ssODN-mediated CRISPR Cas9 System for Gene Deletion, Mutation or TaggingGenome editing precision using ssODN template with CRISPR/Cas9 system
• Create efficient gene knockin or knockout
• Create point mutations
• Modify sequence segments
• Insert small tags
• Add restriction sites into your region of interest
The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system and single-stranded donor oligonucleotides (ssODN) strategy can be applied to carry out precise gene editing quickly and at a low cost. This strategy allows for generating point mutations, defined sequence modifications or insertion of a small tag.
This is a powerful technique that can be used to examine the relevance of DNA alterations in ex vivo and in vivo systems. Using this technique, various nucleotides and amino acids have been proved to be indispensable for promoter or enhancer activity, as well as for the function or regulation of enzymes, transcription factors and signaling molecules. Mutations known or suspected to be relevant to disease can also be studied.
By designing a single-stranded donor oligonucleotides (ssODN) complimentary to CRISPR guide RNA expression plasmids (single-vector or dual-vector system), point mutations can be introduced to create defined genomic modifications at a low cost and in a short time. Both single gRNA/Cas9 (nuclease) and double nicking systems (nickase) are effective tools to evaluate the roles of specific nucleotides or amino acids in the function of the genetic elements or in proteins of interest.
Included in service:
• 3 top ranking gRNA lentiviral vectors targeting human, mouse, or rat genes*
• Cas9 lentiviral expression vector*
• Non-targeting negative control
• ssODN template
Available as bacterial glycerol stock or viral particle format
*Lentiviral vectors available with your choice of selectable marker
CRISPR Cas9-mediated gene editing technique
To perform CRISPR Cas9-mediated gene editing, select the nuclease you will use (e.g. Cas9 nuclease or paired-nickase with Cas9(D10A). transOMIC technologies uses algorithms to choose the 3 highest scoring gRNA sequences appropriate for your Cas9 with the least chance of off target effects, closest to the 5’ end of the gene (unless otherwise required) to create the DNA breaks around the area intended to be mutated or tagged. We then design a ~180 bp oligonucleotide (ssODN) to mutate/tag that region. The ssODN is additionally mutated to be resistant to further cutting by the gRNAs and if possible to incorporate a restriction enzyme site and or tag for downstream analysis. Primers can then be designed to flank the new construct in situ and the presence of a null, heterozygous event or homologous event rapidly determined by PCR and restriction enzyme digestion.
Figure 1: Template clustering of gRNAs
Schematic depicting the clustering of the gRNAs around the region of interest showing PAM sites in red and desired alteration in blue (T529I).
Figure 2: Template alteration
The lower oligo strand is the ssODN incorporating the T529I mutation (ACC to ATC) with PAM sites changed, a restriction HindIII restriction site inserted and PCR primer binding sites.
Figure 3: Expected electrophoretic gel results of PCR amplification and restriction enzyme digestion post knockout/in.
Lane 1 – 100bp Molecular Weight Marker
Lane 2 – null – no knockin
Lane 3 – heterozygous knockin
Lane 4 – homozygous knockin
|ssODN mediated CRISPR Cas9 Nickase Knock-In/Knock-Out Custom Constructs (All-In One Vector System)||TECU1002-GVO-TRI||1 set||1920 €||DETAILS||Add to Cart|
|ssODN mediated CRISPR Cas9 Nickase Knock-In/Knock-Out Custom Constructs (Two Vector System)||TECU1001-GVO-TRI||1 set||1920 €||DETAILS||Add to Cart|
|ssODN mediated CRISPR Cas9 Nuclease Knock-In/Knock-Out Custom Constructs (All-In One Vector System)||TECU1004-GVO-TRI||1 set||1670 €||DETAILS||Add to Cart|
|ssODN mediated CRISPR Cas9 Nuclease Knock-In/Knock-Out Custom Constructs (Two Vector System)||TECU1003-GVO-TRI||1 set||1670 €||DETAILS||Add to Cart|