CRISPR Gene Editing

Cas9 / Cas12a / Cas13a Nucleases

Overview

Cas9 / Cas12a / Cas13a Nucleases for precise RNA-guided DNA or RNA editing and detection

CRISPR 유전자 편집 기술은 유전자 및 세포 치료, 세포주 엔지니어링, 동물 모델 생성, 체외 진단을 포함한 다양한 다운스트림 애플리케이션에서 활용되고 있습니다.
GenScript는 MIT 및 하버드의 유수의 연구실과의 파트너십을 통해 검증된 GMP 등급 및 RUO 등급의 Cas9, Cas12a and Cas13a nuclease, sgRNA 합성 및 HDR donor template 합성서비스 등을 제공하고 있습니다.

Highlights

RNP-mediated CRISPR Gene Editing Consistent high knockout and knock-in efficiency High Lot-to-lot Consistency
• 비바이러스성, 유전자 통합 위험 없음
• 빠른 편집 작용 및 분해
• 낮은 세포 독성 및 면역원성
• 세포주 및 1차 T 세포에서 테스트
• 다양한 electroporation scale
• 대부분의 Ultra Cas9 Nucleases 사용
• In in-vitro cleavage assay
• In gene editing applications

Selection Guide

Applications Recommendations Cat. No. Product Names
Gene KO/KI with high editing efficiency Ultra SpCas9 (wt) Z03621 GenCRISPR™ Ultra NLS-Cas9-Research
Z03623 GenCRISPR™ Ultra NLS-Cas9- basic GMP
Z03623-GMP GenCRISPR™ Ultra NLS-Cas9-GMP
Gene KO/KI with low off-target effects Ultra eSpCas9 (mt) Z03622 GenCRISPR™ Ultra eSpCas9-2NLS-Research
Z03624 GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP
Z03624-GMP GenCRISPR™ Ultra eSpCas9-2NLS-GMP
Gene KI with high KI efficiency SpCas9 with optimized NLS Z03701 GenCRISPR™ Cas9 v1.1
Z03702 GenCRISPR™ Cas9 v1.2
Gene KO/KI following with flow cytometry or other fluorescent assays SpCas9-eGFP fusion protein Z03393 GenCrispr NLS-Cas9-EGFP Nuclease
Z03467
Gene KO/KI with lower MW Cas nucleases AsCas12a Z03502 GenCRISPR™ Cas12a (Cpf1) Nuclease
LbCas12a Z03753 GenCRISPR™ LbCas12a Nuclease
SaCas9 Z03699 GenCRISPR™ SaCas9 2NLS Nuclease
in vitro Diagnostics LwaCas13a Z03486 GenCRISPR™ Cas13a (C2c2) Nuclease
LbuCas13a Z03742 GenCRISPR™ LbuCas13a Nuclease
AsCas12a Z03502 GenCRISPR™ Cas12a (Cpf1) Nuclease
LbCas12a Z03753 GenCRISPR™ LbCas12a Nuclease

Product List

SpCas9 Nuclease (wild type)
Recombinant wild-type S. pyogenes Cas9 nucleases, purified from E. coli expression and contains various nuclear localization sequences (NLSs).

eSpCas9 Nuclease (mutant)
Recombinant mutant S. pyogenes Cas9 nucleases, purified from E. coli expression and contain various nuclear localization sequences (NLSs).

Cas9-eGFP Nuclease
Recombinant S. pyogenes Cas9 nuclease and eGFP fusion protein, purified from E. coli expression, and contains nuclear localization sequence (NLS) and C-terminal 6xHis tag.

SaCas9 Nuclease
Recombinant S. aureus Cas9 nuclease, purified from E. coli expression and contains nuclear localization signals (NLSs) and no tag.

Cat. No. Product Name
Z03699 GenCRISPR™ SaCas9 2NLS Nuclease

Cas9 Nickase D10A
Recombinant S. pyogenes Cas9 Nickase D10A, purified from an E. coli strain expressing the protein, and contains nuclear localization sequences (NLSs).

Cat. No. Product Name
Z03390 GenCrispr NLS-Cas9-D10A Nickase

Cas12a Nuclease
Recombinant Acidaminococcus sp. (strain BV3L6) Cas12a (cpf1) nuclease or Lachnospiraceae bacterium ND2006 Cas12a nuclease, purified from E. coli expression, and contains nuclear localization sequences (NLSs).

Cas13a Nuclease
Recombinant Leptotrichia wadei Cas13a nuclease or Leptotrichia buccalis Cas13a nuclease, purified from E. coli expression.

Case Study

High knockout efficiency in primary T cells (Cat. No. Z03623 and Z03624)

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Figure 1:TRAC knockout in primary T cells using basic GMP Grade GenCRISPR™ Ultra Cas9 or GenCRISPR™ Ultra eSpCas9 with electroporation

The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Cat. No. Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Cat. No. Z03624), and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human TRAC gene knockout by electroporation. After transfection and cell culture, the TRAC knock-out efficiency was analyzed by FACS. This data indicated that both basic GMP Grade GenCRISPR™ Ultra Cas9, and GenCRISPR™ Ultra eSpCas9 enable consistent and high gene knockout efficiency in primary T cells.

Note: No Cargo: No Cas9 protein added (Negative control)
No EP: No electroporation for cell transfection (Negative control)
D3: KO efficiency evaluated after day 3
D6: KO efficiency evaluated after day 6

 

Cell Type: Primary T cells
Transfection Method: Lonza 4D-Nucleofector® Transfection System
Target Gene: TRAC
Cell amount: 1.0 x 106 cells
Cas9: sgRNA ratio: 1:2 (25 pmol : 50 pmol)
Cas9 Nucleases: Z03623 GenCRISPR™ Ultra NLS-Cas9-basic GMP
Z03624 GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP

High knock-in efficiency in primary T cells (Cat. No. Z03623-GMP and Z03624-GMP)

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Figure 2: GFP gene knock-in at TRAC site in primary T cells using GMP Grade GenCRISPR™ Ultra Cas9 or GenCRISPR™ Ultra eSpCas9 with electroporation

The cells were transfected with GMP Grade GenCRISPR™ Ultra NLS-SpCas9-GMP (Cat. No. Z03623-GMP) or GenCRISPR™ Ultra eSpCas9-2NLS-GMP (Cat. No. Z03624-GMP), and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services), as well as dsDNA HDR template (synthesized by GenScript HDR Donor Template Synthesis Services) by electroporation. After transfection and cell culture, the gene knock-in efficiency was analyzed by FACS. Both GenScript GenCRISPR™ Ultra NLS-SpCas9-GMP (Cat. No. Z03623-GMP), and GenCRISPR™ Ultra eSpCas9-2NLS-GMP (Cat. No. Z03624-GMP) maintain high gene knock-in efficiency in primary T cells.

 

Cell Type: Primary T cells
Transfection Method: Lonza 4D-Nucleofector® Transfection System
Target Gene: TRAC
Cell amount: 1.0 x 106 cells
Cas9: sgRNA ratio: 1:2 (25 pmol : 50 pmol)
HDR donor: 2 μg
Cas9 Nucleases: Z03623 GenCRISPR™ Ultra NLS-Cas9-basic GMP
Z03624 GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP

High knock-in efficiency in primary T cells (Cat. No. Z03623-GMP and Z03624-GMP)

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Figure 3: GFP Gene knock-in at TRAC loci in primary T cells by using GenCRISPR™ Cas9 v1.1 or GenCRISPR™ Cas9 v1.2 and sgRNA with electroporation.

The cells were transfected with GenCRISPR™ Cas9 v1.1 (Cat. No. Z03701) or GenCRISPR™ Cas9 v1.2 (Cat. No. Z03702), and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services), as well as dsDNA HDR template (synthesized by GenScript HDR Donor Template Synthesis Services) by electroporation. After transfection and cell culture, the gene knock-in efficiency was analyzed by FACS. Both GenCRISPR™ Cas9 v1.1 and GenCRISPR™ Cas9 v1.2 with optimized NLSs enable high gene knock-in efficiency.

 

Cell Type: Primary T cells
Transfection Method: Lonza 4D-Nucleofector® Transfection System
Target Gene: TRAC
Gene Knocked-in: GFP
Cell amount: 1.0 x 106 cells
Cas9: sgRNA ratio: 1:2 (25 pmol : 50 pmol)
Cas9 Nucleases: Z03623 GenCRISPR™ Ultra NLS-Cas9-basic GMP
Z03624 GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP

High knockout efficiency in Jurkat cells (Cat. No. Z03623-GMP and Z03624-GMP)

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Figure 4: CIITA, GM-CSF and TGFBR2 Gene Knock-out in Jurkat cells by using GMP Grade GenCRISPR™ Ultra NLS-Cas9-GMP or Ultra eSpCas9-2NLS GMP with electroporation

The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-GMP (Cat. No. Z03623-GMP), or GenCRISPR™ Ultra eSpCas9-2NLS-GMP (Cat. No. Z03624-GMP), or a competitor’s product, and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for gene knockout by electroporation. After transfection and cell culture, the knockout efficiency was analyzed by Sanger sequencing. This data shows that both GMP Grade GenCRISPR™ Ultra NLS-Cas9, and GenCRISPR™ Ultra eSpCas9-2NLS enable high gene knockout efficiency in Jurkat cells.

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Cell Type: Jurkat cells
Transfection Method: Neon™ electroporation system
Target Gene: CIITA, GM-CSF, TGFBR2
Cell amount: 0.5 x 106 cells
Cas9: sgRNA ratio: 1:3 (7.5 pmol: 22.5 pmol)
Cas9 Nucleases: Z03623 GenCRISPR™ Ultra NLS-Cas9-basic GMP
Z03624 GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP

High knockout efficiency in NK-92 cells (Cat. No. Z03623)

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Figure 5: CD96 and NKG2A gene knockout in NK-92 cells using GenCRISPR™ Ultra NLS-Cas9-basic GMP with electroporation.

NK-92 cells were cultured for the test. The cells were transfected with GenCRISPR™ Ultra NLS-Cas9-basic GMP (Cat. No. Z03623) and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for CD96 or NKG2A gene knock-out by electroporation. After transfection and cell culture, the gene editing efficiency was analyzed by Sanger sequencing. The results show that GenCRISPR™ Ultra NLS-Cas9-basic GMP (Z03623) has high gene knockout efficiency in NK-92 cells for both genes tested.

 

Cell Type: NK-92 cells
Transfection Method: Lonza 4D-Nucleofector® Transfection System
Target Gene: CD96 , NKG2A
Cell amount: 0.4 x 106 cells
Cas9: sgRNA ratio: 1:1.2
RNP amount: 80 pmol
Cas9 Nucleases: Z03623 GenCRISPR™ Ultra NLS-Cas9-basic GMP
Z03624 GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP

Gene Editing with low off-target effects in 293T cells (Cat. No. Z03623 and Z03624)

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Figure 5: TRAC gene knockout in 293T cells by using GenCRIPSR™ Ultra NLS-Cas9-basic GMP or GenCRIPSR™ Ultra eSpCas9-2NLS-basic GMP and sgRNA with electroporation. A) TRAC gene knockout efficiency analysis. B) Off-target effects analysis on OT1 site. C) Off-target effects analysis on OT2 site.

The cells were transfected separately with either GenCRIPSR™ Ultra NLS-Cas9-basic GMP, or GenCRIPSR™ Ultra eSpCas9-2NLS-basic GMP, or a Competitor’s product, and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for TRAC gene knockout by electroporation. OT1 and OT2 were predicted as the off-target sites and were selected for off-target effects analysis. After transfection and cell culture, the knockout efficiency and off-target effects were analyzed by Sanger sequencing. This data indicates that basic GMP Grade GenCRISPR™ Ultra eSpCas9-2NLS engaged gene editing has low off-target effects, as well as robust on-target editing efficiency.

 

Cell Type: 293T cells
Transfection Method: Neon™ Electroporation Transfection System
Target Gene: TRAC
Cell amount: 0.2 x 106 cells
Cas9: sgRNA ratio: 1:3
Cas9 Nucleases: Z03623 GenCRISPR™ Ultra NLS-Cas9-basic GMP
Z03624 GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP

Resources

CRISPR RNP User Manual
CRISPR RNP User Manual.pdf A guide on how to use GenScript’s RNP products for targeted genome editing
 CRISPR RNP Handbook CRISPR RNP Handbook.pdf Enabling RNP-mediated CRISPR gene editing and transforming life science research
Cas9 and sgRNA Protocol Cas9 and sgRNA Protocol.pdf Simple protocol for CRISPR gene editing using GenCRISPR™ Cas9 nuclease
GenCRISPR gRNA Design Tool GenCRISPR gRNA Design Tool.pdf Interactive CRISPR guide RNA design tool
GenCRISPR Gene Editing Solutions GenCRISPR™ Gene Editing Solutions.pdf All solutions you need for a successful CRISPR gene editing project

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