Precision genome editing using cytosine and adenine base editors in mammalian cells

Tony P. Huang, Gregory A. Newby, David R. Liu

Published: 2021-01-17 DOI: 10.1038/s41596-020-00450-9

single-nucleotide polymorphisms

AI 解读

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Tables 1–6

Additional information on base editor components and a calculator for determining HTS library concentrations.

Supplementary Data 1

A plasmid map annotated with example primer sequences (Table 2) for cloning a base editor into a protein expression vector

Supplementary Data 2

Sequencing files associated with the data shown in Fig. 8, d and e. The raw fastq files are provided, along with a CRISPRessoBatch parameter file for each amplicon in the pre-processing folder. Successfully analyzed files can be found in the post-processing folder.

Supplementary Data 3

Sequencing files associated with the data shown in Fig. 8, f and g. The raw fastq files are provided, along with a CRISPRessoBatch parameter file for each amplicon. Fastq files are named by the delivery method used.

References

Supplementary information

Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160 (1996).
Wolfe, S. A., Nekludova, L. & Pabo, C. O. DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys. Biomol. Struct. 29, 183–212 (2000).
Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).
Miller, J. C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).
Porteus, M. H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).
Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).
Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).
Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).
Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).
Mahfouz, M. M. et al. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc. Natl Acad. Sci. USA 108, 2623–2628 (2011).
Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39, 359–372 (2011).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Jansen, R., Embden, J. D., Gaastra, W. & Schouls, L. M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565–1575 (2002).
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Jeggo, P. A. DNA breakage and repair. in Advances in Genetics Vol. 38 (eds Hall, J. et al.) 185–218 (Academic Press, 1998).
Rouet, P., Smih, F. & Jasin, M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 14, 8096–8106 (1994).
Lukacsovich, T., Yang, D. & Waldman, A. S. Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-Scel. Nucleic Acids Res. 22, 5649–5657 (1994).
Yeh, C. D., Richardson, C. D. & Corn, J. E. Advances in genome editing through control of DNA repair pathways. Nat. Cell Biol. 21, 1468–1478 (2019).
Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).
Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).
Shen, M. W. et al. Predictable and precise template-free CRISPR editing of pathogenic variants. Nature 563, 646–651 (2018).
van Overbeek, M. et al. DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol. Cell 63, 633–646 (2016).
Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera, M. D. C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).
Adikusuma, F. et al. Large deletions induced by Cas9 cleavage. Nature 560, E8–E9 (2018).
Jasin, M. & Rothstein, R. Repair of strand breaks by homologous recombination. Cold Spring Harb. Perspect. Biol. 5, a012740 (2013).
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).
Rees, H. A., Yeh, W.-H. & Liu, D. R. Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks. Nat. Commun. 10, 2212 (2019).
Richardson, C. D., Ray, G. J., Dewitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).
Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).
Landrum, M. J. et al. ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 42, D980–D985 (2013).
Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2015).
Stenson, P. D. et al. The Human Gene Mutation Database: towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Hum. Genet. 136, 665–677 (2017).
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).
Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Mok, B. Y. et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631–637 (2020).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
Huang, T. P. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat. Biotechnol. 37, 626–631 (2019).
Jiang, W. et al. BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity. Cell Res. 28, 855–861 (2018).
Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).
Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016).
Liu, L. D. et al. Intrinsic nucleotide preference of diversifying base editors guides antibody ex vivo affinity maturation. Cell Rep. 25, 884–892.3 (2018).
Wang, Y., Zhou, L., Liu, N. & Yao, S. BE-PIGS: a base-editing tool with deaminases inlaid into Cas9 PI domain significantly expanded the editing scope. Signal Transduct. Target. Ther. 4, 36 (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).
Grünewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).
Tan, J., Zhang, F., Karcher, D. & Bock, R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat. Commun. 10, 439 (2019).
Pluciennik, A. et al. PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair. Proc. Natl Acad. Sci. USA 107, 16066–16071 (2010).
Heller, R. C. & Marians, K. J. Replisome assembly and the direct restart of stalled replication forks. Nat. Rev. Mol. Cell Biol. 7, 932–943 (2006).
Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. http://doi.org/10.1038/s41587-020-0609-x (2020).
Zhao, D. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat. Biotechnol. http://doi.org/10.1038/s41587-020-0592-2 (2020).
Kim, H. S., Jeong, Y. K., Hur, J. K., Kim, J.-S. & Bae, S. Adenine base editors catalyze cytosine conversions in human cells. Nat. Biotechnol. 37, 1145–1148 (2019).
Arbab, M. et al. Determinants of base editing outcomes from target library analysis and machine learning. Cell 182, 463–480.e30 (2020).
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).
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).
Landrum, M. J. et al. ClinVar: improvements to accessing data. Nucleic Acids Res. 48(D1), D835–D844 (2020).
Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).
Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018).
Wang, L. et al. Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor. Cell Res. 27, 1289–1292 (2017).
Zhang, X. et al. Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain. Nat. Cell Biol. 22, 740–750 (2020).
Cheng, T.-L. et al. Expanding C–T base editing toolkit with diversified cytidine deaminases. Nat. Commun. 10, 3612 (2019).
Li, C. et al. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat. Biotechnol. 38, 875–882 (2020).
Sakata, R. C. et al. Base editors for simultaneous introduction of C-to-T and A-to-G mutations. Nat. Biotechnol. 38, 865–869 (2020).
Grünewald, J. et al. A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing. Nat. Biotechnol. 38, 861–864 (2020).
Zhang, X. et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat. Biotechnol. 38, 856–860 (2020).
Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97–110 (2020).
Villiger, L. et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat. Med. 24, 1519–1525 (2018).
Yeh, W.-H. et al. In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness. Sci. Transl. Med. 12, eaay9101 (2020).
Ryu, S.-M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2018).
Yang, L. et al. Amelioration of an inherited metabolic liver disease through creation of a de novo start codon by cytidine base editing. Mol. Ther. 28, 1673–1683 (2020).
Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).
Kleinstiver, B. P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).
Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).
Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290 (2020).
Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020).
Toth, E. et al. Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases. Nucleic Acids Res. 48, 3722–3733 (2020).
Chatterjee, P., Jakimo, N. & Jacobson, J. M. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci. Adv. 4, eaau0766 (2018).
Chatterjee, P. et al. An engineered ScCas9 with broad PAM range and high specificity and activity. Nat. Biotechnol. 38, 1154–1158 (2020).
Chatterjee, P. et al. A Cas9 with PAM recognition for adenine dinucleotides. Nat. Commun. 11, 2474 (2020).
Cox, D. B. T., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Yang, L. et al. Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 9, 814–819 (2019).
Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).
Zhang, Y. et al. Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system. Nat. Commun. 8, 118 (2017).
Chadwick, A. C., Wang, X. & Musunuru, K. In vivo base editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a therapeutic alternative to genome editing. Arterioscler. Thromb. Vasc. Biol. 37, 1741–1747 (2017).
Liu, Z. et al. Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing. Nat. Commun. 9, 2338 (2018).
Wu, Y. et al. Increasing cytosine base editing scope and efficiency with engineered Cas9-PmCDA1 fusions and the modified sgRNA in rice. Front. Genet. 10, 379 (2019).
Ren, B. et al. A CRISPR/Cas9 toolkit for efficient targeted base editing to induce genetic variations in rice. Sci. China Life Sci. 60, 516–519 (2017).
Lee, H. K. et al. Simultaneous targeting of linked loci in mouse embryos using base editing. Sci. Rep. 9, 1662 (2019).
Gapinske, M. et al. CRISPR-SKIP: programmable gene splicing with single base editors. Genome Biol. 19, 107 (2018).
Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).
Zhou, C. et al. Highly efficient base editing in human tripronuclear zygotes. Protein Cell 8, 772–775 (2017).
Yuan, J. et al. Genetic modulation of RNA splicing with a CRISPR-guided cytidine deaminase. Mol. Cell 72, 380–394.e7 (2018).
Hua, K., Tao, X. & Zhu, J.-K. Expanding the base editing scope in rice by using Cas9 variants. Plant Biotechnol. J. 17, 499–504 (2019).
Li, X. et al. Base editing with a Cpf1–cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).
Lee, C. et al. CRISPR-pass: gene rescue of nonsense mutations using adenine base editors. Mol. Ther. 27, 1364–1371 (2019).
Liu, Z. et al. Efficient base editing with expanded targeting scope using an engineered Spy-mac Cas9 variant. Cell Discov. 5, 58 (2019).
Ren, B. et al. Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice. Mol. Plant 12, 1015–1026 (2019).
Zhong, Z. et al. Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG. Mol. Plant 12, 1027–1036 (2019).
Liu, Z. et al. Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions. BMC Biol. 18, 111 (2019).
Liu, Z. et al. Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion. FASEB J. 33, 9210–9219 (2019).
Endo, M. et al. Genome editing in plants by engineered CRISPR–Cas9 recognizing NG PAM. Nat. Plants 5, 14–17 (2019).
Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).
Hu, Z. et al. A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope. PLOS Biol. 18, e3000686 (2020).
Agudelo, D. et al. Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9. Genome Res. 30, 107–117 (2020).
Tan, J., Zhang, F., Karcher, D. & Bock, R. Expanding the genome-targeting scope and the site selectivity of high-precision base editors. Nat. Commun. 11, 629 (2020).
Li, X. et al. Programmable base editing of mutated TERT promoter inhibits brain tumour growth. Nat. Cell Biol. 22, 282–288 (2020).
Wang, X. et al. Cas12a base editors induce efficient and specific editing with low DNA damage response. Cell Rep. 31, 107723 (2020).
Doman, J. L., Raguram, A., Newby, G. A. & Liu, D. R. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat. Biotechnol. 38, 620–628 (2020).
Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).
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).
Xu, W. et al. Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice. BMC Plant Biol. 19, 511 (2019).
Lee, J. K. et al. Directed evolution of CRISPR-Cas9 to increase its specificity. Nat. Commun. 9, 3048 (2018).
Liang, P. et al. Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein Cell 8, 601–611 (2017).
Kim, D., Kim, D.-E., Lee, G., Cho, S.-I. & Kim, J.-S. Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat. Biotechnol. 37, 430–435 (2019).
Hong, R., Ma, S. & Wang, F. Improving the specificity of adenine base editor using high-fidelity Cas9. Preprint at bioRxiv http://doi.org/10.1101/712109 (2019).
Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018).
Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019).
Yu, Y. et al. Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity. Nat. Commun. 11, 2052 (2020).
Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat. Biotechnol. 36, 946–949 (2018).
Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892–900 (2020).
Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).
Rees, H. A., Wilson, C., Doman, J. L. & Liu, D. R. Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci. Adv. 5, eaax5717 (2019).
Liu, Z. et al. Efficient base editing with high precision in rabbits using YFE-BE4max. Cell Death Dis. 11, 36 (2020).
Martin, A. S. et al. A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes. Sci. Rep. 9, 497 (2019).
St. Martin, A. et al. A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells. Nucleic Acids Res. 46, e84 (2018).
Coelho, M. A. et al. BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B. BMC Biol. 16, 150 (2018).
Zuo, E. et al. A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects. Nat. Methods 17, 600–604 (2020).
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Grünewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat. Biotechnol. 37, 1041–1048 (2019).
Wang, T., Badran, A. H., Huang, T. P. & Liu, D. R. Continuous directed evolution of proteins with improved soluble expression. Nat. Chem. Biol. 14, 972–980 (2018).
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).
Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).
Sasaguri, H. et al. Introduction of pathogenic mutations into the mouse Psen1 gene by Base Editor and Target-AID. Nat. Commun. 9, 2892 (2018).
Farzadfard, F. et al. Single-nucleotide-resolution computing and memory in living cells. Mol. Cell 75, 769–780.e4 (2019).
Shevidi, S., Uchida, A., Schudrowitz, N., Wessel, G. M. & Yajima, M. Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo. Dev. Dyn. 246, 1036–1046 (2017).
Banno, S., Nishida, K., Arazoe, T., Mitsunobu, H. & Kondo, A. Deaminase-mediated multiplex genome editing in Escherichia coli. Nat. Microbiol. 3, 423–429 (2018).
Wang, Y. et al. MACBETH: multiplex automated Corynebacterium glutamicum base editing method. Metab. Eng. 47, 200–210 (2018).
Xie, J. et al. Efficient base editing for multiple genes and loci in pigs using base editors. Nat. Commun. 10, 2852 (2019).
Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).
Gammage, P. A., Moraes, C. T. & Minczuk, M. Mitochondrial genome engineering: the revolution may not be CRISPR-ized. Trends Genet. 34, 101–110 (2018).
Molla, K. A. & Yang, Y. CRISPR/Cas-mediated base editing: technical considerations and practical applications. Trends Biotechnol. 37, 1121–1142 (2019).
Hess, G. T., Tycko, J., Yao, D. & Bassik, M. C. Methods and applications of CRISPR-mediated base editing in eukaryotic genomes. Mol. Cell 68, 26–43 (2017).
Liu, Z. et al. Highly efficient RNA-guided base editing in rabbit. Nat. Commun. 9, 2717 (2018).
Li, Q. et al. CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development. Nat. Cell Biol. 20, 1315–1325 (2018).
Yeh, W.-H., Chiang, H., Rees, H. A., Edge, A. S. B. & Liu, D. R. In vivo base editing of post-mitotic sensory cells. Nat. Commun. 9, 2184 (2018).
Zeng, Y. et al. Correction of the Marfan syndrome pathogenic FBN1 mutation by base editing in human cells and heterozygous embryos. Mol. Ther. 26, 2631–2637 (2018).
Li, G. et al. Highly efficient and precise base editing in discarded human tripronuclear embryos. Protein Cell 8, 776–779 (2017).
Liang, P. et al. Correction of β-thalassemia mutant by base editor in human embryos. Protein Cell 8, 811–822 (2017).
Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).
Lu, Y. & Zhu, J. K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Mol. Plant 10, 523–525 (2017).
Park, D.-S. et al. Targeted base editing via RNA-guided cytidine deaminases in Xenopus laevis embryos. Mol. Cells 40, 823–827 (2017).
Rossidis, A. C. et al. In utero CRISPR-mediated therapeutic editing of metabolic genes. Nat. Med. 24, 1513–1518 (2018).
Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).
Tanaka, S. et al. In vivo targeted single-nucleotide editing in zebrafish. Sci. Rep. 8, 1–11 (2018).
Qin, L. et al. High-efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system. Plant Biotechnol. J. 18, 45–56 (2020).
Chen, Y. et al. CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis. Sci. China Life Sci. 60, 520–523 (2017).
Li, J., Sun, Y., Du, J., Zhao, Y. & Xia, L. Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Mol. Plant 10, 526–529 (2017).
Li, Y. et al. Programmable single and multiplex base-editing in Bombyx mori using RNA-guided cytidine deaminases. G3 (Bethesda) 8, 1701–1709 (2018).
Qin, W. et al. Precise A•T to G•C base editing in the zebrafish genome. BMC Biol. 16, 1–8 (2018).
Li, C. et al. Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion. Genome Biol. 19, 59 (2018).
Liang, P. et al. Effective and precise adenine base editing in mouse zygotes. Protein Cell 9, 808–813 (2018).
Ma, Y. et al. Highly efficient and precise base editing by engineered dCas9-guide tRNA adenosine deaminase in rats. Cell Discov. 4, 1–4 (2018).
Kang, B.-C. et al. Precision genome engineering through adenine base editing in plants. Nat. Plants 4, 427–431 (2018).
Hua, K., Tao, X., Yuan, F., Wang, D. & Zhu, J.-K. Precise A· T to G· C base editing in the rice genome. Mol. Plant 11, 627–630 (2018).
Song, C.-Q. et al. Adenine base editing in an adult mouse model of tyrosinaemia. Nat. Biomed. Eng. 4, 125–130 (2020).
Yan, F. et al. Highly efficient A· T to G· C base editing by Cas9n-guided tRNA adenosine deaminase in rice. Mol. Plant 11, 631–634 (2018).
Kuscu, C. et al. CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations. Nat. Methods 14, 710–712 (2017).
Billon, P. et al. CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons. Mol. Cell 67, 1068–1079.e4 (2017).
Tang, W. & Liu, D. R. Rewritable multi-event analog recording in bacterial and mammalian cells. Science 360, eaap8992 (2018).
Hwang, B. et al. Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements. Nat. Commun. 10, 1234 (2019).
Yin, K., Gao, C. & Qiu, J.-L. Progress and prospects in plant genome editing. Nat. Plants 3, 17107 (2017).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Heyer, W.-D., Ehmsen, K. T. & Liu, J. Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–139 (2010).
Moynahan, M. E. & Jasin, M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207 (2010).
Jiang, T. et al. Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat. Commun. 11, 1979 (2020).
Zeng, J. et al. Therapeutic base editing of human hematopoietic stem cells. Nat. Med. 26, 535–541 (2020).
Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Chen, J. S. et al. Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature 550, 407–410 (2017).
Kim, J. et al. Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase. Biochemistry 45, 6407–6416 (2006).
Gao, X. et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553, 217–221 (2018).
Ma, X. et al. Analysis of error profiles in deep next-generation sequencing data. Genome Biol. 20, 50 (2019).
Petrackova, A. et al. Standardization of sequencing coverage depth in NGS: recommendation for detection of clonal and subclonal mutations in cancer diagnostics. Front. Oncol. 9, 851 (2019).
Hwang, G.-H. et al. Web-based design and analysis tools for CRISPR base editing. BMC Bioinformatics 19, 542 (2018).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Kluesner, M. G. et al. EditR: a method to quantify base editing from Sanger sequencing. CRISPR J. 1, 239–250 (2018).
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).
Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
Gao, Z., Harwig, A., Berkhout, B. & Herrera-Carrillo, E. Mutation of nucleotides around the +1 position of type 3 polymerase III promoters: the effect on transcriptional activity and start site usage. Transcription 8, 275–287 (2017).
Kim, S., Bae, T., Hwang, J. & Kim, J.-S. Rescue of high-specificity Cas9 variants using sgRNAs with matched 5′ nucleotides. Genome Biol. 18, 218 (2017).
Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B. & Valen, E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res. 44, W272–W276 (2016).
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).
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).