INDEL Detection by Amplicon Analysis (IDAA)
The IDAA method is an in-expensive, fast and efficient High-Throughput Screening (HTS) method used to detect insertions and deletions (indels) in cell lines, tissues, animals or therapeutics settings induced by CRISPRs, ZFNs or TALENs.
By using the IDAA method you will reduce the cost and workload in number of cells, cell clones or animals you will need to screen to obtain your desired outcome. IDAA is highly amenable to HTS and can shorten the timeline and importantly reduce the workload of cell line editing in both basic science, the biomedical drug discovery field and demanding genome editing applications such as therapeutic indel profiling.
The IDAA method is part of the CRISPR INDEL Profiling Platform (CIPP).
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IDAA™ is a trademark of Cobo Technologies
IDAA - Methodology
The IDAA methodology serves an unmet need in the precise editing field and enables fast, precise, robust and cost efficient identification of insertions and deletions (indels), down to the single base, created by a variety of precise gene-targeting approaches that have recently become available in the field such as; TALEN’s1, ZFN’s2,3, Meganucleases4 and CRISPR5. No description for use of this methodology described here, for the detection of precise gene targeting induced indels, have previously been reported and thus IDAA™ may serve as a paradigm shift in precise targeting induced indel detection.
Currently, detection of indels induced by precise gene targeting approaches is performed by laborious assays based on nuclease cleavage of heteroduplex DNA and/or cloning and sequencing of amplicons derived from the target locus6. Both currently used approaches introduce serious risk of detecting artefacts due to cross contamination issues and are time and labor intensive. IDAA eliminates the pitfalls connected with currently used detection of indels and describes a semi-automated procedure applicable to high throughput screening procedures highly warranted in the field.
IDAA - Principle
Recent improvements in a variety of precise gene targeting technologies have enabled, that any gene can be precisely edited in cells and whole organisms. These technological developments have great implications in academic research and biomedicine and show great translational and therapeutic potential in clinical research. The bottleneck in current gene targeting approaches lies in the cost and workload connected with screening and identification of correctly gene edited cells6.
IDAA combines the use of a simple, quick DNA extraction method and a tri-primer single step amplification protocol with precise gene targeting regimes where detection of indels down to 1bp are warranted7. The methodology provides a simple, rapid, cost effective, accurate and robust methodology highly applicable for high throughput identification of precise gene targeting events in cells. The whole IDAA workflow has been outlined and utilized in recent high impact publications6,7,8,9,10.
Overview - Indel Detection Analysis in Genome Editing
Indel Detection Analysis in Genome Editing
Assay time and performance for four indel detection methods: NGS, Sanger sequencing, EMC and IDAA. Time in days on axis shown below for the required steps (blue hexagons) for each method from genome targeted cell pool (grey hexagon) to insight. QC (white triangle) represents necessary quality control steps. The table shown to the right displays the performance (high or low) of the respective methods with regard to sensitivity, resolution, high through-put amenability (HTRP) and cost. Preferred choices are shown in bold font..
Publication: Solving an Unmet Need in Genome Targeting – Fast, Precise and Efficient Indel Detection by Amplicon Analysis -BioZoom 2016 Number 4
IDAA - Selected References
- Mussolino, C. et al.A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39, 9283–93 (2011).
- Santiago, Y. et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. PNAS. 105, 5809–5814 (2008).
- Beumer, K. J. et al. Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila. G3 (Bethesda). 3,1717 25 (2013).
- Marcaida, M. J. et al. Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering. Proc. Natl. Acad. Sci. U. S. A. 105, 16888–93 (2008).
- Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–6 (2013).
- Bennett EP, Jacobi AM, Rettig GR and Behlke MA. Genome Editing & Engineering: “Detection of insertion/deletion (indel) events after genome targeting: Pro’s and con’s of the available methods”, Cambridge University Press, (2017).
- Yang Z, Steentoft C, Hauge C, Hansen L, Thomsen AL, Niola F, Vester-Christensen MB, Frödin M, Clausen H, Wandall HH, Bennett EP. Fast and sensitive detection of indels induced by precise gene targeting. Nucleic Acids Res. 43, e59;1-8 (2015).
- Steentoft C, Bennett EP, Schjoldager KT, Vakhrushev SY, Wandall HH, Clausen H. Precision genome editing: a small revolution for glycobiology. Glycobiology. 2014 Aug;24(8):663-80.
- Yang Z, Wang S, Halim A, Schulz MA, Frodin M, Rahman SH, Vester-Christensen MB, Behrens C, Kristensen C, Vakhrushev SY, Bennett EP, Wandall HH, Clausen H. Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat. Biotechnol. , 2014–2017 (2015).
- Lonowsk, LA, Narimatsu Y, Riaz A, Delay C, Niola F, Duda K, Yang Z, Clausen H, Wandall HH, Hansen SH, Bennett EP and Frödin M. Genome editing using FACS enrichment of nuclease expressing cells and Indel Detection by Amplicon Analysis (IDAA). Nat. Protoc. 12, 581-603 (2017).