Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation

By John G Doench, E. Hartenian, D. Graham, Z. Tothova, Mudra Hegde, Ian Smith, Meagan E. Sullender, Benjamin L. Ebert, Ramnik J. Xavier, D. Root. 2014

1,545 citations recorded by the source corpus.

Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.

Subfields: Molecular Biology, Computational Biology & Bioinformatics

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Citations in the corpus

Discriminative Embeddings of Latent Variable Models for Structured Data (2016, positive): “ovided by CRISPR/Cas9 system, on which the task it to tell whether the guide RNA will direct Cas9 to target DNA. There are 5310 guides included in the dataset. Details of this dataset can be found in Doench et al. (2014); Fusi et al. (2015). We use two variants for spectrum string kernel: 1) kmer-si”
Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. (2016, positive): “Six gRNA sequences targeting the lacZ gene (Doench et al., 2014) inserted into plasmid pAG_TS1_KS001 were used for the CRISPR/Cas9 freezedried assay (Figure S5A).”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “(142) made a library of all possible sgRNAs, tiling across a handful of genes.”
WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system (2015, positive): “Consistent with previous findings [21], there was a strong bias against U and C at the end of functional gRNAs.”
CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering (2016, background): “A study from Doench et al . produced a large dataset to calculate efficiencies across a wide range of sgRNAs (14), and the rules for computationally-aided sgRNA design were recently further refined by the same group (13).”
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR (2016, methodology): “[8] was used as rank-percent values as provided; we also converted parts”