このレポートに関連する利害の関係はありません。

すべての動物の世話と実験手順は、国立衛生研究所と動物介護施設使用委員会 (IACUC)、ミシガン大学からガイドラインを追った。RNase フリー ソリューションを使用して、ピペットのプロトコル全体のヒント。RNase 除染液が製造元のガイドライン (表を参照)、次に作業領域、ピペット、チューブ、および遠心分離機をきれい。
1. sgRNA ターゲットの選択
<ol><li>Web ブラウザーを使用して web ページを開く: http://crispor.tefor.net8<ol>
<li>興味の目的の編集を並ぶシーケンスの ~ 60 塩基対 (bp) を入力 (つまり30 bp 5' と 3' の目的の編集)。</li>
		<li>線虫ゲノムをプルダウン メニューから選択します。</li>
		<li>Protospacer 隣接するモチーフを選択 (20 bp NGG - SpCas9、SpCas9-HF1、eSPCas9 1.1) プルダウン メニューから。</li>
		<li>[送信] をクリックします。結果ページで、関心の編集に最も近い上位ランク ターゲット シーケンスを選択します。並ぶ 60 bp 入力シーケンス内の適切なターゲット ・ サイトがない場合は 100 までクエリ シーケンスを展開 bp (すなわち50 bp 目的のいずれかの側を編集)。</li>
	</ol>
</li>
	<li>利用可能なソース、例えばsynthego から以下の仕様で 20 mer ターゲット sgRNA を取得: 3 nmol;変更はありません。</li>
	<li>受領後、遠心チューブを含む凍結乾燥 sgRNA > 室温で 1 分間 12,000 × g。チューブにヌクレアーゼ フリーのテの 60 μ L を追加し、軽く P200 ピペットを使用して 15-20 回上下ピペッティング再停止します。最終濃度は 50 μ M です。</li>
</ol>2 相同監督修復テンプレート デザイン
<ol><li>SsODN の HDR テンプレート含む設計: 最初と最後の変異を並ぶ 4) 50 bp 5' と 3' の相同性腕 3) NGG PAM シーケンス内で Gs の一方または両方のサイレント突然変異、2) ユニークなフレームに制限酵素サイト 1) 関心の突然変異。(図 1)9,10。<ol>
<li>NGG PAM 一連の突然変異が可能でない場合は、ssODN ヘッダー テンプレートの sgRNA を介した胸の谷間を防ぐために sgRNA ターゲット認識シーケンス内で 5-6 サイレント変異をご紹介します。</li>
	</ol>
</li>
	<li>使用可能なソース、例えばidtdna から次のパラメーターを「Ultramer DNA オリゴヌクレオチド」を取得: スケール: 4 nmol;定式化: どれも;浄化: 標準脱塩。</li>
	<li>受領後、遠心チューブを含む凍結乾燥 ssODN > 室温で 1 分間 12,000 × g。軽く P200 ピペットを使用して 15-20 回上下ピペッティングによるにヌクレアーゼ フリー ddH2O の最終濃度 100 μ M の ssODN を中断再。</li>
</ol>3. 設計遺伝子型プライマー
<ol><li>400 ~ 700 bp PCR 増幅11を生成するゲノム修飾を側面に設定プライマーを設計します。</li>
	<li>シングル、特定私アンプリコン11を生成する PCR サイクリング条件を最適化します。</li>
</ol>4. 注入ミックスを準備します。
<ol><li>2 分 4 ° C で最大速度でテーブル 1試薬を遠心分離します。</li>
	<li>順番は、ヌクレアーゼ フリー PCR チューブに表 1試薬を追加します。</li>
	<li>P20 ピペットでピペッティング 10 回上下に優しくによって徹底的にミックスします。</li>
	<li>注入ミックス室温で 10 分間、インキュベートします。</li>
</ol>5. 注入のプロトコル
<ol><li>注入ミックス12の約半分をインジェクション ピペットをロードします。注射針は下駄や中断に備えて注入ミックスの残りを保存します。</li>
	<li>20 ~ 30 p.s.i. は、段階的な流れるソリューション12を生成できるように、マイクロ ピペット チップを破る。 注: より大きい直径のヒント否定的動物の健康に影響を与えるし、F1後代の収量を減少させます。</li>
	<li>注入 10-15 若い成虫は両方生殖腕の中で可能であれば12。ない場合は、片腕生殖腺への注入で十分です。</li>
	<li>OP50 シード 35 線虫成長メディア (NGM) プレートに注入された動物 (P0) 室温で 1-2 時間を回復するを許可します。その後、白金線ワームを使用して個々 の OP50 シード 35 mm NGM プレートに単一の注入 P0動物は12を選ぶ。</li>
</ol>6. 画面 P0板と単一 mCherry(+) F1s
<ol><li>2 日間投与後、蛍光顕微鏡を用いた咽頭で mCherry を表現する F1子孫を含んでいる P0版を識別する (図 1一日 2-3)。ほとんどの mCherry(+) F1動物を含む 3 つの P0プレートを選択します。</li>
	<li>シングル 24 36 mCherry(+) F1のワームを使用しての合計のための 8-12 mCherry(+) F1動物選ぶ、3 つの選択した P0プレートから (図 1一日 2-3)。 注: mCherry(-) 動物はこれらの P0プレートからも指摘することができますが、正しく識別する確率はあまり動物の編集 (図 2 a) を下げます。</li>
	<li>個々 の F1s を self-fertilize し、室温で 1 〜 2 日のための卵を産みます。</li>
</ol>7. シングル ワーム PCR および遺伝子型解析
<ol><li>産卵の 1-2 日後に、ワーム ピック (表 2図 1一日 4-5) を使用してワームの換散バッファーの 7 μ L を含む個別 PCR ストリップ管キャップに F1s を転送します。</li>
	<li>遠心チューブ下部に動物をもたらす、-80 ° C 1 時間で管を固定するために室温で 1 分最大速度で PCR チューブ。 注: ワームが-80 ° C で不明確に保存されます。</li>
	<li>次のプログラムを使用してたちで冷凍ワームを溶解: 95 ° C、15 分、60 分の 60 ° C 4 ° C を保持します。</li>
	<li>セットアップ PCR マスター ミックス表 3に示すように。</li>
	<li>きれいな PCR チューブに PCR マスター ミックスの 21 μ L を追加し、手順 3 からワームの換散の 4 μ L を追加します。ピペットを使用してミックスし、PCR プログラム (材料表参照) 製造業者の指針を次を実行します。</li>
	<li>製造元の指示に従って、DNA 清潔濃縮キットを使用して PCR 反応を浄化 (材料表を参照してください)。スピンの列に 10 μ L の水を追加することによって DNA を溶出します。</li>
	<li>表 4に示すように、セットアップ制限酵素マスターをミックス。</li>
	<li>各洗浄の PCR 反応に制限酵素マスターミックスプロの 10 μ L を追加し、37 ° C13で 1-2 時間インキュベートします。</li>
	<li>個別は、120 V 1 × トリス-酢酸-EDTA (TAE) バッファー14の使用で実行 1.5% の agarose のゲルの PCR の製品を消化しました。</li>
</ol>8. 識別と編集された動物のシーケンスの検証
<ol><li>潜在的な編集動物14 (図 14-5 日) を示すバンドの存在のため消化酵素サンプルを確認します。 注: は、バンドのサイズおよび/またはバンディング パターン予期しない、複雑な制限の酵素の消化力の変化として観察することができます潜在的な塩基変異を識別するために N2 コントロールを読み込みます。</li>
	<li>潜在的な編集した動物を識別した後ワーム換散の残りの 3 μ L を使用し、PCR 反応を繰り返します。きれいにしていないか、プログラムが完了した後、制限酵素は PCR 反応を消化します。別 1% の agarose のゲルの PCR の反作用をロードし、ゲル抽出キット15を使用してバンドを抽出 (材料表を参照してください)。</li>
	<li>サンガー シーケンス16ゲルが F1 のプライマーを使用して正しく編集した動物 (図 1 a) を識別する DNA を抽出 注: 読み取りは、ヘテロ野生型の存在のためのピークをオーバーレイし、対立遺伝子を設計しました。</li>
	<li>取得してホモ動物検証ヘテロ接合体の編集された動物、単一 8 122動物 7-8 の手順に従います。</li>
</ol>

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頼むこの原稿の批判的研究室のメンバーに感謝いたします。系統は、線虫の遺伝学センター研究インフラストラクチャ プログラム (P40 OD010440) の NIH のオフィスによって資金が供給されるによって提供されました。頼む研究室での研究は NIH (R01 NS094678) や筋ジストロフィー協会 (MDA382300) からの補助金によってサポートされて A.A.B. に

CRISPR Cas9 システムのモデル生物のゲノムを正確に変更するための強力かつ効果的なツールです。ここでは、そのキメラ sgRNAs20と相まって ssODN HDR 有効テンプレートに線虫の遺伝子の点変異の高効率生成を示します。重要なは、示す共同選択マーカーとして使用する蛍光 RNP 配信が編集効率を生成する使いやすさと手法の信頼性を強調.
線虫ゲ...

最近の技術の進歩が根本的に形質転換し、正確にエンジニアのゲノムに能力を加速します。特に、RNA 誘導エンドヌクレアーゼの興味のターゲット シーケンスに近い二重鎖切断 (DSB) を誘導する Cas9 に頼る、CRISPR Cas9 システムは広くモデル有機体の使用の大半のゲノムを正確にエンジニアに使用されています生物医学研究1,2,3,4。大幅に CRISPR Cas9 の使用は、ゲノムはc. の elegans5のような困難な種でも編集ロックを解除が。種に関係なく編集システム基づく CRISPR Cas9 ゲノムと点突然変異を生成する 3 つのコア コンポーネントに依存しています: 1) Cas9 エンドヌクレアーゼ、ターゲット シーケンスと 3) 設計ユーザーに Cas9 エンドヌクレアーゼを指示する 2) シングル ガイド RNA (sgRNA)2興味の目的の編集を含むホモロジー監督修理 (HDR) テンプレートです。
ターゲットの sgRNA と Cas9 を紹介するために使用できるいくつかの方法があるプラスミッド、RNA ウイルス ベースの配信方法6などのセルにヌクレアーゼ。最近、中古複合 sgRNA Cas9 Ribonucleoproteins (結合) の直接配信は、CRISPR ベース Cas9 ゲノム編集7で強力で効率的なツールとして浮上しています。中古複合 CRISPR Cas9 結合の直接配信はいくつかのメリット、すなわち: 1) 結合細胞のトランスクリプションのための必要性のバイパス、翻訳、2) 結合が急速にクリアされ、特異性を増加するには可能性があります利用可能な時間の短縮オフ ターゲット胸の谷間、そして 3) 結合に非ネイティブのシーケンスをランダムな統合により宿主ゲノムに導入を回避する外国の DNA ・ RNA 要素が含まれていません。一緒に、これらの属性の可能性が高いターゲットに CRISPR 編集オフターゲット効果を最小限に抑えながらの短いバーストを提供します。
線虫 c. エレガンスのサイト固有の genomic 変更を導入するための簡単かつ効率的なプロトコルについて述べる。このプロトコルでは、ターゲットに正しく編集した動物を明確に同定のジェノタイピング戦略と配信、sgRNA Cas9 RNP 錯単一鎖オリゴヌクレオチド (ssODN) HDR テンプレート デザイン sgRNA 含まれています。この戦略を使用すると、目的のサイト固有の変更を回復できる、だけでなく他の非特定塩基の変異を回復も可能性があります。したがって、当社の戦略は、1 つの戦略を使用してモノラル対立、bi 対立、対立シリーズの生成を許可、F1世代で塩基変異を生成できます。

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">CRISPRevolution sgRNA&#160; EZ Kit</td>
			<td style="width:216px;">Synthego Inc.</td>
			<td style="width:344px;"></td>
			<td style="width:227px;">chimeric sgRNA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nuclease-free TE</td>
			<td>Synthego Inc.</td>
			<td>provided with the sgRNA kit EZ kit</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nuclease-free water</td>
			<td>Synthego Inc.</td>
			<td>provided with the sgRNA kit EZ kit</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4 nmole Ultramer DNA Oligo</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>ssODN HDR template</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alt-R S.p. HiFi Cas9 Nuclease 3NLS</td>
			<td>Integrated DNA Technologies</td>
			<td>1078728</td>
			<td>Cas9 protein</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">pCFJ90&#160;</td>
			<td>Addgene</td>
			<td>19327</td>
			<td>Pmyo-2::mCherry Marker Plasmid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNA Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>D4004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Zymoclean Gel DNA Recovery Kit</td>
			<td>Zymo Research</td>
			<td>D4002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Q5 Hot Start High-Fidelity 2X Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0494L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass Borosilicate Glass Micropipettes</td>
			<td>Sutter Instruments</td>
			<td>BF100-78-10</td>
			<td>OD: 1.0mm. ID: 0.78mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma Hydrochloride</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MgCl2</td>
			<td>Sigma</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NP-40</td>
			<td>Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RNaseZap Decontamination Solution</td>
			<td>Fisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and facile pipeline for generating genomic point mutants in c. elegans using crispr/cas9 ribonucleoproteins

クラスター化された定期的に散在させていて回文繰り返し (CRISPR) - CRISPR - に関連したタンパク質 9 (Cas9) 原核生物の適応免疫の防衛システムが抱き込まれてしまった正確な真核生物ゲノム工学のための強力なツールとして線虫のゲノムの点変異の効率的で正確な世代キメラ シングル ガイド Rna (sgRNA) と CRISPR Cas9 Ribonucleoproteins (結合) を使用して迅速かつ簡単な方法を紹介します。SgRNA ターゲットの選択、ホモロジー監督修復 (HDR) テンプレート デザイン、CRISPR Cas9 RNP 錯化と配信、および正しく編集した動物の堅牢かつ迅速な同定を可能にする遺伝子型戦略のパイプラインについて述べる。我々 のアプローチは、変異動物安易な世代とゲノムの任意の場所の同定が可能だけでなく、高効率および減らされたスクリーニング作業負荷で約 4-5 日で他の複雑な塩基の対立遺伝子の検出を容易にも。

人間スーパーオキシドジスムターゼ 1 (SOD1) の変異は、家族性筋萎縮性側索硬化症、麻痺と死の17に必ずや壊滅的な神経変性疾患の 10 ~ 20% を占めます。ヒト SOD 1 です (図 1 b) c. の elegans SOD 1 タンパク質と 55% のアイデンティティおよび 70% の類似性を共有進化上保存されたタンパク質です。シンプルさ、可能性、CRISP...

Watch this Scientific Journal Video about A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins at JoVE.com

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

迅速かつ安易なパイプラインを生成する突然変異点ゲノムにc. の elegansを用いた CRISPR/Cas9 Ribonucleoproteins

ここでは、CRISPR Cas9 ribonucleoproteins と相同性依存修理テンプレートを使用して線虫のゲノム エンジニア リング方法を提示します。

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

a-rapid-facile-pipeline-for-generating-genomic-point-mutants-c

Research

JoVE Journal

Genetics

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

7.5K ビュー.  University of Michigan. ここでは、CRISPR Cas9 ribonucleoproteins と相同性依存修理テンプレートを使用して線虫のゲノム エンジニア リング方法を提示します。

ビデオ: A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

でヒストンminiSOGを使用した光遺伝学ランダム突然変異誘発<em&gt; C。エレガンス</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

遺伝学

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 ...

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 ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

アフリカツメガエルのリープフロッギング CRISPR/Cas9 ベースの始原生殖細胞の移植

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

点突然変異修復 CRISPR/Cas9 とひと細胞での SsODN によって触媒の機能として敷地内変異原性を調べるための標準方法論

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9 仲介の対象となる統合生体内で相同性を介した終わり参加ベースの戦略を使用して

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

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. ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

作る条件付き変異体の人間のマラリア原虫マラリアに CRISPR/Cas9 遺伝子の編集

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

人間でタグ内因性のタンパク質誘導多能性幹細胞 CRISPR/Cas9 を使用して

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

CRISPR/Cas9リボヌクレオタンパク質媒介精密遺伝子編集チューブエレクトロポレーション

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...