An apobec3a-cas9 base editor with minimized bystander and off-target activities

Nature Biotechnology

Volumn 36, Issue 10, 2018, Pages 977-

  Gehrke, Jason M ^a,b   Cervantes, Oliver ^a   Clement, M Kendell ^a,c   Wu, Yuxuan ^d   Zeng, Jing ^d   Bauer, Daniel E ^d   Pinello, Luca ^a,c   Joung, J Keith ^a,c  

^a MASSACHUSETTS GENERAL HOSPITAL   (United States)

^b HARVARD UNIVERSITY   (United States)

^c HARVARD MEDICAL SCHOOL   (United States)

^d HARVARD STEM CELL INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BASE PAIRS; CYTIDINE DEAMINASE; ENZYMATIC ACTIVITIES; GENOMIC LOCUS; HUMAN CELLS; MUTATION FREQUENCY; TARGET ACTIVITY; TARGET SITES;

BIOTECHNOLOGY;

APOBEC3A PROTEIN; CYTIDINE DEAMINASE; NUCLEOTIDE; PROTEIN; PROTEIN CAS9; UNCLASSIFIED DRUG; APOBEC3A PROTEIN, HUMAN; DNA;

BASE PAIRING; BETA THALASSEMIA; CONTROLLED STUDY; ENZYME ACTIVITY; GENE EXPRESSION; GENE MUTATION; HUMAN; HUMAN CELL; LETTER; MUTATION RATE; PRIORITY JOURNAL; PROMOTER REGION; CRISPR CAS SYSTEM; GENE EDITING; GENETICS; GENOME; MUTATION; NUCLEOTIDE SEQUENCE; PROCEDURES;

BASE SEQUENCE; BETA-THALASSEMIA; CRISPR-ASSOCIATED PROTEIN 9; CRISPR-CAS SYSTEMS; CYTIDINE DEAMINASE; DNA; GENE EDITING; GENOME; HUMANS; MUTATION; PROMOTER REGIONS, GENETIC; PROTEINS;

EID: 85054153501     PISSN: 10870156     EISSN: 15461696     Source Type: Journal    
DOI: 10.1038/nbt.4199     Document Type: Article

References (33)

1. 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).
2. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).
3. Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).
4. Rees, H.A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).
5. Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).
6. Hess, G.T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016).
7. Doudna, J.A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
8. Cox, D.B.T., Platt, R.J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
9. Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).
10. 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).
11. 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).
12. Logue, E.C. et al. A DNA sequence recognition loop on APOBEC3A controls substrate specificity. PLoS One 9, e97062 (2014).
13. Shi, K. et al. Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B. Nat. Struct. Mol. Biol. 24, 131–139 (2017).
14. Kouno, T. et al. Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity. Nat. Commun. 8, 15024 (2017).
15. Bohn, M.-F. et al. The ssDNA mutator APOBEC3A is regulated by cooperative dimerization. Structure 23, 903–911 (2015).
16. Wang, M., Yang, Z., Rada, C. & Neuberger, M.S. AID upmutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity. Nat. Struct. Mol. Biol. 16, 769–776 (2009).
17. Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).
18. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
19. 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).
20. Kleinstiver, B.P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).
21. Chen, J.S. et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550, 407–410 (2017).
22. Wang, L. et al. Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor. Cell Res. 27, 1289–1292 (2017).
23. Cao, A. & Galanello, R. Beta-thalassemia. Genet. Med. 12, 61–76 (2010).
24. Liang, P. et al. Correction of β-thalassemia mutant by base editor in human embryos. Protein Cell 8, 811–822 (2017).
25. Eng, B. et al. Three new beta-globin gene promoter mutations identified through newborn screening. Hemoglobin 31, 129–134 (2007).
26. Li, Z. et al. A novel promoter mutation (HBB: c.-75G>T) was identified as a cause of β(+)-thalassemia. Hemoglobin 39, 115–120 (2015).
27. Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
28. Hu, J.H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).
29. Rathore, A. et al. The local dinucleotide preference of APOBEC3G can be altered from 5′-CC to 5′-TC by a single amino acid substitution. J. Mol. Biol. 425, 4442–4454 (2013).
30. Pinello, L. et al. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat. Biotechnol. 34, 695–697 (2016).
31. Kulcsár, P.I. et al. Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. Genome Biol. 18, 190 (2017).
32. Kim, S., Bae, T., Hwang, J. & Kim, J.-S. Rescueof high-specificity Cas9 variants using sgRNAs with matched 5′ nucleotides. Genome Biol. 18, 218 (2017).
33. Ye, L. et al. Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia. Proc. Natl. Acad. Sci. USA 113, 10661–10665 (2016).