CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

By P. Mali, John D Aach, P. Benjamin Stranges, Kevin M. Esvelt, Mark Moosburner, Sriram Kosuri, Luhan Yang, George M. Church. 2013

1,784 citations recorded by the source corpus.

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

Subfields: Molecular Biology

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Citations in the corpus

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila (2014, background): “Simultaneous expression of two gRNAs can be used to create defined deletions (5, 8), to target two genes simultaneously (20), or to mutagenize a single gene by offset nicking (21, 22).”
The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells (2016, positive): “10 20 Current strategies to reduce CRISPR-Cas9 off-target effects include the use of partially inactivated Cas9 nickases 21,22 and a catalytically dead Cas9 fused to the FokI endonuclease domain, 23–25 both of which require the design and delivery of two gRNAs.”
A protein tagging system for signal amplification in gene expression and fluorescence imaging (2014, positive): “…Cas9 (dCas9), which can be targeted to any sequence in the mammalian genome using sequence-specific small guide RNAs (sgRNAs) (Cheng et al., 2013; Farzadfard et al., 2013; Gilbert et al., 2013; Hu et al., 2014; Kearns et al., 2014; Maeder et al., 2013; Mali et al., 2013; PerezPinera et al., 2013).”
Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing (2014, background): “As noted above, an important weakness of the paired Cas9 nickase approach is that single monomeric nickases can introduce indel mutations with high frequencies at certain target sites 4, 10, 11, 13 .”
CasOT: a genome-wide Cas9/gRNA off-target searching tool (2014, background): “For example, Cas9/gRNA seems to have considerable offtarget effects in cultured human cells (Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013) and in several organisms (Fujii et al., 2013; Li et al., 2013; Shan et al., 2013).”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “When targeted to paired adjacent sequences, the FokI domains of these programmable, site-specific nucleases form a dimer that activates the nuclease activity, thus creating a DSB near their binding sites (Figure 1a).”
Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins (2014, background): “…exploited this system to develop RNA-guided endonucleases or engineered nucleases (RGENs) that enable targeted genome editing in cultured human cells (Cho et al. 2013a; Cong et al. 2013; Jinek et al. 2013; Mali et al. 2013b), zebrafish embryos (Hwang et al. 2013), and bacteria (Jiang et al. 2013).”
sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites (2014, methodology): “In order to enhance genome editing specificity, hspCas9 D10A is used in complex with paired-gRNA to generate double nicking with a 59 overhang [3,28].”