する、2つのエクソン内に位置することができた場合でも、一方または両方のallelESは削除されませんでした。またはペアは、特定のアイソフォームの破壊を可能にするために特定のエクソンに隣接することがあります。 PIM1に使用し、削除戦略は、全遺伝子本体、8キロバイトの削除（ 図2）を削除するフランキングsgRNAsを設計することでした。この戦略により、PIM1遺伝子の比較的小さいサイズに部分的に選択した。この例では、ボトム（クリック）鎖上のトップ（ワトソン）鎖と1 PAM上の1つのPAM（緑）を示している。しかし、DSBは、上部または下部の鎖にPAMシーケンスローカリゼーションとは無関係です。 sgRNA対はボトム鎖上に、上の鎖の両方に、両方のPAM配列を有する、または各一ができる。上述のプロトコルを使用して、2つのsgRNAを設計pX330発現ベクターにクローニングし、そしてGFPレポーター（ 図2A）に沿ってエレクトロポレーションによりMEL細胞に送達した。 GFP +細胞のトップ3％、ポストエレクトロポ二日ソートして、限界希釈でクローン的にプレーティングした。スクリーンステップ2および図2に示されるように説明したようるプライマーを設計した。PCR条件は親MEL細胞から及び「バルク」ソートされた細胞から単離されたgDNAを用いて最適化した。 10日めっき後、gDNAを、すべてのクローンから単離し、非削除（ND）、削除（D）アンプリコンのパターンに応じて、非削除、アレル及び対立遺伝子の欠失クローンを同定したPCRによる欠失についてスクリーニングした（ 図3） 。非欠失クローンは、非削除アンプリコンの存在および削除リコンの有無を有すると同定された。アレルクローンは非欠失および欠失のアンプリコンの両方の存在を有すると同定された。対立遺伝子クローンは、非削除リコンの有無および欠失アンプリコンの存在を有すると同定された。この削除のために、400個のクローンをスクリーニングした126アレル欠失クローンと32二対立DELETを特定している<html>イオンクローン（これは、削除周波数が削除サイズ12によって変化することに留意することが重要である）。対立遺伝子の欠失クローンを選択し、メディア8 mlのフラスコに移した。拡張のために5日間与えた後、各クローンは、二対立遺伝子欠失及びアンプリコンが正確な欠失（ 図2B）を識別するサンガー配列決定に供した確認のためのgDNA PCRにより再試験した。削除アンプリコン内の不均一性は不完全なインデル形成NHEJ修復を反映している。多くの場合インデルを発見monoallelelic欠失クローン非欠失対立遺伝子の配列決定、アレル及び対立遺伝子の欠失クローンの両方が要求される用途のために重要であり得る、偶数非削除対立遺伝子が頻繁CRISPR / Cas9によって編集されることを実証。 RNAは、二対立遺伝子欠失クローンから単離し、PIM1発現（ 図4）の消失を確認するために、RT-qPCRにより分析した。 方法は &quot;&gt; <img alt="図1" src="/files/ftp_upload/52118/52118fig1highres.jpg" /> 削除の可能性戦略の図1.。（A）は 、ゲノムの欠失のための2つの例sgRNAペア（それぞれ黒とオレンジ色で表示）。青い矢印は、非削除リコンを検出するためのプライマーを示し、赤い矢印は、削除リコンを検出するためのプライマーを示している。 sgRNAは17位置および18で赤と青で強調表示されsgRNA-サイト-1/2と位置17との間の予測Cas9切断を示す赤い線でsgRNA-サイト-B 1 / B 2で紫とオレンジ色で強調表示されますおよび18（B）CRISPR指向の切断は、縦の黒い線で示されている。青い矢印は、非削除リコンを検出するためのプライマーを示し、赤い矢印は、削除リコンを検出するためのプライマーを示す。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52118/52118fig1large.jpg" target="_blank">見るにはこちらをクリックしてください</a>この図の拡大版。 <img alt="図2" src="/files/ftp_upload/52118/52118fig2highres.jpg" /> シークエンシングの削除と非欠失アレルを含む、PIM1の欠失を生成し、検出するために、図2の戦略。PIM1 欠失クローンを同定するためのPCRベースのスクリーニング戦略の（A）の回路図。一つのプライマー対が欠失（青い矢印）と一つのプライマー対の内部に配置されているが欠失（赤矢印）の外部に局在している。 sgRNA配列（protospacer配列）が紫色で示されている。縦の赤い線は17位とsgRNA系列の18との間の予測Cas9切断を示している。（B）サンガーシーケンシングがsgRNA認識サイトでインデル形成を明らかにしている。 sgRNA配列は、緑、紫、およびPAM配列に示されている。削除イベントはshにあるダッシュマークと挿入の同等の数によって自身が青で強調表示されます。赤い縦線は17位とsgRNAの18との間に、予測された切断部位を示す。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52118/52118fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/52118/52118fig3highres.jpg" /> 図3。非削除、アレル、および対立遺伝子クローンを同定する代表ゲル。各クローンについて、左車線が非削除アンプリコン（青で「ND」）と右車線を表し、赤の欠失アンプリコン（「D」を表し）。個々のクローンは、点線で分離されている。 3アレル欠失クローンは、クローン5、6であり、そして配列決定データに見られるように、2 3対立遺伝子の欠失クローンは、cに対する欠失バンドクローン15,17、および13である<html>唯一の13は、欠失接合部に大きな欠失によるサイズが小さい。 <img alt="図4" src="/files/ftp_upload/52118/52118fig4highres.jpg" /> 対立遺伝子の欠失クローンでPIM1式図4.損失。PIM1発現はRT-qPCRにより2対立遺伝子の欠失クローンを算出した。 ΔCtの方法-データは2を使用して、GAPDHに対して正規化した。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> Protospacerシーケンス</td></tr><tr><td> sgRNA-A </td><td> 5&#39;-TAGGAAAATGACTCGTCACT-3 &#39; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;-GTCTATCCTATCTCGATAAA-3 &#39; </td></tr></tbody></table> PIM1の削除の2 sgRNA表1. 20-merのprotospacerシーケンス。 543 &quot;&gt; <tbody><tr height="23"><td height="23" style="height:23px;width:101px;"></td><td style="width:443px;"> Protospacerシーケンスの逆相補</td></tr><tr height="23"><td height="23" style="height:23px;"> sgRNA-A-RC </td><td> 5&#39;-AGTGACGAGTCATTTTCCTA-3 &#39; </td></tr><tr height="23"><td height="23" style="height:23px;"> sgRNA-B-RC </td><td> 5&#39;-TTTATCGAGATAGGATAGAC-3 &#39; </td></tr></tbody></table> 表1から2 sgRNAためのprotospacer系列の表2.逆相補。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td>シーケンス</td></tr><tr><td> sgRNA-A </td><td> 5&#39;-CACCTAGGAAAATGACTCGTCACT-3 &#39; </td></tr><tr><td> sgRNA-A-RC </td><td> 5&#39;-AAACAGTGACGAGTCATTTTCCTA-3 &#39; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;-CACCGTCTATCCTATCTCGATAAA-3 &#39; </td></tr><tr><td> sgRNA-B-RC </td><td> 5&#39;-AAACTTTATCGAGATAGGATAGAC-3 &#39; </td></tr></tbody></table> 表3. Protospacer配列およびそれらの逆が「CACC」と「AAAC」で補完するには、BbsIで制限酵素を使用してpX330ベクターへのクローニングのために追加。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td>シーケンス</td></tr><tr><td> sgRNA-A </td><td> 5&#39;- CACCGTAGGAAAATGACTCGTCACT-3 &#39; </td></tr><tr><td> sgRNA-A-RC </td><td> 5&#39;- AAACAGTGACGAGTCATTTTCCTAC-3 &#39; </td></tr><tr><td> sgRNA-B </td><td> 5&#39;- CACCGTCTATCCTATCTCGATAAA-3 &#39; </td></tr><tr><td> sgRNA-B-RC </td><td> 5&#39;- AAACTTTATCGAGATAGGATAGAC-3 &#39; </td></tr></tbody></table> pX330ベクトルのU6プロモーターから表4 sgRNA発現はCACC配列の後および前のGヌクレオチドの添加によって増強される20マー。余分のGヌクレオチドの付加は、逆相補オリゴの3 &#39;末端（ 例えば、sgRNA-A）におけるCヌクレオチドの添加を必要とする。 20マー（protospacer配列）の最初の位置は既にGヌクレオチドである場合は、別のGを追加する必要がない（ 例えば、sgRNA-B）および逆相補体の最終位置にCを追加する必要なしオリゴ。

Watch this Scientific Journal Video about Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 at JoVE.com

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

CRISPR / Cas9経由哺乳動物細胞株におけるゲノム欠失の生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

generation-of-genomic-deletions-in-mammalian-cell-lines-via-crisprcas9

Universidad de Cartagena UDEC

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Unit 2

Analyzing Gene Expression and Function

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95.2K ビュー.  Harvard Medical School. CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

ビデオ: Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

ハイスループットフォーマットでの遺伝子型CRISPR / Cas9媒介ノックアウト変異体に蛍光PCR-キャピラリーゲル電気泳動法を用いました

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

遺伝学

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

哺乳類細胞におけるCRISPR / Cas9媒介遺伝子ノックアウトの選択依存性および独立した世代

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9媒介1アレル欠失マウス胚性幹細胞でエンハンサー機能を研究するために、

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

発生生物学

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 によるマウスおよび人間の造血前駆細胞の高効率遺伝子破壊

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

CRISPR Cas を用いた哺乳類細胞におけるゲノム編集

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

哺乳類細胞におけるCRISPRベースの遺伝子スクリーンのプール

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

ヒト多能性幹細胞におけるCRISPR-CAS9を用いて定義されたゲノム修飾の生成

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

マイクロRNAクラスターネットワーク解析のためのCRISPR遺伝子編集ツール

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

生物学

CRISPR/Cas9システムを用いたセントロメア関連Protein-E遺伝子編集

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

著者スポットライト: CENP-E ノックアウトHeLa細胞の確立 – キネシン-7 CENP-E 生物学とその阻害剤を研究するための新しいアプローチ

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

CRISPR/Cas9システムを用いたセントロメア関連プロテイン-E CENP-E-/-ノックアウト細胞株の作製

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...