Analysis and minimization of cellular RNA editing by DNA adenine base editors

We report cellular RNA editing by adenine base editors and new ABE variants with reduced RNA editing activity. Adenine base editors (ABEs) enable precise and efficient conversion of target A•T base pairs to G•C base pairs in genomic DNA with a minimum of by-products. While ABEs have been reported to exhibit minimal off-target DNA editing, off-target editing of cellular RNA by ABEs has not been examined in depth. Here, we demonstrate that a current ABE generates low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs. Using structure-guided principles to design mutations in both deaminase domains, we developed new ABE variants that retain their ability to edit DNA efficiently but show greatly reduced RNA editing activity, as well as lower off-target DNA editing activity and reduced indel by-product formation, in three mammalian cell lines. By decoupling DNA and RNA editing activities, these ABE variants increase the precision of adenine base editing by minimizing both RNA and DNA off-target editing activity.

[1]  G. Verdine,et al.  Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA , 2006, Nature Structural &Molecular Biology.

[2]  David R. Liu,et al.  In vivo base editing of post-mitotic sensory cells , 2018, Nature Communications.

[3]  Beum-Chang Kang,et al.  Precision genome engineering through adenine base editing in plants , 2018, Nature Plants.

[4]  Brenda L Bass,et al.  RNA editing by adenosine deaminases that act on RNA. , 2002, Annual review of biochemistry.

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

[6]  Martin J. Aryee,et al.  GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases , 2014, Nature Biotechnology.

[7]  David R. Liu,et al.  Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction , 2018, Nature Biotechnology.

[8]  Q. Gao,et al.  Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice , 2019, Science.

[9]  David R. Liu,et al.  Base editing: precision chemistry on the genome and transcriptome of living cells , 2018, Nature Reviews Genetics.

[10]  David R. Liu,et al.  Publisher Correction: Base editing: precision chemistry on the genome and transcriptome of living cells , 2018, Nature Reviews Genetics.

[11]  Lihua Julie Zhu,et al.  Adenine base editing in an adult mouse model of tyrosinemia , 2019, Nature Biomedical Engineering.

[12]  John G Doench,et al.  Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade , 2018, Nature.

[13]  Eli Eisenberg,et al.  A-to-I RNA editing — immune protector and transcriptome diversifier , 2018, Nature Reviews Genetics.

[14]  Qiang Sun,et al.  Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing , 2018, Nature Communications.

[15]  Emma M Schatoff,et al.  Optimized base editors enable efficient editing in cells, organoids and mice , 2018, Nature Biotechnology.

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

[17]  David R. Liu,et al.  Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery , 2017, Nature Communications.

[18]  T. Lindahl.  Instability and decay of the primary structure of DNA , 1993, Nature.

[19]  V. Schramm,et al.  Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase. , 2006, Biochemistry.

[20]  Mingyao Liu,et al.  Correction to: Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants , 2018, Protein & Cell.

[21]  J. Pal,et al.  Role of 5′‐ and 3′‐untranslated regions of mRNAs in human diseases , 2009, Biology of the cell.

[22]  David R. Liu,et al.  Targeting fidelity of adenine and cytosine base editors in mouse embryos , 2018, Nature Communications.

[23]  L. Steinmetz,et al.  Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos , 2019, Science.

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

[25]  Matthew C. Canver,et al.  CRISPResso2 provides accurate and rapid genome editing sequence analysis , 2019, Nature Biotechnology.

[26]  Ping Wang,et al.  Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance , 2016 .

[27]  Dan Liu,et al.  Genome-wide profiling of adenine base editor specificity by EndoV-seq , 2019, Nature Communications.

[28]  Mingyao Liu,et al.  Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants , 2018, Protein & Cell.

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

[30]  Daesik Kim,et al.  Genome-wide target specificity of CRISPR RNA-guided adenine base editors , 2019, Nature Biotechnology.

[31]  L. Lai,et al.  Highly efficient RNA-guided base editing in rabbit , 2018, Nature Communications.

[32]  Shuo Lin,et al.  Precise A•T to G•C base editing in the zebrafish genome , 2018, BMC Biology.

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