Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
John G. Doench,Nicolo Fusi,Meagan E Sullender,Mudra Hegde,Emma W Vaimberg,Katherine F Donovan,Ian Smith,Zuzana Tothova,Zuzana Tothova,Craig B. Wilen,Robert C. Orchard,Herbert W. Virgin,Jennifer Listgarten,David E. Root +13 more
TLDR
Recently devised sgRNA design rules are used to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results, and a metric to predict off-target sites is developed.Abstract:
CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.read more
Citations
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Journal ArticleDOI
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.
Maximilian Haeussler,Kai Schönig,Helene Eckert,Alexis Eschstruth,Joffrey Mianné,Jean-Baptiste Renaud,Sylvie Schneider-Maunoury,Alena Shkumatava,Lydia Teboul,Jim Kent,Jean-Stéphane Joly,Jean-Paul Concordet +11 more
TL;DR: It is found that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro, and it is demonstrated that the best predictions can significantly reduce the time spent on guide screening.
Journal ArticleDOI
Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells.
Robin M. Meyers,Jordan Bryan,James M. McFarland,Barbara A. Weir,Ann E. Sizemore,Han Xu,Neekesh V. Dharia,Phillip G. Montgomery,Glenn S. Cowley,Sasha Pantel,Amy Goodale,Yenarae Lee,Levi D. Ali,Guozhi Jiang,Rakela Lubonja,William F. Harrington,Matthew Strickland,Ting Wu,Derek C. Hawes,Victor A. Zhivich,Meghan R. Wyatt,Zohra Kalani,Jaime J. Chang,Michael Okamoto,Kimberly Stegmaier,Todd R. Golub,Jesse S. Boehm,Francisca Vazquez,Francisca Vazquez,David E. Root,William C. Hahn,Aviad Tsherniak +31 more
TL;DR: CERES, a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens while accounting for the copy number–specific effect, is developed and found that CERES decreased false-positive results and estimated sgRNA activity for both this data set and previously published screens performed with different sg RNA libraries.
Journal ArticleDOI
Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors
Andrew V. Anzalone,Luke W. Koblan,Luke W. Koblan,Luke W. Koblan,David R. Liu,David R. Liu,David R. Liu +6 more
TL;DR: This work analyzes key considerations when choosing genome editing agents and identifies opportunities for future improvements and applications in basic research and therapeutics.
Journal ArticleDOI
CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing
Kornel Labun,Tessa G. Montague,Maximilian Krause,Yamila N. Torres Cleuren,Håkon Tjeldnes,Eivind Valen +5 more
TL;DR: This major update of CHOPCHOP introduces functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions, and incorporates new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes.
Journal ArticleDOI
CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens.
TL;DR: CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets.
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Journal ArticleDOI
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
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Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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