Reagent Kits

AATI's capillary electrophoresis instruments utilize specially designed reagent kits to analyze diverse nucleic acid samples, from small RNA to genomic DNA, and NGS library quality control.

Reagent Kit Box

CRISPR Discovery Gel Kit

CRISPR Discovery Gel Kit
1000 Sample CRISPR Discovery Gel Kit

The CRISPR Discovery Gel Kit was designed for the automated screening of CRISPR-induced mutation events within a target fragment. Cleavage products from heteroduplexed fragments are separated and measured with a high degree of precision. Post-separation analysis is simplified with the CRISPR Plugin for PROSize® which efficiently calculates the mutation efficiency with unmatched precision and accuracy.

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Components

1,000 Sample Kit
dsDNA 810 Separation Gel 500 mL (DNF-810-0500)
Intercalating Dye 30 µL x 2 (DNF-600-U030)
5X 930 dsDNA Inlet Buffer 300 mL (DNF-355-0300)
5X Capillary Conditioning Solution 100 mL (DNF-475-0100)
1 bp and 6,000 bp Markers 3.2 mL (FA-MRK915F-0003)
Mutation Detection DNA Ladder 1.2 mL x 2 (FS-SLR480-0001)
Mineral Oil 15 mL (FS-SMO15)
Dilution Buffer E 0.1X 60 mL (FS-CLKDBE)

Specifications

Specifications Description
Sample Volume Required 2 µL
Diluent Marker Volume Required 22 µL
DNA Sizing Range 100 bp – 6,000 bp (defined by lower/upper marker)
Separation Resolution Ultra-Short Array: 10 bp @ 500 bp
Short Array: 8 bp @ 500 bp
DNA Sizing Accuracy ± 5% or better
DNA Sizing Precision 2% CV
DNA Fragment Concentration Range 0.005 ng/µL – 2 ng/µL input DNA
Total Electrophoresis Run Time 35 min (Ultra-Short Array, 22-47); 50 min (Short Array, 33-55)

Examples

Fast and accurate screening of large libraries of CRISPR modified lines serves to accelerate discovery and push gene editing forward. The CRISPR Discovery Gel Kit (DNF-910CP) provides researchers with a tool to not only rapidly and accurately identify lines positive for CRISPR induced mutations, but begin to elucidate the nature of the modifications made.

A quality ladder is necessary for all kits designed for the Fragment Analyzer, and the CRISPR Discovery Gel Kit is not an exception. Carefully selected fragments in the Mutation Detection DNA Ladder (FS-SLR480-0001) provide the necessary power to accurately measure cleavage fragments and parent amplicons for the determination of CRISPR mutation success and zygosity.

Figure 1
Figure 1. Separation of the Mutation Detection DNA Ladder (FS-SLR480-0001) performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55) using the CRISPR Discovery Gel Kit (DNF-910CP).

Heteroduplex Cleavage (HDC) assays efficiently identify CRISPR induced mutation events in both pooled and single cell lines. Notably, HDC assays provide a cost-effective method to distinguish between all diploid zygosity events, monoallelic, diallelic homozygous, and diallelic heterozygous. Paired with the Fragment Analyzer, HDC assays provide an automated solution to screening samples for CRISPR mutation events.

Other methods used to screen for CRISPR induced mutations are often time and money consuming. Techniques include: Large scale sequencing – NGS and Sanger – ddPCR, and qPCR. Sequencing provides excellent results but at a high monetary and time cost for a screening method. Digital Droplet PCR (ddPCR), while able to detect HDR and InDels, is unable to determine zygosity. Notably, ddPCR has a high upfront cost for instrumentation as well as a high cost per reaction. While a viable option for screening for mutation events, qPCR has several issues including result quality dependent no primer dimers, exact identity of a mutation cannot be determined, and the mutation frequency of pooled samples cannot be calculated.

Large scale and efficient screening of CRISPR induced mutation events is made possible through the CRISPR Plugin for PROSize® (Developed in conjunction with IDT). The plugin determines mutation success and zygosity for diploid lines via the application of statistical models to the relative abundance of the cleavage products and parent amplicon. Notably, the statistical models employed can measure pooled samples and individual lines equally well. A color coded well layout – purple for positive, white for negative – provides an intuitive graphical user interface to rapidly identify lines positive for gene editing events. As an added benefit the data processed by the CRISPR Plugin can be exported as an easy to read report, in PDF or CSV format.

The CRISPR Plugin is able to not only identify mutation events, but elaborate on the nature of the events. Successful CRISPR induced mutations in a diploid organism have three potential zygosity outcomes: diallelic homozygous, diallelic heterozygous, and monoallelic.

Figure 2 exhibits the CRISPR Plugin electropherogram of a diallelic homozygous CRISPR modified line, with fragments (red and green) and parent amplicon (blue) highlighted for easy reference. Diallelic homozygous lines are characterized by identical CRISPR induced mutations on both alleles of the target gene or genes.

Figure 2
Figure 2. A diallelic homozygous modified line. Both alleles of the target gene have identical modifications following the users CRISPR/Cas9 strategy. The cleavage products are labeled as follows: Fragment 1 > Red, Fragment 2 > Green, and the Parent Amplicon > Blue. The well layout in the upper right corner quickly identifies what lines have had successful modifications, purple wells have modified lines, white wells did not have a gene editing event, while the green well is the selected well. The table on the right provides the relative quantification for the parent amplicon and cleavage products and the percent cleavage.

Figure 3 depicts the CRISPR Plugin electropherogram of a diallelic heterozygous line. Diallelic heterozygous lines are characterized by the successful mutation of both alleles of the target gene by CRISPR/Cas9, with a different modification on each allele. The cleavage products (red and green) and the parent amplicon (blue) are highlighted for easy reference.

Figure 3
Figure 3. A diallelic heterozygous modified line. Two alleles of the target gene have been modified with different mutations following the users CRISPR/Cas9 strategy. The peaks are labeled as followed, Fragment 1 > Red, Fragment 2 > Green, and the Parent Amplicon > Blue. The well layout in the upper right corner quickly identifies what lines have had successful modifications, purple wells have modified lines, white wells did not have a gene editing event, while the green well is the selected well. The table on the right provides the relative quantification for the parent amplicon and cleavage products and the percent cleavage.

The final zygosity possibility for a modified diploid organism is monoallelic, characterized by the successful editing of only one allele of the target gene. Figure 4 shows the CRISPR Plugin electropherogram of a monoallelic line, with the fragments (red and green) and parent amplicon (blue) highlighted for easy reference.

Figure 4
Figure 4. A monoallelic modified line. Only one allele has been modified following the users CRISPR/Cas9 strategy. The peaks are labeled as followed, Fragment 1 > Red, Fragment 2 > Green, and the Parent Amplicon > Blue. The well layout in the upper right corner quickly identifies what lines have had successful modifications, purple wells have modified lines, white wells did not have a gene editing event, while the green well is the selected well. The table on the right provides the relative quantification for the parent amplicon and cleavage products and the percent cleavage.
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