Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.

Abstract:

:CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.

journal_name

Nat Biotechnol

journal_title

Nature biotechnology

authors

Doench JG,Fusi N,Sullender M,Hegde M,Vaimberg EW,Donovan KF,Smith I,Tothova Z,Wilen C,Orchard R,Virgin HW,Listgarten J,Root DE

doi

10.1038/nbt.3437

subject

Has Abstract

pub_date

2016-02-01 00:00:00

pages

184-191

issue

2

eissn

1087-0156

issn

1546-1696

journal_volume

34

pub_type

杂志文章
  • 1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life.

    abstract::We present 1,003 reference genomes that were sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) initiative, selected to maximize sequence coverage of phylogenetic space. These genomes double the number of existing type strains and expand their overall phylogenetic diversity by 25%. Comparativ...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.3886

    authors: Mukherjee S,Seshadri R,Varghese NJ,Eloe-Fadrosh EA,Meier-Kolthoff JP,Göker M,Coates RC,Hadjithomas M,Pavlopoulos GA,Paez-Espino D,Yoshikuni Y,Visel A,Whitman WB,Garrity GM,Eisen JA,Hugenholtz P,Pati A,Ivanova NN,Woyke

    更新日期:2017-07-01 00:00:00

  • Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation.

    abstract::Whole-genome mRNA quantitation can be used to identify the genes that are most responsive to environmental or genotypic change. By searching for mutually similar DNA elements among the upstream non-coding DNA sequences of these genes, we can identify candidate regulatory motifs and corresponding candidate sets of core...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt1098-939

    authors: Roth FP,Hughes JD,Estep PW,Church GM

    更新日期:1998-10-01 00:00:00

  • Selective amplification of protein-coding regions of large sets of genes using statistically designed primer sets.

    abstract::We describe a novel approach to design a set of primers selective for large groups of genes. This method is based on the distribution frequency of all nucleotide combinations (octa- to decanucleotides), and the combined ability of primer pairs, based on these oligonucleotides, to detect genes. By analyzing 1000 human ...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt0796-857

    authors: López-Nieto CE,Nigam SK

    更新日期:1996-07-01 00:00:00

  • Epigenome-wide association data implicate DNA methylation as an intermediary of genetic risk in rheumatoid arthritis.

    abstract::Epigenetic mechanisms integrate genetic and environmental causes of disease, but comprehensive genome-wide analyses of epigenetic modifications have not yet demonstrated robust association with common diseases. Using Illumina HumanMethylation450 arrays on 354 anti-citrullinated protein antibody-associated rheumatoid a...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.2487

    authors: Liu Y,Aryee MJ,Padyukov L,Fallin MD,Hesselberg E,Runarsson A,Reinius L,Acevedo N,Taub M,Ronninger M,Shchetynsky K,Scheynius A,Kere J,Alfredsson L,Klareskog L,Ekström TJ,Feinberg AP

    更新日期:2013-02-01 00:00:00

  • Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.

    abstract::Development of a cell therapy for diabetes would be greatly aided by a renewable supply of human beta-cells. Here we show that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice. Upon glucose stimulation of the implant...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt1393

    authors: Kroon E,Martinson LA,Kadoya K,Bang AG,Kelly OG,Eliazer S,Young H,Richardson M,Smart NG,Cunningham J,Agulnick AD,D'Amour KA,Carpenter MK,Baetge EE

    更新日期:2008-04-01 00:00:00

  • Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

    abstract::We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing mic...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/76469

    authors: Brenner S,Johnson M,Bridgham J,Golda G,Lloyd DH,Johnson D,Luo S,McCurdy S,Foy M,Ewan M,Roth R,George D,Eletr S,Albrecht G,Vermaas E,Williams SR,Moon K,Burcham T,Pallas M,DuBridge RB,Kirchner J,Fearon K,Mao J,

    更新日期:2000-06-01 00:00:00

  • Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases.

    abstract::Genetic studies of human disease have traditionally focused on the detection of disease-causing mutations in afflicted individuals. Here we describe a complementary approach that seeks to identify healthy individuals resilient to highly penetrant forms of genetic childhood disorders. A comprehensive screen of 874 gene...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.3514

    authors: Chen R,Shi L,Hakenberg J,Naughton B,Sklar P,Zhang J,Zhou H,Tian L,Prakash O,Lemire M,Sleiman P,Cheng WY,Chen W,Shah H,Shen Y,Fromer M,Omberg L,Deardorff MA,Zackai E,Bobe JR,Levin E,Hudson TJ,Groop L,Wang J,

    更新日期:2016-05-01 00:00:00

  • Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.

    abstract::Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nucle...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.3408

    authors: Wang J,Exline CM,DeClercq JJ,Llewellyn GN,Hayward SB,Li PW,Shivak DA,Surosky RT,Gregory PD,Holmes MC,Cannon PM

    更新日期:2015-12-01 00:00:00

  • Biomagnetic isolation of antigen-specific CD8+ T cells usable in immunotherapy.

    abstract::Isolating antigen-specific T lymphocytes is hampered by the low frequency of the cells and the low affinity between T-cell receptors (TCR) and antigen. We describe the isolation and purification of antigen-specific CD8+ T lymphocytes from mixed T-cell populations. Magnetic beads coated with major histocompatibility co...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt0398-281

    authors: Luxembourg AT,Borrow P,Teyton L,Brunmark AB,Peterson PA,Jackson MR

    更新日期:1998-03-01 00:00:00

  • Growth factors regulate the survival and fate of cells derived from human neurospheres.

    abstract::Cells isolated from the embryonic, neonatal, and adult rodent central nervous system divide in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2), while retaining the ability to differentiate into neurons and glia. These cultures can be grown in aggregates termed neurospheres, which conta...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/88158

    authors: Caldwell MA,He X,Wilkie N,Pollack S,Marshall G,Wafford KA,Svendsen CN

    更新日期:2001-05-01 00:00:00

  • Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model.

    abstract::MicroRNAs (miRNAs) are increasingly implicated in the regulation of metastasis. Despite their potential as targets for anti-metastatic therapy, miRNAs have only been silenced in normal tissues of rodents and nonhuman primates. Therefore, the development of effective approaches for sequence-specific inhibition of miRNA...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.1618

    authors: Ma L,Reinhardt F,Pan E,Soutschek J,Bhat B,Marcusson EG,Teruya-Feldstein J,Bell GW,Weinberg RA

    更新日期:2010-04-01 00:00:00

  • Resolution of cutaneous inflammation after local elimination of macrophages.

    abstract::We constructed an immunotoxin, composed of an antibody directed against the high-affinity IgG receptor CD64 and Ricin-A, with the aim of resolving chronic inflammation through elimination of activated macrophages. In vitro, this immunotoxin proved very efficient in inducing apoptosis in activated macrophages, leaving ...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/71908

    authors: Thepen T,van Vuuren AJ,Kiekens RC,Damen CA,Vooijs WC,van De Winkel JG

    更新日期:2000-01-01 00:00:00

  • Sensitive digital quantification of DNA methylation in clinical samples.

    abstract::Analysis of abnormally methylated genes is increasingly important in basic research and in the development of cancer biomarkers. We have developed methyl-BEAMing technology to enable absolute quantification of the number of methylated molecules in a sample. Individual DNA fragments are amplified and analyzed either by...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.1559

    authors: Li M,Chen WD,Papadopoulos N,Goodman SN,Bjerregaard NC,Laurberg S,Levin B,Juhl H,Arber N,Moinova H,Durkee K,Schmidt K,He Y,Diehl F,Velculescu VE,Zhou S,Diaz LA Jr,Kinzler KW,Markowitz SD,Vogelstein B

    更新日期:2009-09-01 00:00:00

  • Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein.

    abstract::RNA interference (RNAi) induced by short interfering RNA (siRNA) allows for discovery research and large-scale screening; however, owing to their size and anionic charge, siRNAs do not readily enter cells. Current approaches do not deliver siRNAs into a high percentage of primary cells without cytotoxicity. Here we re...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.1541

    authors: Eguchi A,Meade BR,Chang YC,Fredrickson CT,Willert K,Puri N,Dowdy SF

    更新日期:2009-06-01 00:00:00

  • Recent Supreme Court decisions and licensing power.

    abstract::The recently decided eBay and MedImmune cases interject both reassurance and uncertainty into the present patent licensing landscape, affecting the strategic decisions to be considered during negotiations. ...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt0208-183

    authors: Giordano-Coltart J,Calkins CW

    更新日期:2008-02-01 00:00:00

  • Functional interrogation and mining of natively paired human VH:VL antibody repertoires.

    abstract::We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly ...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.4052

    authors: Wang B,DeKosky BJ,Timm MR,Lee J,Normandin E,Misasi J,Kong R,McDaniel JR,Delidakis G,Leigh KE,Niezold T,Choi CW,Viox EG,Fahad A,Cagigi A,Ploquin A,Leung K,Yang ES,Kong WP,Voss WN,Schmidt AG,Moody MA,Ambrozak DR

    更新日期:2018-02-01 00:00:00

  • Targeted RNA sequencing reveals the deep complexity of the human transcriptome.

    abstract::Transcriptomic analyses have revealed an unexpected complexity to the human transcriptome, whose breadth and depth exceeds current RNA sequencing capability. Using tiling arrays to target and sequence select portions of the transcriptome, we identify and characterize unannotated transcripts whose rare or transient exp...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.2024

    authors: Mercer TR,Gerhardt DJ,Dinger ME,Crawford J,Trapnell C,Jeddeloh JA,Mattick JS,Rinn JL

    更新日期:2011-11-13 00:00:00

  • At the heart of genetic testing.

    abstract::Genetic testing for rare heart conditions might someday expand to more common cardiac ailments. Already there are signs testing is dramatically changing how some conditions are treated and doctors' definition of who a patient is. Stephen Strauss reports. ...

    journal_title:Nature biotechnology

    pub_type: 新闻

    doi:10.1038/nbt1010-1003

    authors: Strauss S

    更新日期:2010-10-01 00:00:00

  • Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

    abstract::Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional sa...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.4004

    authors: Schmitt TM,Aggen DH,Ishida-Tsubota K,Ochsenreither S,Kranz DM,Greenberg PD

    更新日期:2017-12-01 00:00:00

  • Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart.

    abstract::Systemic gene delivery into muscle has been a major challenge for muscular dystrophy gene therapy, with capillary blood vessels posing the principle barrier and limiting vector dissemination. Previous efforts to deliver genes into multiple muscles have relied on isolated vessel perfusion or pharmacological interventio...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt1073

    authors: Wang Z,Zhu T,Qiao C,Zhou L,Wang B,Zhang J,Chen C,Li J,Xiao X

    更新日期:2005-03-01 00:00:00

  • Mutation detection by electrocatalysis at DNA-modified electrodes.

    abstract::Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified w...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/80301

    authors: Boon EM,Ceres DM,Drummond TG,Hill MG,Barton JK

    更新日期:2000-10-01 00:00:00

  • Microarray reality checks in the context of a complex disease.

    abstract::A problem in analyzing microarray-based gene expression data is the separation of genes causally involved in a disease from innocent bystander genes, whose expression levels have been secondarily altered by primary changes elsewhere. To investigate this issue systematically in the context of a class of complex human d...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt965

    authors: Miklos GL,Maleszka R

    更新日期:2004-05-01 00:00:00

  • Development and function of human innate immune cells in a humanized mouse model.

    abstract::Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strai...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt.2858

    authors: Rongvaux A,Willinger T,Martinek J,Strowig T,Gearty SV,Teichmann LL,Saito Y,Marches F,Halene S,Palucka AK,Manz MG,Flavell RA

    更新日期:2014-04-01 00:00:00

  • Large multiple sequence alignments with a root-to-leaf regressive method.

    abstract::Multiple sequence alignments (MSAs) are used for structural1,2 and evolutionary predictions1,2, but the complexity of aligning large datasets requires the use of approximate solutions3, including the progressive algorithm4. Progressive MSA methods start by aligning the most similar sequences and subsequently incorpora...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/s41587-019-0333-6

    authors: Garriga E,Di Tommaso P,Magis C,Erb I,Mansouri L,Baltzis A,Laayouni H,Kondrashov F,Floden E,Notredame C

    更新日期:2019-12-01 00:00:00

  • Ordered surface carbons distinguish antifreeze proteins and their ice-binding regions.

    abstract::Antifreeze proteins (AFPs) are found in cold-adapted organisms and have the unusual ability to bind to and inhibit the growth of ice crystals. However, the underlying molecular basis of their ice-binding activity is unclear because of the difficulty of studying the AFP-ice interaction directly and the lack of a common...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt1224

    authors: Doxey AC,Yaish MW,Griffith M,McConkey BJ

    更新日期:2006-07-01 00:00:00

  • Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

    abstract::Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transp...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt0998-857

    authors: Pooga M,Soomets U,Hällbrink M,Valkna A,Saar K,Rezaei K,Kahl U,Hao JX,Xu XJ,Wiesenfeld-Hallin Z,Hökfelt T,Bartfai T,Langel U

    更新日期:1998-09-01 00:00:00

  • The chemical evolution of oligonucleotide therapies of clinical utility.

    abstract::After nearly 40 years of development, oligonucleotide therapeutics are nearing meaningful clinical productivity. One of the key advantages of oligonucleotide drugs is that their delivery and potency are derived primarily from the chemical structure of the oligonucleotide whereas their target is defined by the base seq...

    journal_title:Nature biotechnology

    pub_type: 杂志文章,评审

    doi:10.1038/nbt.3765

    authors: Khvorova A,Watts JK

    更新日期:2017-03-01 00:00:00

  • Efficient epitope mapping by bacteriophage lambda surface display.

    abstract::A bacteriophage lambda surface expression system, lambda foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase 1 to make random epitope libraries. The libraries were s...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt0197-74

    authors: Kuwabara I,Maruyama H,Mikawa YG,Zuberi RI,Liu FT,Maruyama IN

    更新日期:1997-01-01 00:00:00

  • Design of stable biologically active recombinant lutropin analogs.

    abstract::Glycoprotein hormones are noncovalent heterodimers comprised of a common alpha subunit and a hormone-specific beta subunit. Secretion and biologic action of these hormones are dependent on the formation of the heterodimer. The human LH beta subunit is unique among the other beta subunits in that it assembles inefficie...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt0797-663

    authors: Garcia-Campayo V,Sato A,Hirsch B,Sugahara T,Muyan M,Hsueh AJ,Boime I

    更新日期:1997-07-01 00:00:00

  • Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library.

    abstract::This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial An...

    journal_title:Nature biotechnology

    pub_type: 杂志文章

    doi:10.1038/nbt828

    authors: Cesaro-Tadic S,Lagos D,Honegger A,Rickard JH,Partridge LJ,Blackburn GM,Plückthun A

    更新日期:2003-06-01 00:00:00