Highly Parallel Profiling of Cas9 Variant Specificity.

CRISPR-Associated Protein 9 / genetics CRISPR-Cas Systems DNA Breaks, Double-Stranded Gene Editing Genetic Variation HEK293 Cells Humans K562 Cells RNA, Guide, Kinetoplastida / genetics
CRISPR-Cas9 Cas9 specificity variants double-strand break detection off-target analysis tagmentation

Journal

Molecular cell
ISSN: 1097-4164
Titre abrégé: Mol Cell
Pays: United States
ID NLM: 9802571

Informations de publication

Date de publication:
21 05 2020
Historique:
received: 29 08 2019
revised: 24 01 2020
accepted: 27 02 2020
pubmed: 19 3 2020
medline: 25 8 2020
entrez: 19 3 2020
Statut: ppublish

Résumé

Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

Identifiants

DOI: 10.1016/j.molcel.2020.02.023 PMID: 32187529 PMC: PMC7370240
pubmed: 32187529
pii: S1097-2765(20)30143-X
doi: 10.1016/j.molcel.2020.02.023
pmc: PMC7370240
mid: NIHMS1577774
pii:
doi:

Substances chimiques

RNA, Guide 0
CRISPR-Associated Protein 9 EC 3.1.-

Types de publication

Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

794-800.e8

Subventions

Organisme : NHLBI NIH HHS
ID : DP1 HL141201
Pays : United States
Organisme : NHGRI NIH HHS
ID : R01 HG009761
Pays : United States
Organisme : NIMH NIH HHS
ID : R01 MH110049
Pays : United States
Organisme : NHGRI NIH HHS
ID : RM1 HG006193
Pays : United States
Organisme : Howard Hughes Medical Institute
Pays : United States

Informations de copyright

Copyright © 2020 Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of Interests The Broad Institute has filed patent applications related to the work presented here. F.Z. is a co-founder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Sherlock Biosciences.

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Auteurs

Jonathan L Schmid-Burgk (JL)

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Linyi Gao (L)

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

David Li (D)

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Zachary Gardner (Z)

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Jonathan Strecker (J)

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Blake Lash (B)

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Feng Zhang (F)

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Cambridge, MA 02139, USA. Electronic address: zhang@broadinstitute.org.

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Classifications MeSH

questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines associées aux CRISPR › Protéine-9 associée à CRISPR › Highly Parallel Profiling of Cas9 Variant Specificity.
questionsmedicales.fr › Phénomènes génétiques › Régulation de l'expression des gènes › Épigenèse génétique › Extinction de l'expression des gènes › Systèmes CRISPR-Cas › Highly Parallel Profiling of Cas9 Variant Specificity.
questionsmedicales.fr › Phénomènes génétiques › Altération de l'ADN › Cassures de l'ADN › Cassures double-brin de l'ADN › Highly Parallel Profiling of Cas9 Variant Specificity.
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Génie génétique › Édition de gène › Highly Parallel Profiling of Cas9 Variant Specificity.
questionsmedicales.fr › Phénomènes génétiques › Variation génétique › Highly Parallel Profiling of Cas9 Variant Specificity.
questionsmedicales.fr › Cellules › Cellules épithéliales › Cellules HEK293 › Highly Parallel Profiling of Cas9 Variant Specificity.
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › Highly Parallel Profiling of Cas9 Variant Specificity.
questionsmedicales.fr › Cellules › Cellules érythroïdes › Cellules K562 › Highly Parallel Profiling of Cas9 Variant Specificity.
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › ARN non traduit › Petit ARN non traduit › ARN guide › Highly Parallel Profiling of Cas9 Variant Specificity.