pengyu | GeneCopoeia™

Cat #	Product Name	Ex/Em (nm)	Applications
C047	LysoBeacon™ Blue	370/425	Lysosome labeling
C048	LysoBeacon™ Green	443/505	Lysosome labeling

Introduction

T7 Endonuclease I (T7EI) is a structure-selective enzyme that recognizes and cleaves mismatched DNA, cruciform DNA structures, Holliday structures or junctions, heteroduplex DNA, as well as nicked double-stranded DNA. This enzyme has a preference for single stranded over double stranded DNA with cleavage occurring at the first, second or third phosphodiester bond that is 5′ to the mismatch. In CRISPR/TALEN experiments, T7E1 can accurately recognize insertions and deletions of ≥2 bases that are generated by non-homologous end joining (NHEJ) activity.

GeneCopoeia’s T7 endonuclease I assay kits, which contain T7E1 and positive controls for both target PCR and indel mismatch cleavage, provide a simple and fast workflow for CRISPR/TALEN validation and screening. T7E1 is compatible with a broad range of PCR buffers and does not usually require purification of the PCR product prior to digestion. By incubating re-annealed PCR fragments with T7 endonuclease I, the presence of two shorter bands of predicted sizes usually means that CRISPR/TALEN has successfully introduced indel mutations at the targeted chromosomal site.

T7 endonuclease I assay kits can also be used with our Target site PCR kits and Target site PCR cloning kits for IndelCheck™ CRISPR/TALEN insertion or deletion (indel) detection system.

Advantages

More accuracy with high-quality T7E1
Easy to use with optimized conditions and positive control
Can be a complete system to simplify your CRISPR/TALEN validation and edited clone screening by IndelCheck™ detection system.

Applications

Applications for T7 Endonuclease I (T7E1) include:

Validate the function of CRISPR sgRNAs or TALENs (Figure 1)
Screen for genome-edited cell line clones
Resolve four-way junction or branched DNA
Detect or cleave heteroduplex and nicked DNA
Randomly cleave linear DNA for shotgun cloning

CRISPR sgRNA/TALEN Functional Validation

Figure 1. CRISPR or TALEN functional validation using the IndelCheck™ system. Cells transfected with CRISPR or TALEN plasmids are harvested in bulk, followed by generation of a PCR product using primers flanking the target site with the Target site PCR kits. The PCR product is denatured, followed by re-annealing, leading to a population of double strand fragments, some of which contain mismatches. These mismatches are detected by the T7 endonuclease I Assay kits. If CRISPR or TALEN is active in the cell, then cleavage products will be visible on an agarose gel.

A			B
M	–	+	M	–	+


Figure 2. T7 Endonuclease I digestion of genomic amplicons modified by Cas9-sgRNA. Control cells (-) should only have a single band corresponding to uncut amplicon. Amplicons from sample cells (+) transfected with active Cas9-sgRNA yield 3 bands: 1 unmodified + 2 cleavage products of predicted sizes.

Resources

User manuals IndelCheck^TM Insertion or Deletion Detection System V2.0 (includes T7 Endonuclease I Assay Kit) Technical Notes IndelCheck^TM: A Powerful CRISPR/TALEN Validation & Screening Tool Genome Editing In Mammalian Cells: What Do I Do Next? Material Safety Data Sheet IndelCheck^TM Insertion or Deletion Detection System MSDS Ver 2.0 (includes T7 Endonuclease I Assay Kit)

Related Services

Validation service

GeneCopoeia also offers CRISPR/TALEN validation services using the IndelCheck™ CRISPR/TALEN insertion and deletion detection system. We can validate the CRISPR/TALEN you purchased from us using HEK293 cells (human), NIH3T3 cells (mouse), or your specific cell line(s). Please contact us at inquiry@genecopoeia.com or 1-301-762-0888.