title: Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.
abstract: The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments. This was used with a functional assay for protein disruption to perform high-throughput analysis of crRNA activity and specificity. We performed a comprehensive test of target cleavage using crRNAs that contain one and two nucleotide mismatches to the DNA target in the 20mer targeting region of the crRNA, allowing for the evaluation of hundreds of potential mismatched target sites without the requirement for the off-target sequences and their adjacent PAMs to be present in the genome. Our results demonstrate that while many crRNAs are functional, less than 5% of crRNAs with two mismatches to their target are effective in gene editing; this suggests an overall high level of functionality but low level of off-targeting.
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Emily M
authors.initials: EM
authors.lastname: Anderson
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Haupt
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Schiel
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Eldon
authors.initials: E
authors.lastname: Chou
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Hidevaldo B
authors.initials: HB
authors.lastname: Machado
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Žaklina
authors.initials: Ž
authors.lastname: Strezoska
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: Lenger
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Shawn
authors.initials: S
authors.lastname: McClelland
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Birmingham
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA.
authors.forename: Annaleen
authors.initials: A
authors.lastname: Vermeulen
authors.affiliation: Dharmacon, Part of GE Healthcare, Lafayette, CO 80026, USA. Electronic address: anja.smith@ge.com.
authors.forename: Anja van Brabant
authors.initials: Av
authors.lastname: Smith
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D045744:Cell Line, Tumor; D018390:Gene Targeting; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D012313:RNA / Q000235:genetics; D017393:RNA Editing / Q000235:genetics
chemical_list: D049452:Green Fluorescent Proteins; D012313:RNA
keywords: CRISPR-Cas9; Functional assay; Gene editing; Genome engineering; Specificity; crRNA