title: CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.
abstract: 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.
authors.affiliation: 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2].
authors.forename: Prashant
authors.initials: P
authors.lastname: Mali
authors.forename: John
authors.initials: J
authors.lastname: Aach
authors.forename: P Benjamin
authors.initials: PB
authors.lastname: Stranges
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Esvelt
authors.forename: Mark
authors.initials: M
authors.lastname: Moosburner
authors.forename: Sriram
authors.initials: S
authors.lastname: Kosuri
authors.forename: Luhan
authors.initials: L
authors.lastname: Yang
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D015534:Trans-Activators / Q000235:genetics*; D058727:RNA, Small Untranslated
chemical_list: D064130:CRISPR-Associated Proteins; D015534:Trans-Activators; D003850:Deoxyribonuclease I; D058727:RNA, Small Untranslated
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