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Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles.

Nature methods (2020-06-10)
Georg Michlits, Julian Jude, Matthias Hinterndorfer, Melanie de Almeida, Gintautas Vainorius, Maria Hubmann, Tobias Neumann, Alexander Schleiffer, Thomas Rainer Burkard, Michaela Fellner, Max Gijsbertsen, Anna Traunbauer, Johannes Zuber, Ulrich Elling
ABSTRACT

CRISPR-Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a 'Bioscore' and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR-Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.

MATERIALS
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Sigma-Aldrich
L-Glutamine solution, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
Penicillin-Streptomycin, with 10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, 0.1 μm filtered, BioReagent, suitable for cell culture
SAFC
MEM Non-essential Amino Acid Solution (100×), without L-glutamine, liquid, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Sodium pyruvate solution, 100 mM, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, HEPES Modification, With 4500 mg/L glucose, L-glutamine, and 25 mM HEPES, without sodium bicarbonate and pyruvate, powder, suitable for cell culture
Sigma-Aldrich
2-Mercaptoethanol, for synthesis