title: Synthesis of an arrayed sgRNA library targeting the human genome.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) can be employed to introduce double stand breaks into mammalian genomes at user-defined loci. The endonuclease activity of the Cas complex can be targeted to a specific genomic region using a single guide RNA (sgRNA). We developed a ligation-independent cloning (LIC) assembly method for efficient and bias-free generation of large sgRNA libraries. Using this system, we performed an iterative shotgun cloning approach to generate an arrayed sgRNA library that targets one critical exon of almost every protein-coding human gene. An orthogonal mixing and deconvolution approach was used to obtain 19,506 unique sequence-validated sgRNAs (91.4% coverage). As tested in HEK 293T cells, constructs of this library have a median genome editing activity of 54.6% and employing sgRNAs of this library to generate knockout cells was successful for 19 out of 19 genes tested.
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Schmidt
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Schmid-Burgk
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.
authors.forename: Veit
authors.initials: V
authors.lastname: Hornung
mesh_terms: D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D005091:Exons; D055786:Gene Knockout Techniques / Q000379:methods*; D015723:Gene Library; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems