iGUIDE Method for CRISPR Off-Target Detection
With the advent of genome editing technologies, scientists have recognized that these technologies can be prone to nonspecific or off-target activity. As many areas of the genome are sensitive and can give rise to abnormalities if mutated, it is imperativ
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Introduction Following the development of CRISPR-based technologies and other designer nucleases, a number of methods were developed to measure off-target activity, or the amount and location of DNA editing not intended by design [1–7]. These methods centered around quantifying the distributions of DNA double-strand breaks (DSBs) by different means, leading to each having their own strengths and limitations. Initial methods to identify DSBs either used whole genome sequencing approaches or recognized marking of large foreign DNA could indicate regions of DSBs (such as with integration defective retroviral vectors or adeno-associated viral vectors) [1, 3]. Other methods identify DSBs by fixing cells and their DNA to isolate factors associated with DSBs or DNA-repair [2, 5, 6]. Lastly but similar to previous work, small protected oligonucleotides were found to incorporate into DSBs at a higher frequency than large DNA, providing a high-throughput targeted approach at identifying DSBs over the course of exposure to designer nucleases [3, 7]. The GUIDE-seq method was originally developed by the laboratory of J. Keith Joung, MD, Ph.D. and the
Mario Andrea Marchisio (ed.), Computational Methods in Synthetic Biology, Methods in Molecular Biology, vol. 2189, https://doi.org/10.1007/978-1-0716-0822-7_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Christopher L. Nobles
protocol was further improved upon by the laboratory of Frederic D. Bushman, Ph.D., by applying practices developed for unbiasedtargeted sequencing, resulting in iGUIDE [3, 7]. While experimental-based off-target analysis is commonly applied to CRISPR-gRNA selection at later stages of research, its value can be applied much earlier in gRNA selection within the appropriate model system. Discordance can often be observed between in silico methods of off-target analysis and experimental or in vivo methods [3]. The latter typically revealing fewer off-target sites associated with a specific gRNA, likely due to cellular-based restrictive factors associated with the DNA of a cell within eukaryotic organism [8, 9]. Therefore, experimental-based off-target analyses typically carry more relevant results that could have a significant impact on the decision to use a gRNA. Both GUIDE-seq and iGUIDE share similar protocols. GUIDE-seq uses a 34-bp double-stranded oligo-dinucleotide (dsODN) while iGUIDE uses a 46-bp dsODN to mark DSBs, and both methods use a nested-PCR approach to enrich for incorporated dsODN and identify the flanking DNA by pairedend sequencing. The ODN marker is included during delivery of targeted-nucleases (plasmid, mRNA, or enzyme-gRNA complex) to cells or tissue. The marker incorporates into DSB induced by nucleases (and other sources of DSBs) and will persist within cells when it has terminal phosphorothioate bonds, preventing exonuclease digestion [3, 9, 10]. Genomic DNA is harvested from the cells or tissue after a recovery period, typically hours to days. This genomic DNA is then sheared into small fragments
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