Stay on-target with your genome editing projects

For pre-clinical and other applications where you need to minimize off-target Cas9 activity and are using transfectable cells, All-in-one Cas9 SmartNickase plasmids are an excellent choice. Unlike our newer Cas9 constructs, this first generation Cas9 SmartNickase—EF1α-hspCas9-nickase-H1-gRNA All-in-one Cas9 SmartNickase Plasmid—does not contain a selectable marker for identifying transfected cells and is now available as an intact, circular plasmid.Unlike the wildtype Cas9 protein which introduces double-strand breaks (DSBs), the Cas9 SmartNickase introduces paired nicks at the gRNA-directed site. Creating nicks favors the higher-fidelity homologous recombination process over non-homologous end joining (NHEJ), with paired nicking shown to reduce off-target activity by 50- to 1,500-fold in cell lines, and to facilitate gene knockout in mice without losing on-target cleavage efficiency1.

• Conveniently deliver Cas9 Nickase and gRNA with a single vector
• Reduce off-target activity
• Drive Cas9 Nickase expression with the EF1α promoter, which provides medium expression levels in most cell types, including primary cells and stem cells
• Express gRNA from the H1 promoter for maximum specificity and choice of targets
• Ensure efficient import of Cas9 Nickase to the nucleus with N-term and C-term nuclear localization signals (NLSs)
• Boost Cas9 Nickase gene expression and stabilize the transcript via the WPRE regulatory element after the C-term NLS
• Easily detect and/or purify the Cas9 Nickase protein with the N-term myc-tag

As with all of our Cas9 Nickase delivery options, the EF1α-hspCas9-nickase-H1-gRNA Plasmid is functionally validated and comes backed by our expert technical support team—if you’ve got a genome engineering question just ask by emailing tech@systembio.com.

Why an HR targeting vector is a recommended

Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. HR donors can supply elements for positive or negative selection ensuring easier identification of successful mutation events. In addition, HR donors can include up to 6-8 kb of open reading frame for gene knock-ins or tagging, and, when small mutations are included in either 5’ or 3’ homology arms, can make specific, targeted gene edits.

Not sure whether you need a CRISPR/Cas9 plasmid, purified protein, or mRNA?

For This Application	In these types of cells

Use These ProductsMODIFYING ORGANISMS

• Gene tagging


• Transgenic organism generation


• Model organism engineering

Embryos—to create transgenic animalsInjectable Cas9 mRNA & gRNA Synthesis Kits
Cas9 Protein
EGFP-labeled Cas9 ProteinAnimals models—in vivo genome editingAAV-Cas9 Vectors  
Cas9 Protein
EGFP-labeled Cas9 ProteinMODIFYING CELL LINES

• Stable KO, KI, and genome editing of
  somatic cells


• Transgenic cell line generation


• Cell-based disease models

Cells that are transfectableCas9 Plasmids
Cas9 Protein
EGFP-labeled Cas9 ProteinDifficult-to-transfect cell lines:

• Primary cells


• Hematopoietic cells


• Stem cells

AAV-Cas9 Vectors 
Lenti Cas9 SystemsSCREENING

• Genome-wide surveys


• gRNA library screens


• Functional screens

All cell types requiring stable Cas9 overexpressionLenti Cas9 Systems
AAVS1 Safe Harbor Site Cas9
Gene Knock-in System
Cas9 Protein
EGFP-labeled Cas9 ProteinPRE-CLINICAL APPLICATIONS

• Off-target events are of highest concern

All cell types and applicationsCas9 Nickase, available in all delivery formats
Cas9 Protein
EGFP-labeled Cas9 ProteinSIMULTANEOUS ENGINEERING OF MULTIPLE MUTATIONSAll cell types and applicationsMultiplex gRNA cloning kit, compatible with all Cas9 delivery options

References

1. Ran, FA. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Oct 24; 8:2281-2308. PMCID: PMC3969860.

美国SBI代理 System Biosciences,简称SBI，美国加州湾区新成立的生技公司，致力于独特、创新生物技术之开发，以研发利于基因及蛋白质功能鉴定、研究之崭新方法和工具为宗旨。 现阶段研发重心为RNA干扰（RNAi）研究之相关工具。 System Biosciences (SBI) 致力于开发独特、革新的技术，为客户研究蛋白组学和基因组学功能提供研究工具。SBI 是专业的慢病毒产品公司，提供基于慢病毒 美国SBI代理 ExoQuick and ExoQuick-TC Products Product Size Catalog # ExoQuick serum exosome precipitation solution (5 ml) 75 reactions EXOQ5A-1 ExoQuick Plasma prep and Exosome precipitation kit (5 ml ExoQuick plus 500 ul Thrombin at 500U/mL), replaces EXOQ5TD-1 product 75 reactions EXOQ5TM-1 Thrombin Plasma prep for Exosome precipitation (500 ul at 500U/mL), replaces TDEXO-1 product 100 reactions TMEXO-1 ExoQuick serum exosome precipitation solution (20 ml) 300 reactions EXOQ20A-1 ExoQuick-TC for Tissue Culture Media and Urine (10ml) 10 reactions EXOTC10A-1 ExoQuick-TC for Tissue Culture Media and Urine (50ml) 50 reactions EXOTC50A-1 Exosome-depleted FBS Media Supplement - USA Certified 50 ml EXO-FBS-50A-1 Exosome-depleted FBS Media Supplement - USA Certified 250 ml EXO-FBS-250A-1 ExoQuick-LP Lipoprotein Pre-Clear & Exosome Precipitation Kit 5 reactions EXOLP5A-1