title: Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion.
abstract: CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.|Institut Hospital del Mar d'Investigacions Mediques (IMIM), Barcelona, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Pulido-Quetglas
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.|Institut Hospital del Mar d'Investigacions Mediques (IMIM), Barcelona, Spain.
authors.forename: Estel
authors.initials: E
authors.lastname: Aparicio-Prat
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.|Institut Hospital del Mar d'Investigacions Mediques (IMIM), Barcelona, Spain.
authors.forename: Carme
authors.identifier: 0000-0002-7431-2088
authors.initials: C
authors.lastname: Arnan
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.|Institut Hospital del Mar d'Investigacions Mediques (IMIM), Barcelona, Spain.
authors.forename: Taisia
authors.initials: T
authors.lastname: Polidori
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.
authors.forename: Toni
authors.identifier: 0000-0003-2016-6465
authors.initials: T
authors.lastname: Hermoso
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.|Institut Hospital del Mar d'Investigacions Mediques (IMIM), Barcelona, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: Palumbo
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.
authors.forename: Julia
authors.identifier: 0000-0002-1477-9444
authors.initials: J
authors.lastname: Ponomarenko
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.|Institut Hospital del Mar d'Investigacions Mediques (IMIM), Barcelona, Spain.
authors.forename: Roderic
authors.initials: R
authors.lastname: Guigo
authors.affiliation: Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.|Institut Hospital del Mar d'Investigacions Mediques (IMIM), Barcelona, Spain.|Department of Clinical Research, University of Bern, Bern, Switzerland.|Department of Medical Oncology, Inselspital, University Hospital and University of Bern, Bern, Switzerland.
authors.forename: Rory
authors.identifier: 0000-0003-4607-2782
authors.initials: R
authors.lastname: Johnson
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics*; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D055785:Gene Knockdown Techniques / Q000379:methods*; D017393:RNA Editing / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems