A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects.
Journal
Nature methods
ISSN: 1548-7105
Titre abrégé: Nat Methods
Pays: United States
ID NLM: 101215604
Informations de publication
Date de publication:
06 2020
06 2020
Historique:
received:
18
01
2020
accepted:
13
04
2020
pubmed:
20
5
2020
medline:
12
9
2020
entrez:
20
5
2020
Statut:
ppublish
Résumé
Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant-YE1-BE3-FNLS-that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits.
Identifiants
pubmed: 32424272
doi: 10.1038/s41592-020-0832-x
pii: 10.1038/s41592-020-0832-x
doi:
Substances chimiques
RNA
63231-63-0
Cytosine
8J337D1HZY
DNA
9007-49-2
CRISPR-Associated Protein 9
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
600-604Références
Rees, H.A. & Liu, D.R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).
Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).
doi: 10.1038/s41586-019-1161-z
Grünewald, J. et al. CRISPR adenine and cytosine base editors with reduced RNA off-target activities. 37, 1041–1048 (2019).
Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 37, 1041–1048 (2019).
Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).
doi: 10.1038/nbt.3803
Holden, L. G. et al. Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications. Nature 456, 121–124 (2008).
doi: 10.1038/nature07357
Chen, K. M. et al. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G. Nature 452, 116–119 (2008).
doi: 10.1038/nature06638
Teng, B. B. et al. Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1). structure-function relationships of RNA editing and dimerization. J. Lipid Res. 40, 623–635 (1999).
pubmed: 10191286
Teng, B., Burant, C. F. & Davidson, N. O. Molecular cloning of an apolipoprotein B messenger RNA editing protein. Science 260, 1816–1819 (1993).
doi: 10.1126/science.8511591
Gehrke, J. M. et al. An APOBEC3A–Cas9 base editor with minimized bystander and off-target activities. Nat Biotechnol. 36, 977–982 (2018).
Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat Biotechnol. 36, 946–949 (2018).
doi: 10.1038/nbt.4198
Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat Biotechnol. 36, 888–893 (2018).
Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).
doi: 10.1038/nbt.4172
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
doi: 10.1038/nature17946
Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).
Doman, J. L., Raguram, A., Newby, G. A. & Liu, D. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat. Biotechnol. (in the press).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
doi: 10.1038/s41587-019-0032-3
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
doi: 10.1016/S0022-2836(05)80360-2
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
doi: 10.1093/bioinformatics/btp324
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
doi: 10.1038/nmeth.1923
Park, J., Lim, K., Kim, J. S. & Bae, S. Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33, 286–288 (2017).
doi: 10.1093/bioinformatics/btw561
Wang, X. et al. CRISPR–DAV: CRISPR NGS data analysis and visualization pipeline. Bioinformatics 33, 3811–3812 (2017).
doi: 10.1093/bioinformatics/btx518
Cibulskis, K. et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat. Biotechnol. 31, 213–219 (2013).
doi: 10.1038/nbt.2514
Wilm, A. et al. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res. 40, 11189–11201 (2012).
doi: 10.1093/nar/gks918
Saunders, C. T. et al. Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs. Bioinformatics 28, 1811–1817 (2012).
Fang, H. et al. Indel variant analysis of short-read sequencing data with Scalpel. Nat. Protoc. 11, 2529–2548 (2016).
doi: 10.1038/nprot.2016.150
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
doi: 10.1093/bioinformatics/btu048
Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).
doi: 10.1186/s13059-016-1012-2
Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).
doi: 10.1093/nar/gkq603
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
doi: 10.1093/bioinformatics/btu170
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
doi: 10.1101/gr.107524.110
Chen, C. C., Hwang, J. K. & Yang, J. M. (PS)2: protein structure prediction server. Nucleic Acids Res. 34, W152–157 (2006).
Huang, T. T. et al. (PS)2: protein structure prediction server version 3.0. Nucleic Acids Res. 43, W338–342 (2015).
doi: 10.1093/nar/gkv454