CRISPRa workflow diagram with stable dCas9-VPR expression

CRISPR activation workflow with Lentiviral dCas9-VPR and synthetic crRNA:tracrRNA (right side) or Lentiviral expressed sgRNA (left side).

Tighter control of CRISPR activation with the Strict-R Inducible Lentiviral CRISPRa System

Figure 1: K562 cells were transduced with lentiviral particles containing the respective dCas9-VPR constructs at MOIs <0.3. Cells were selected with 10 µg/ mL blasticidin for two weeks with passaging every 3 to 4 days. After blasticidin selection, stable cell lines were transduced with either a CRISPRmod CRISPRa lentiviral sgRNA targeting POU5F1 or a CRISPRmod CRISPRa lentiviral non-targeting control (NTC) at MOIs of 0.3. After 48 hours, cells were selected with 2.5 µg/µL puromycin for 5 days. Cells were plated in 96-well plates at 20,000 cells/well and stimulated with 0.5 µg/ mL doxycycline and 0.25 nM Shield1 for 24 hours prior to harvest. Total RNA was isolated and relative gene expression was measured using RT-qPCR. The relative expression of POU5F1 was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC. The combination of the TREG3G system and the FKBP12-derived destabilizing domain (DD) used in the Strict-R Inducible Lentiviral CRISPRa System significantly reduces background target gene activation (leakiness, purple) in the unstimulated state while enabling potent target activation with the addition of two highly cell-permeable small molecules.

Robust activation and minimal leakiness with the Strict-R Inducible Lentiviral CRISPRa System across cell lines and gene targets

Figure 2: U2OS, Hela, and human induced pluripotent stem cells (hiPSCs) expressing either the Strict-R Inducible Lentiviral CRISPRa System or dCas9-VPR under control of the constitutive hEF1α promoter (const. VPR, in black) were transduced with CRISPRmod™ CRISPRa lentiviral sgRNA targeting EGFR or ASCL1 or a CRISPRmod CRISPRa lentiviral non-targeting control (NTC) at MOIs of 0.3. After 48 hours, cells were selected with 2.5 µg/µL puromycin for 5 to 7 days. Cells were plated in 96-well plates at 20,000 cells/well and stimulated with 0.5 µg/ mL doxycycline and 0.25 nM Shield1 for 48 hours prior to harvest. Total RNA was isolated and relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC. Robust target gene upregulation was observed with the Strict-R Inducible Lentiviral CRISPRa System after 48 hours of stimulation, comparable to levels achieved with the constitutive dCas9-VPR system, with minimal background target gene activation (leakiness) observed in the unstimulated cell populations.

FACS can be used to select for Strict-R Inducible Lentiviral CRISPRa positive cells with high EGFP co-expression to enrich for CRISPRa activity in Jurkat cells

Figure 3: Jurkat cells were transduced with Strict-R Inducible Lentiviral CRISPRa EGFP particles at an MOI <0.3. Cells were expanded and then FACS was performed to sort the cells into two categories, GFP low and GFP hi (sic), based on fluorescence intensity. Following sorting, cells were replated in 6-well dishes and allowed to recover and expand. Cells were then transduced with CRISPRmod CRISPRa lentiviral sgRNA targeting EGFR or POU5F1 or a CRISPRmod CRISPRa lentiviral non-targeting control (NTC) at MOIs of 0.3. After 48 hours, cells were selected with 2.5 µg/µL puromycin for 5 days. Cells were plated in 96-well plates at 20,000 cells/well and stimulated with 0.5 µg/ mL doxycycline and 0.25 nM Shield1 for 48 hours prior to harvest. Total RNA was isolated and relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC. Robust target gene upregulation was observed after 48 hours of stimulation with substantially more target activation in the GFP hi populations. Importantly, minimal background target gene activation (leakiness) was detected in both GFP low and GFP hi populations of unstimulated cells indicating enrichment for high effector expression does not significantly increase background activation.

Drug-induced activation of ASCL1 leads to upregulation of downstream targets DLL1 and DLL3 in hiPSCs

Figure 4:  Human induced pluripotent stem cells (hiPSCs) expressing the Strict-R Inducible Lentiviral CRISPRa System were transduced with CRISPRmod CRISPRa lentiviral sgRNA targeting ASCL1 or a CRISPRmod CRISPRa lentiviral non-targeting control (NTC) at MOIs of 0.3. After 48 hours, cells were selected with 2.5 µg/µL puromycin for 7 days. Cells were plated in 96-well plates at 20,000 cells/well and stimulated with 0.5 µg/ mL doxycycline and 0.25 nM Shield1 for 48 hours prior to harvest. Total RNA was isolated and the relative gene expression was measured using RT-qPCR for ASCL1 and its downstream Delta gene targets DLL1 and DLL3. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC. Activation of Delta genes by proneural transcription factors such as ASCL1 is an evolutionarily conserved step in neurogenesis that results in activation of Notch signaling and maintenance of an undifferentiated state in a subset of neural progenitors. Stimulation with doxycycline and Shield1 induced potent activation of ASCL1 which resulted in marked upregulation of both DLL3 and DLL1. Crucially, low level background activation of ASCL1 did not significantly impact DLL1 or DLL3 expression.

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LentiBOOST™レンチウイルストランスダクションエンハンサーは、レンチウイルススピンフェクションの有無にかかわらず使用可能な独自処方のトランスダクション試薬です。細胞生存率を維持しながら、ウイルス導入効率を向上させることができます。患者由来の貴重な初代細胞や複雑な動物モデルの構築において特に有用であり、トランスダクション効率の向上により、各編集・導入ステップの成功率を高め、時間およびコスト削減につながります。また、LentiBOOST技術はすでに複数の臨床段階治療法の製造に使用されており、臨床応用を見据えたワークフローへの適用可能性を示しています。

LentiBOOSTはDharmacon試薬カタログから購入可能です。

LentiBOOST技術の詳細については、Revvity LentiBOOSTウェブページをご覧ください。

Supporting Data

LentiBOOST™ Lentivirus Transduction Enhancerを用いたCD8+ T細胞SMARTvector™ shRNAレンチウイルスシステムのトランスダクション効率向上

100,000個のヒト初代CD8+ T細胞に、NTCまたはPPIBを標的とするSMARTvector™ mCMV tGFPレンチウイルスコントロール粒子を、30,000 TU（MOI 3、緑）または70,000 TU（MOI 7、紫）で、1:100希釈のLentiBOOSTトランスダクションエンハンサーとともに導入しました。細胞は32 °C、800 x gで1時間遠心後、4時間培養し、その後レンチウイルス粒子およびトランスダクションエンハンサーを除去しました。トランスダクション効率（生細胞中のGFP陽性率）および細胞生存率は、トランスダクション5日後にフローサイトメトリーで評価しました。LentiBOOST技術の添加により、細胞生存率に大きな影響を与えることなく、トランスダクション効率が顕著に向上しました。

LentiBOOST™ Lentivirus Transduction Enhancerを用いたCD4+およびCD8+ T細胞Edit R™ All in one sgRNA/Cas9レンチウイルスシステムのトランスダクション効率向上

2名のドナー由来の100,000個のヒト初代CD4+およびCD8+ T細胞に、Edit R GFP DeliveryコントロールmCMVを250,000 TUで、1:100希釈のLentiBOOSTトランスダクションエンハンサーとともに導入しました。細胞は32 °C、800 x gで1時間遠心後、一晩培養し、その後レンチウイルス粒子およびトランスダクションエンハンサーを除去しました。トランスダクション効率および細胞生存率は、トランスダクション72時間後にフローサイトメトリーで評価しました。LentiBOOST技術の添加により、細胞生存率に大きな影響を与えることなく、トランスダクション効率が顕著に向上しました。

LentiBOOST™ Lentivirus Transduction Enhancerを用いたStrict R™ Inducible CRISPRaレンチウイルスシステムによるヒト人工多能性幹細胞（hiPSC）のトランスダクション効率向上

10,000個のWTC hiPSCに、Strict R™ Inducible EGFP dCas9 VPRレンチウイルス粒子を20,000 TU（MOI 2、緑）または40,000 TU（MOI 4、紫）で、1:100希釈のLentiBOOSTトランスダクションエンハンサーとともに導入しました。細胞は32 °C、800 x gで1時間遠心後、一晩培養し、その後レンチウイルス粒子およびトランスダクションエンハンサーを除去しました。トランスダクション効率および細胞生存率は、トランスダクション72時間後にフローサイトメトリーで評価しました。LentiBOOSTの添加により、細胞生存率に大きな影響を与えることなく、トランスダクション効率が顕著に向上しました。