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 border="0" cellpadding="0" cellspacing="0" width="842">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:388px;">Name of Material/ Equipment</td>
			<td style="width:168px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase (with associated ligation buffer)</td>
			<td>New England Biolabs</td>
			<td>M0202T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#39;-Triphosphate (ATP)</td>
			<td>New England Biolabs</td>
			<td>P0756S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA, Molecular Biology Grade</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BbsI Restriction Enzyme (with associated NEB Buffer 2.1)</td>
			<td>New England Biolabs</td>
			<td>R0539S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pSpCas9(BB) (pX330)</td>
			<td>Addgene</td>
			<td>42230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S.O.C. Medium</td>
			<td>Life Technologies</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BTX ECM 830</td>
			<td>Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BTX Solution and 2mm Cuvettes</td>
			<td>Harvard Apparatus</td>
			<td>45-0803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pmaxGFP Plasmid</td>
			<td>Lonza</td>
			<td>VPA-1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickExtract DNA Extraction Solution</td>
			<td>Epicentre</td>
			<td>QE09050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStarTaq PCR Master Mix Kit</td>
			<td>Qiagen</td>
			<td>203443</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zero Blunt PCR Cloning Kit</td>
			<td>Life Technologies</td>
			<td>K2700-20</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://www.jove.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://www.jove.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

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Analyzing Gene Expression and Function

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94.9K 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

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

哺乳動物細胞におけるRNAiスクリーニングのためのミッションesiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

生物学

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

複数の遺伝子欠失を持つ酵母菌株の世代のためのグリーンモンスタープロセス

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

テトラサイクリン誘導性（テトオン）shRNAまたはcDNA発現で安定ヒト細胞株の世代

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

哺乳類セルラインの嫌気性成長と維持

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

火花のサイレンシング:弱い電気魚のCRISPR/Cas9ゲノム編集

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

CRISPR/Cas9を用いた初等ヒトB細胞のゲノム工学

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&amp;#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

TGF β媒介性間葉転移(EndMT)およびCRISPR/Cas9遺伝子編集を用いたEndMTエフェクターの機能評価

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

CRISPR/Cas9技術を用いた渡りバッタのホモ接合変異体の構築

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

遺伝子治療アプリケーションのための造血幹細胞および前駆細胞のCRISPR/Cas9遺伝子編集

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9を介したゼブラフィッシュハートの正確なノックイン編集

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...