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Method Article
CRISPR-UMI Step by Step: A protocol for robust CRISPR screening
Ulrich Elling
Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, 1030 Vienna, Austria.
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
CRISPR-UMI extends the existing repertoire of CRSIPR-screening methods. It circumvents cell heterogeneity, a consequence of Cas9 genome editing, by scoring single cell derived clones individually. The strength of this new CRISPR screening method is its robustness towards clonal heterogeneity and clonal outliers and is therefore expected to be most useful in challenging biological screens with strong bottlenecks and clonal effects such as organoid or in-vivo screens.
This step-by-step protocol is an addition to the publication CRISPR-UMI: Single cell lineage tracing of pooled CRISPR/Cas9 screens doi: 10.1038/nmeth.4466. It contains a detailed description for pooled CRISPR screening using CRISPR-UMI. It especially highlights the steps which are critical and unique to the use of CRISPR-UMI. Those critical steps are library preparation at very high complexity of up to 100million unique plasmids, and data analysis were unique guide-UMI pairs are evaluated separately.
Keywords
CRISPR, Screen, Etoposide, iPS, reprogramming, UMI
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.docx
Main document 1 CRISPR-UMI Step by Step Main Document This is the main document for the CRISPR-UMI Step by Step protocol.
- supplement0.zip
Zip_archive 2 Scripts 1-23 A zip archive. contains all Scripts (perl, python, R, sh) mentioned in the Step by Step protocol.
- supplement0.xlsx
Table 1 Primers_oligos table Excel file contains short Sequences Primers and oligo mentioned in the Step by Step protocol
- supplement0.zip
Zip_arichive 3 Sequences 1-4 Vector maps and Sequence files in gbk format. mentioned in the Step by Step protocol.
- supplement0.zip
Zip_archive 1 Files 1-20 A zip archive. contains all Files (Data Files, Intermediate Files and results) mentioned in the Step by Step protocols
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Biological techniques
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This is a preprint. It has not completed peer review.
Method Article
CRISPR-UMI Step by Step: A protocol for robust CRISPR screening
Ulrich Elling
Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, 1030 Vienna, Austria.
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 17 Oct, 2017
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
CRISPR-UMI extends the existing repertoire of CRSIPR-screening methods. It circumvents cell heterogeneity, a consequence of Cas9 genome editing, by scoring single cell derived clones individually. The strength of this new CRISPR screening method is its robustness towards clonal heterogeneity and clonal outliers and is therefore expected to be most useful in challenging biological screens with strong bottlenecks and clonal effects such as organoid or in-vivo screens.
This step-by-step protocol is an addition to the publication CRISPR-UMI: Single cell lineage tracing of pooled CRISPR/Cas9 screens doi: 10.1038/nmeth.4466. It contains a detailed description for pooled CRISPR screening using CRISPR-UMI. It especially highlights the steps which are critical and unique to the use of CRISPR-UMI. Those critical steps are library preparation at very high complexity of up to 100million unique plasmids, and data analysis were unique guide-UMI pairs are evaluated separately.