GMP Cas9 Nucleases

Product List

Stringent Manufacturing and Release Specifications

• Manufacturing Specifications

  Test
  		GenCRISPR™ Ultra Cas9 and eSpCas9 Nucleases
  		GMP Grade*	Basic GMP Grade**	RUO Grade
  Facility		GMP Compliant	Non-GMP Compliant	Non-GMP Compliant
  cGMP Guidelines		+++	++	+
  ISO9001		/	/	+++
  ISO13485		+++	+++	/
  Quality Control		+++	+++	+
  Documentation		+++	++	+

  Note:
  *GMP Grade is a specific term that GenScript uses to describe the Cas9 nucleases manufactured in GMP-complaint facility and in compliance with guidelines of Current Good Manufacturing Practice (cGMP), ISO 13485 quality management system standards with stringent process controls and complete documentation records. GenScript is capable of providing documents, site audits, and other support to help with the applications of your projects in specific regions.
  
  **Basic GMP Grade is a branding term that GenScript uses to describe reagents manufactured in compliance with ISO 13485 quality management system standards and with more stringent process controls and relatively complete documentation records. GenScript is capable of providing documents, site audit and other support to help with the applications of your projects in specific regions.
  

• Release Specifications

  Test
  		GenCRISPR™ Ultra Cas9 and eSpCas9 Nucleases
  		GMP Grade	Basic GMP Grade	RUO Grade
  Concentration		10.0 mg/mL±10%	10.0 mg/mL±10%	10.0 mg/mL±10%
  Appearance		Clear, colorless	Clear, colorless	Clear, colorless
  Purity	SDS-PAGE	≥ 95 %	≥ 95 %	≥ 90 %
  	SEC-HPLC	≥ 95 %	≥ 95 %	≥ 95 %
  Activity		≥ 90 % (SpCas9) ≥ 85 % (eSpCas9)	≥ 90 % (SpCas9) ≥ 85 % (eSpCas9)	≥ 90 % (SpCas9) ≥ 85 % (eSpCas9)
  Endotoxin		< 10 EU/mg	< 10 EU/mg	< 10 EU/mg
  Residual DNase		≤ 10 ng/mg	≤ 10 ng/mg	Non-detectable Qualitative Method
  Residual RNase		≤ 1 ng/mg	≤ 1 ng/mg	Non-detectable Qualitative Method
  Residual HCP		< 10 ng/mg	< 10 ng/mg
  Residual HCDNA		≤ 1 ng/mg	≤ 1 ng/mg
  Mycoplasma		< LOD	< LOD
  Sterility		Sterile	Sterile

Case Study

• Consistent high knockout efficiency in primary T cells using different grade Cas9 nucleases
  

  Figure 1: TRAC knock-out in primary T cells using GMP, basic GMP and RUO Grade GenCRISPR™ Ultra Cas9 or GenCRISPR™ Ultra eSpCas9 with electroporation

  The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-Research (Cat. No. Z03621), GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Cat. No. Z03623), GenCRISPR™ Ultra NLS-SpCas9-GMP (Cat. No. Z03623-GMP) or GenCRISPR™ Ultra eSpCas9-2NLS-Research (Cat. No. Z03622), GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Cat. No. Z03624), GenCRISPR™ Ultra eSpCas9-2NLS-GMP (Cat. No. Z03624-GMP) and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services ) for human TRAC gene knock-out by electroporation. After transfection and cell culture, the TRAC knock-out efficiency was measured by FACS. This data indicated that GMP, basic GMP and RUO Grade GenCRISPR™ Ultra Cas9 or GenCRISPR™ Ultra eSpCas9 enable consistent high gene editing efficiency in primary T cells.

  Cell Type:	Primary T cells
  Transfection Method:	Electroporation
  Target Gene:	TRAC
  Cell amount:	1.0 x 106 cells
  Cas9:sgRNA ratio:	1:2
  RNP amount:	25 pmol

• Consistent high editing efficiency in primary T cells in different electroporation scale
  

  Figure 2: TRAC knock-out in primary T cells using GMP Grade GenCRISPR™ Ultra Cas9 or GenCRISPR™ Ultra eSpCas9 with electroporation in different electroporation scale

  Human primary T cells were cultured for the test. 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 Cas9 Nucleases from other suppliers, and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human TRAC gene knock-out by electroporation in a small electroporation scale of 20 μl and a large scale of 100 μl. After transfection and cell culture, the TRAC knock-out efficiency was measured and analyzed by FACS. The data indicated that GMP Grade GenCRISPR™ Ultra Cas9 and GenCRISPR™ Ultra eSpCas9 maintain consistent high editing efficiency in different electroporation scale.

  Electroporation Scale	20 μl	100 μl
  Cas9 nuclease amount	4 μg	20 μg
  Cell amount	1.0 × 106	5.0 × 106
  Cas9: sgRNA (molar ratio)	1:2	1:2
  RNP amount	25 pmol	125 pmol

• High knock-in efficiency in primary T cells using GMP Grade GenCRISPR™ Ultra Cas9 and Ultra eSpCas9
  

  Figure 3: 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 measured and 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:	Electroporation
  Target Gene:	TRAC
  Gene Knocked-in:	GFP
  Cell amount：	1.0 x 106 cells
  Cas9:sgRNA ratio:	1:2
  RNP amount:	25 pmol

• High knockout efficiency in cell lines using GMP Grade GenCRISPR™ Ultra Cas9 and Ultra eSpCas9
  

  Figure 4: CIITA, GM-CSF, TGFBR2 knock-out in Jurkat cells using GenCRISPR™ Ultra SpCas9 or GenCRISPR™ Ultra eSpCas9 with electroporation

  Jurkat cells were cultured for the test. The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-GMP (Z03623-GMP) or GenCRISPR™ Ultra eSpCas9-2NLS-GMP (Z03624-GMP) or wild type Cas9 protein from other supplier and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human CIITA, GM-CSF and TGFBR2 gene knock-out by electroporation. After transfection and cell culture, the gene editing efficiency was analyzed by Sanger sequencing. Both GenScript GenCRISPR™ Ultra NLS-SpCas9-GMP (Z03623-GMP) and GenCRISPR™ Ultra eSpCas9-2NLS-GMP (Z03624-GMP) show high gene knock-out efficiency.

  Cell Type:	Jurkat cells
  Transfection Method:	Electroporation
  Target Gene:	CIITA, GM-CSF and TGFBR2
  Cell amount:	0.5 x 106 cells
  Cas9:sgRNA ratio:	1:3
  RNP amount:	7.5 pmol

Resources

CRISPR RNP User Manual

A guide on how to use GenScript's RNP products for targeted genome editing

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CRISPR RNP Handbook

Enabling RNP-mediated CRISPR gene editing and transforming life science research

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Cas9 and sgRNA Protocol

Simple protocol for CRISPR gene editing using GenCrisprTM Cas9 nuclease

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GenCRISPR gRNA Design Tool

Interactive CRISPR guide RNA design tool

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GenCRISPR™ Gene Editing Solutions

All solutions you need for a successful CRISPR gene editing project

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