Ice crispr analysis tool

Manufactured by Synthego

Sourced in United States

The ICE CRISPR Analysis Tool is a software application developed by Synthego to analyze CRISPR gene editing experiments. The tool provides a comprehensive analysis of CRISPR-mediated modifications, including indel (insertion/deletion) frequencies and patterns. The ICE CRISPR Analysis Tool offers a streamlined and efficient way to evaluate the outcomes of CRISPR gene editing experiments.

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Lab products found in correlation

Dna extract all reagents kit, by Thermo Fisher Scientific (1 mentions) Evagreen mix, by Bio-Rad (1 mentions) Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions) Quick dna rna miniprep, by Zymo Research (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Sybr gold dna stain, by Thermo Fisher Scientific (1 mentions) T7 endonuclease 1, by New England Biolabs (1 mentions) Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions) P3 buffer, by Lonza (1 mentions) Kod hot start dna polymerase, by Merck Group (1 mentions) 4d nucleofector, by Lonza (1 mentions) Gotaq hot start green master mix, by Promega (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) Snapgene, by GSL Biotech (1 mentions)

5 protocols using ice crispr analysis tool

Genome Editing Analysis in Erythroid Cells

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Genome editing was analyzed in HUDEP-2 cells at days 0 and 9 of erythroid differentiation and in CB and adult mobilized HSPC-derived erythroid cells at days 6 and 14 of erythroid differentiation, respectively. Genomic DNA was extracted from control and edited cells using the PureLink Genomic DNA Mini Kit (Life Technologies), Quick-DNA/RNA Miniprep (ZYMO Research), or DNA Extract All Reagents Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. To evaluate NHEJ efficiency at gRNA target sites, we performed PCR followed by Sanger sequencing and TIDE analysis (tracking of InDels by decomposition) (49 (link)) or ICE CRISPR Analysis Tool (Synthego) (Table 2) (50 (link)).
Digital droplet PCR was performed using EvaGreen mix (Bio-Rad) to quantify the frequency of the 4.9-kb deletion. Short (~1 min) elongation time allowed the PCR amplification of the genomic region harboring the deletion. Control primers annealing to a genomic region on the same chromosome (chr 11) were used as DNA loading control (Table 3).

Weber L., Frati G., Felix T., Hardouin G., Casini A., Wollenschlaeger C., Meneghini V., Masson C., De Cian A., Chalumeau A., Mavilio F., Amendola M., Andre-Schmutz I., Cereseto A., El Nemer W., Concordet J.P., Giovannangeli C., Cavazzana M, & Miccio A. (2020). Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype. Science Advances, 6(7), eaay9392.

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CRISPR-Cas9 Editing Evaluation in Dmd Mice

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CRISPR-Cas9 editing using the single and combo guide strategies was evaluated using the online ICE CRISPR analysis tool (Synthego). The 337-bp region encompassing the in55g1_SD-mediated cleavage site was amplified using the primers indicated in Table 2 using tissue-specific DNA from treated and untreated Dmd Δ52–54 mice. The amplicons were purified using the QIAquick PCR Purification Kit (QIAGEN) and sequenced with Sanger sequencing using the sa_ex55g1_fw primer. Sanger sequencing data from treated mice and untreated mice (control) were uploaded into ICE, which determined the frequency and nature of indel formation.

Rok M., Wong T.W., Maino E., Ahmed A., Yang G., Hyatt E., Lindsay K., Fatehi S., Marks R., Delgado-Olguín P., Ivakine E.A, & Cohn R.D. (2023). Prevention of early-onset cardiomyopathy in Dmd exon 52–54 deletion mice by CRISPR-Cas9-mediated exon skipping. Molecular Therapy. Methods & Clinical Development, 30, 246-258.

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Quantifying Genome Editing Efficiency via T7 Endonuclease I Assay

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Genomic DNA from modified cells was isolated according to the manufacturer’s protocol of DNeasy Blood & Tissue Kit (Qiagen). 200 ng of isolated amplified genomic DNA (20 μl of purified PCR product in 1x NEB 2. Buffer (NEB)) was then denatured and reannealed to form heteroduplexes by using the next program on Veriti Thermal Cycler (life Technologies): 95 °C for 10 min; 95 °C to 85 °C ramping at –2 °C/s; 85 °C to 25 °C at – 0.25 °C/s; and 25 °C hold for 1 min. After reannealing process, products were treated with 1 μl of T7 Endonuclease I (NEB) and were analyzed on 10–15 % native PAGE gel. Gels were stained with SYBR Gold DNA stain (Life Technologies) for 30 min and were imaged with a DNA bioimaging system (Bio-Rad). Quantification was based on relative band intensities. Indel percentage was determined: 100 x (1 – (1 – (b + c)/(a + b + c))^1/2), where a is the integrated intensity (determined by using ImageJ 1.53k software) of the undigested PCR product, wherein b and c are integrated intensities of cleaved PCR product.
Some PCR amplicons were also send for Sanger sequencing and then subjected to TIDE analysis^{70 (link)} or ICE CRISPR Analysis Tool from Synthego to determine the degree of genome editing.

Lainšček D., Forstnerič V., Mikolič V., Malenšek Š., Pečan P., Benčina M., Sever M., Podgornik H, & Jerala R. (2022). Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing. Nature Communications, 13, 3604.

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CRISPR Ribonucleoprotein Delivery and Indel Analysis

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sgRNA (Synthego, 50 μM) and NLS-Cas9 protein (Synthego, 20μM) were mixed at 2.5:1 molar ratio (1 μL sgRNA + 1 μL protein) for 10 min at room temperature. 1 × 10⁶ cells were resuspended with 18 μL electroporation buffer, P3 buffer (Lonza, V4XP-3032) for primary cells, SG buffer (Lonza, V4XC-3032) for Raji cells, and SF buffer (Lonza, V4XC-2032) for Nalm6 cells and K562 cells. 2 μL of Cas9/sgRNA complex was added to 18 μL of cells. Cells were electroporated on a Lonza 4D-Nucleofector with X Unit using vendor’s recommended settings for each cell type. Cells were transferred to flasks with warmed media immediately after electroporation. To verify gene editing, DNA was extracted from CRISPR edited cells using DNsasy Blood & Tissue Kit (QIAGEN, 69506). DNA samples were PCR amplified using KOD Hot Start DNA Polymerase (MilliporeSigma, 71842) with the primers listed in Supplementary Table 1 and the PCR product was subjected to Sanger sequencing. Indel frequencies were quantified using the ICE CRISPR Analysis Tool (Synthego).

Yi F., Cohen T., Zimmerman N., Dündar F., Zumbo P., Eltilib R., Brophy E.J., Arkin H., Feucht J., Gormally M.V., Hackett C.S., Kropp K.N., Etxeberria I., Chandran S.S., Park J.H., Hsu K.C., Sadelain M., Betel D, & Klebanoff C.A. (2024). CAR-engineered lymphocyte persistence is governed by a FAS ligand/FAS auto-regulatory circuit. bioRxiv.

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Single-Embryo Mutation Analysis Protocol

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Single embryos were collected at morula (Experiment 5) or blastocyst (Experiments 3–4) stage and lysed in 10 μL lysis buffer (Lucigen, Palo Alto, CA, United States) at 65°C for 6 min and 98°C for 2 min. PCR reactions were performed in two rounds with 35 cycles each. First PCR was composed of 9.2 μL embryo lysis and 10 μL Master Mix (GoTaq Hot Start Green Master Mix, Promega, Madison, United States) at 0.8 μL of 10 μM primers (Table 1) in DNAse/RNAse free water. Second round of PCR was composed of 5 μL from first PCR, 4.2 μL of water, 10 μL Master Mix and 0.8 μL of 10 μM primers in DNAse/RNAse free water. PCR conditions included one cycle at 95°C for 3 min followed by 35 cycles of 95°C for 30 s, primer annealing temperature for 30 s (ZFX: 60°C; OCT4: 54°C) and elongation at 72°C for 30 s, and then 1 cycle at 72°C for 5 min. PCR products were run in a 1% agarose gel and bands were extracted and purified (Qiaquick Gel extraction kit, Qiagen, Hilden, Germany) for Sanger sequencing. Sequencing was performed by services provided by Genewiz (South Plainfield, NJ, United States). Mutations were analyzed by ICE CRISPR Analysis Tool (Synthego) and multiple sequence alignment (SNAPGene, GSL Biotech LLC, Chicago, United States). Indel rate was calculated based on the proportion of embryos with insertions/deletions vs. embryos sequenced.

Camargo L.S., Owen J.R., Van Eenennaam A.L, & Ross P.J. (2020). Efficient One-Step Knockout by Electroporation of Ribonucleoproteins Into Zona-Intact Bovine Embryos. Frontiers in Genetics, 11, 570069.

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