Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction

Base editors enable targeted single-nucleotide conversions in genomic DNA. Here we show that expression levels are a bottleneck in base-editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals (NLS) and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.

[1]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[2]  A. R. Wagner.  Molecular Biology and Evolution , 2001 .

[3]  G. Raymond,et al.  MPDU1 mutations underlie a novel human congenital disorder of glycosylation, designated type If. , 2001, The Journal of clinical investigation.

[4]  F. Drabløs,et al.  Uracil in DNA – occurrence, consequences and repair , 2002, Oncogene.

[5]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[6]  Ziheng Yang.  PAML 4: phylogenetic analysis by maximum likelihood. , 2007, Molecular biology and evolution.

[7]  Internation Journal of laboratory Hematology. , 2008 .

[8]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[9]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[10]  H. Park,et al.  High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice , 2011, PloS one.

[11]  K. Katoh,et al.  MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability , 2013, Molecular biology and evolution.

[12]  J. A. Gavira,et al.  Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian β-lactamases. , 2013, Journal of the American Chemical Society.

[13]  M. Harms,et al.  Evolutionary biochemistry: revealing the historical and physical causes of protein properties , 2013, Nature Reviews Genetics.

[14]  Hollis G. Potter,et al.  Author Manuscript , 2013 .

[15]  P. Grisanti,et al.  Interpreting elevated fetal hemoglobin in pathology and health at the basic laboratory level: new and known γ‐ gene mutations associated with hereditary persistence of fetal hemoglobin , 2014, International journal of laboratory hematology.

[16]  C. Woods,et al.  Painful and painless channelopathies , 2014, The Lancet Neurology.

[17]  A. von Haeseler,et al.  IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies , 2014, Molecular biology and evolution.

[18]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[19]  David R. Liu,et al.  Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage , 2016, Nature.

[20]  David R. Liu,et al.  Continuous evolution of B. thuringiensis toxins overcomes insect resistance , 2016, Nature.

[21]  Ricardo Villamarín-Salomón,et al.  ClinVar: public archive of interpretations of clinically relevant variants , 2015, Nucleic Acids Res..

[22]  Lucas C Wheeler,et al.  The thermostability and specificity of ancient proteins. , 2016, Current opinion in structural biology.

[23]  Edward M. Callaway,et al.  In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration , 2016, Nature.

[24]  Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage , 2017, Nature.

[25]  Dan Liu,et al.  Correction of β-thalassemia mutant by base editor in human embryos , 2017, Protein & Cell.

[26]  Gaelen T. Hess,et al.  Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes. , 2017, Molecular cell.

[27]  Thomas K. F. Wong,et al.  ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates , 2017, Nature Methods.

[28]  Kevin T. Zhao,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 , 2017, Science Advances.

[29]  Guanglei Li,et al.  Highly efficient and precise base editing in discarded human tripronuclear embryos , 2017, Protein & Cell.

[30]  Kevin T. Zhao,et al.  Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions , 2017, Nature Biotechnology.

[31]  David R. Liu,et al.  Evolved Cas9 variants with broad PAM compatibility and high DNA specificity , 2018, Nature.

[32]  Eugene Chung,et al.  Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy , 2018, Nature Biotechnology.

[33]  A. von Haeseler,et al.  UFBoot2: Improving the Ultrafast Bootstrap Approximation , 2017, bioRxiv.

[34]  The Uniprot Consortium.  UniProt: the universal protein knowledgebase , 2018, Nucleic acids research.

[35]  J. Coller,et al.  Codon optimality, bias and usage in translation and mRNA decay , 2017, Nature Reviews Molecular Cell Biology.

[36]  Martha L. Bulyk,et al.  Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch , 2018, Cell.