دورية أكاديمية
CRISPR technologies for genome, epigenome and transcriptome editing.
العنوان: | CRISPR technologies for genome, epigenome and transcriptome editing. |
---|---|
المؤلفون: | Villiger L; McGovern Institute for Brain Research, Massachusetts Institute of Technology Cambridge, Cambridge, MA, USA., Joung J; Whitehead Institute for Biomedical Research, Cambridge, MA, USA.; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA., Koblan L; Whitehead Institute for Biomedical Research, Cambridge, MA, USA.; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA., Weissman J; Whitehead Institute for Biomedical Research, Cambridge, MA, USA.; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA.; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA., Abudayyeh OO; McGovern Institute for Brain Research, Massachusetts Institute of Technology Cambridge, Cambridge, MA, USA. omar@abudayyeh.science., Gootenberg JS; McGovern Institute for Brain Research, Massachusetts Institute of Technology Cambridge, Cambridge, MA, USA. jgoot@mit.edu. |
المصدر: | Nature reviews. Molecular cell biology [Nat Rev Mol Cell Biol] 2024 Jun; Vol. 25 (6), pp. 464-487. Date of Electronic Publication: 2024 Feb 02. |
نوع المنشور: | Journal Article; Review |
اللغة: | English |
بيانات الدورية: | Publisher: Nature Pub. Group Country of Publication: England NLM ID: 100962782 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1471-0080 (Electronic) Linking ISSN: 14710072 NLM ISO Abbreviation: Nat Rev Mol Cell Biol Subsets: MEDLINE |
أسماء مطبوعة: | Original Publication: London, UK : Nature Pub. Group, [2000- |
مواضيع طبية MeSH: | Gene Editing*/methods , CRISPR-Cas Systems*/genetics , Epigenome*/genetics , Transcriptome*/genetics, Humans ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome/genetics |
مستخلص: | Our ability to edit genomes lags behind our capacity to sequence them, but the growing understanding of CRISPR biology and its application to genome, epigenome and transcriptome engineering is narrowing this gap. In this Review, we discuss recent developments of various CRISPR-based systems that can transiently or permanently modify the genome and the transcriptome. The discovery of further CRISPR enzymes and systems through functional metagenomics has meaningfully broadened the applicability of CRISPR-based editing. Engineered Cas variants offer diverse capabilities such as base editing, prime editing, gene insertion and gene regulation, thereby providing a panoply of tools for the scientific community. We highlight the strengths and weaknesses of current CRISPR tools, considering their efficiency, precision, specificity, reliance on cellular DNA repair mechanisms and their applications in both fundamental biology and therapeutics. Finally, we discuss ongoing clinical trials that illustrate the potential impact of CRISPR systems on human health. (© 2024. Springer Nature Limited.) |
التعليقات: | Erratum in: Nat Rev Mol Cell Biol. 2024 May 13;:. (PMID: 38740926) |
References: | Wang, J. Y. & Doudna, J. A. CRISPR technology: a decade of genome editing is only the beginning. Science 379, eadd8643 (2023). (PMID: 3665694210.1126/science.add8643) Koonin, E. V., Gootenberg, J. S. & Abudayyeh, O. O. Discovery of diverse CRISPR-Cas systems and expansion of the genome engineering toolbox. Biochemistry 62, 3465–3487 (2023). (PMID: 3719209910.1021/acs.biochem.3c00159) Wang, J. Y., Pausch, P. & Doudna, J. A. Structural biology of CRISPR-Cas immunity and genome editing enzymes. Nat. Rev. Microbiol. 20, 641–656 (2022). (PMID: 3556242710.1038/s41579-022-00739-4) Hille, F. et al. The biology of CRISPR-Cas: backward and forward. Cell 172, 1239–1259 (2018). (PMID: 2952274510.1016/j.cell.2017.11.032) Zhang, F. Development of CRISPR-Cas systems for genome editing and beyond. Q. Rev. Biophys. 52, e6 (2019). (PMID: 10.1017/S0033583519000052) Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). First description of Cas9 reprogrammability biochemically. (PMID: 22745249628614810.1126/science.1225829) Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001). (PMID: 111132038880210.1128/MCB.21.1.289-297.2001) Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003). (PMID: 1273059410.1126/science.1079512) Bibikova, M., Golic, M., Golic, K. G. & Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002). (PMID: 12136019146216610.1093/genetics/161.3.1169) Porteus, M. H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003). (PMID: 1273059310.1126/science.1078395) Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005). (PMID: 1580609710.1038/nature03556) Joung, J. K. & Sander, J. D. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14, 49–55 (2013). (PMID: 2316946610.1038/nrm3486) Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011). (PMID: 21493687313029110.1093/nar/gkr218) Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011). (PMID: 21248753308453310.1038/nbt.1775) Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010). (PMID: 20660643294287010.1534/genetics.110.120717) Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013). Together with ref. 17, this article provides the first demonstration of mammalian genome editing using RNA-programmable Cas9. (PMID: 23287718379541110.1126/science.1231143) Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013). Together with ref. 16, this article provides the first demonstration of mammalian genome editing using RNA-programmable Cas9. (PMID: 23287722371262810.1126/science.1232033) Jinek, M. et al. RNA-programmed genome editing in human cells. Elife 2, e00471 (2013). (PMID: 23386978355790510.7554/eLife.00471) Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013). This article introduces CRISPRi for transcription repression. (PMID: 23452860366429010.1016/j.cell.2013.02.022) 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 USA 109, E2579–E2586 (2012). 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). (PMID: 3179237610.1038/s41556-019-0425-z) Hussmann, J. A. et al. Mapping the genetic landscape of DNA double-strand break repair. Cell 184, 5653–5669.e25 (2021). (PMID: 34672952907446710.1016/j.cell.2021.10.002) Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016). (PMID: 27851729533178510.1038/nature20565) Lieber, M. R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Ann. Rev. Biochem. 79, 181–211 (2010). (PMID: 2019275910.1146/annurev.biochem.052308.093131) Mao, Z., Bozzella, M., Seluanov, A. & Gorbunova, V. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair 7, 1765–1771 (2008). (PMID: 18675941269599310.1016/j.dnarep.2008.06.018) Smith, J., Baldeyron, C., De Oliveira, I., Sala-Trepat, M. & Papadopoulo, D. The influence of DNA double-strand break structure on end-joining in human cells. Nucleic Acids Res. 29, 4783–4792 (2001). (PMID: 117266879670610.1093/nar/29.23.4783) Chen, S. et al. Accurate in vitro end joining of a DNA double strand break with partially cohesive 3’-overhangs and 3’-phosphoglycolate termini: effect of Ku on repair fidelity. J. Biol. Chem. 276, 24323–24330 (2001). (PMID: 1130937910.1074/jbc.M010544200) Waters, C. A. et al. The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining. Nat. Commun. 5, 4286 (2014). (PMID: 2498932410.1038/ncomms5286) Song, B., Yang, S., Hwang, G.-H., Yu, J. & Bae, S. Analysis of NHEJ-based DNA repair after CRISPR-mediated DNA cleavage. Int. J. Mol. Sci. 22, 6397 (2021). (PMID: 34203807823268710.3390/ijms22126397) van Overbeek, M. et al. DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol. Cell 63, 633–646 (2016). (PMID: 2749929510.1016/j.molcel.2016.06.037) Deriano, L. & Roth, D. B. Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage. Annu. Rev. Genet. 47, 433–455 (2013). (PMID: 2405018010.1146/annurev-genet-110711-155540) Rudin, N., Sugarman, E. & Haber, J. E. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics 122, 519–534 (1989). (PMID: 2668114120372610.1093/genetics/122.3.519) Plessis, A., Perrin, A., Haber, J. E. & Dujon, B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics 130, 451–460 (1992). (PMID: 1551570120486410.1093/genetics/130.3.451) Amoasii, L. et al. Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy. Sci. Transl. Med. 9, eaan8081 (2017). (PMID: 29187645574940610.1126/scitranslmed.aan8081) Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015). (PMID: 26375006464410110.1038/nature15521) Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). This article introduces SaCas9 and AAV-mediated Cas9 editing in mice. (PMID: 25830891439336010.1038/nature14299) Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013). (PMID: 24076762384486910.1038/nmeth.2681) Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl Acad. Sci. USA 110, 15644–15649 (2013). (PMID: 23940360378573110.1073/pnas.1313587110) Edraki, A. et al. A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing. Mol. Cell 73, 714–726.e4 (2019). (PMID: 3058114410.1016/j.molcel.2018.12.003) Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017). (PMID: 28220790547364010.1038/ncomms14500) Hu, Z. et al. A compact Cas9 ortholog from Staphylococcus auricularis (SauriCas9) expands the DNA targeting scope. PLoS Biol. 18, e3000686 (2020). (PMID: 32226015714527010.1371/journal.pbio.3000686) Chatterjee, P., Jakimo, N. & Jacobson, J. M. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci. Adv. 4, eaau0766 (2018). (PMID: 30397647620036310.1126/sciadv.aau0766) Hirano, H. et al. Structure and engineering of francisella novicida Cas9. Cell 164, 950–961 (2016). (PMID: 26875867489997210.1016/j.cell.2016.01.039) Harrington, L. B. et al. A thermostable Cas9 with increased lifetime in human plasma. Nat. Commun. 8, 1424 (2017). (PMID: 29127284568153910.1038/s41467-017-01408-4) Chylinski, K., Makarova, K. S., Charpentier, E. & Koonin, E. V. Classification and evolution of type II CRISPR-Cas systems. Nucleic Acids Res. 42, 6091–6105 (2014). (PMID: 24728998404141610.1093/nar/gku241) Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015). This article introduces RNA-programmable Cas12a for mammalian genome editing. (PMID: 26422227463822010.1016/j.cell.2015.09.038) Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016). This article introduces RNA-programmable RNA-targeting Cas13 systems. (PMID: 27256883512778410.1126/science.aaf5573) East-Seletsky, A. et al. Two distinct RNase activities of CRISPR–C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270–273 (2016). (PMID: 27669025557636310.1038/nature19802) Brouns, S. J. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008). (PMID: 18703739589823510.1126/science.1159689) Hale, C. R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009). (PMID: 19945378295126510.1016/j.cell.2009.07.040) Jiang, F. & Doudna, J. A. CRISPR–Cas9 structures and mechanisms. Annu. Rev. Biophys. 46, 505–529 (2017). (PMID: 2837573110.1146/annurev-biophys-062215-010822) Müller, M. et al. Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome. Mol. Ther. 24, 636–644 (2016). (PMID: 26658966478691710.1038/mt.2015.218) Chatterjee, P. et al. A Cas9 with PAM recognition for adenine dinucleotides. Nat. Commun. 11, 2474 (2020). (PMID: 32424114723524910.1038/s41467-020-16117-8) Karvelis, T. et al. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements. Genome Biol. 16, 253 (2015). (PMID: 26585795465388010.1186/s13059-015-0818-7) Gasiunas, G. et al. A catalogue of biochemically diverse CRISPR–Cas9 orthologs. Nat. Commun. 11, 5512 (2020). (PMID: 33139742760646410.1038/s41467-020-19344-1) Hirano, S., Nishimasu, H., Ishitani, R. & Nureki, O. Structural basis for the altered PAM specificities of engineered CRISPR-Cas9. Mol. Cell 61, 886–894 (2016). (PMID: 2699099110.1016/j.molcel.2016.02.018) Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290–296 (2020). (PMID: 32217751729704310.1126/science.aba8853) Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018). (PMID: 30166441636845210.1126/science.aas9129) Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015). This article reports engineered Cas9 molecules that recognize different PAMs. (PMID: 26098369454023810.1038/nature14592) Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018). This work describes phage-assisted continuous evolution for Cas9. (PMID: 29512652595163310.1038/nature26155) Chatterjee, P. et al. An engineered ScCas9 with broad PAM range and high specificity and activity. Nat. Biotechnol. 38, 1154–1158 (2020). (PMID: 3239382210.1038/s41587-020-0517-0) Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020). (PMID: 32042170714574410.1038/s41587-020-0412-8) Legut, M. et al. High-throughput screens of PAM-flexible Cas9 variants for gene knockout and transcriptional modulation. Cell Rep. 30, 2859–2868.e5 (2020). (PMID: 32130891755843510.1016/j.celrep.2020.02.010) Kim, D., Luk, K., Wolfe, S. A. & Kim, J.-S. Evaluating and enhancing target specificity of gene-editing nucleases and deaminases. Annu. Rev. Biochem. 88, 191–220 (2019). (PMID: 3088319610.1146/annurev-biochem-013118-111730) 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). (PMID: 2551378210.1038/nbt.3117) Enache, O. M. et al. Cas9 activates the p53 pathway and selects for p53-inactivating mutations. Nat. Genet. 52, 662–668 (2020). (PMID: 32424350734361210.1038/s41588-020-0623-4) 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). (PMID: 2453556810.1038/nbt.2800) 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). This study introduces CBEs that use rat APOBEC1 fused to nCas9. (PMID: 27096365487337110.1038/nature17946) 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). Development of laboratory-evolved TadA deaminase to achieve A-to-G editing. (PMID: 29160308572655510.1038/nature24644) Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016). This study introduces base editors that use AID fused to nCas9. (PMID: 2749247410.1126/science.aaf8729) Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). First work to describe prime editing, using reverse transcription for Cas9-targeted genome editing. (PMID: 31634902690707410.1038/s41586-019-1711-4) Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019). First description of CASTs, together with ref. 73. (PMID: 31171706665911810.1126/science.aax9181) Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature 571, 219–225 (2019). First description of CASTs, together with ref. 72. (PMID: 3118917710.1038/s41586-019-1323-z) Lampe, G. D. et al. Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases. Nat. Biotechnol. 42, 87–98 (2023). (PMID: 369911121062001510.1038/s41587-023-01748-1) Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013). This study demonstrates transcription repression by KREB fused to dCas9. (PMID: 23849981377014510.1016/j.cell.2013.06.044) Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014). (PMID: 25307932425385910.1016/j.cell.2014.09.029) Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. 41, 500–512 (2022). (PMID: 364244891025735110.1038/s41587-022-01527-4) Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2021). (PMID: 34887556911739310.1038/s41587-021-01133-w) Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9–based transcription factors. Nat. Methods 10, 973–976 (2013). This study demonstrates transcription activation by VP64 fused to dCas9. (PMID: 23892895391178510.1038/nmeth.2600) Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013). (PMID: 23892898379405810.1038/nmeth.2598) Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015). (PMID: 2549420210.1038/nature14136) Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014). (PMID: 25307933425260810.1016/j.cell.2014.09.039) Nuñez, J. K. et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell 184, 2503–2519.e17 (2021). This study demonstrates Cas9-mediated heritable epigenome editing with CRISPRoff. (PMID: 33838111837608310.1016/j.cell.2021.03.025) Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2016). (PMID: 538950710.1093/nar/gkw1112) Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016). (PMID: 27662091506260910.1016/j.cell.2016.08.056) Abudayyeh, O. O. et al. RNA targeting with CRISPR–Cas13. Nature 550, 280–284 (2017). (PMID: 28976959570665810.1038/nature24049) Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017). This report introduces Cas13-based RNA A-to-I editing. (PMID: 29070703579385910.1126/science.aaq0180) Konermann, S. et al. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell 173, 665–676.e14 (2018). (PMID: 29551272591025510.1016/j.cell.2018.02.033) Özcan, A. et al. Programmable RNA targeting with the single-protein CRISPR effector Cas7–11. Nature 597, 720–725 (2021). (PMID: 3448959410.1038/s41586-021-03886-5) Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019). (PMID: 31147612707920710.1038/s41580-019-0131-5) Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018). (PMID: 29765029595393110.1038/s41467-018-04252-2) Thakore, P. I., Black, J. B., Hilton, I. B. & Gersbach, C. A. Editing the epigenome: technologies for programmable transcription and epigenetic modulation. Nat. Methods 13, 127–137 (2016). (PMID: 26820547492263810.1038/nmeth.3733) 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). (PMID: 30323312653518110.1038/s41576-018-0059-1) 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). (PMID: 3257226910.1038/s41587-020-0561-9) 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). (PMID: 28875174557687610.1126/sciadv.aao4774) 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). (PMID: 3248336510.1038/s41587-020-0509-0) Grünewald, J. et al. A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing. Nat. Biotechnol. 38, 861–864 (2020). (PMID: 32483364772351810.1038/s41587-020-0535-y) Zhang, X. et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat. Biotechnol. 38, 856–860 (2020). (PMID: 3248336310.1038/s41587-020-0527-y) Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41–46 (2021). (PMID: 3269097110.1038/s41587-020-0609-x) Zhao, D. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat. Biotechnol. 39, 35–40 (2021). (PMID: 3269097010.1038/s41587-020-0592-2) Chen, L. et al. Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat. Commun. 12, 1384 (2021). (PMID: 33654077792552710.1038/s41467-021-21559-9) Koblan, L. W. et al. Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning. Nat. Biotechnol. 39, 1414–1425 (2021). (PMID: 34183861898552010.1038/s41587-021-00938-z) Chen, L. et al. Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01821-9 (2023). Tong, H. et al. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase. Nat. Biotechnol. 41, 1–5 (2023). (PMID: 10.1038/s41587-022-01595-6) Yang, C. et al. HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing. Nat. Commun. 14, 2430 (2023). (PMID: 371059761014017710.1038/s41467-023-38193-2) Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018). (PMID: 29813047612694710.1038/nbt.4172) Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018). (PMID: 29969439613088910.1038/nbt.4194) Wang, L. et al. Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor. Cell Res. 27, 1289–1292 (2017). (PMID: 28849781563067710.1038/cr.2017.111) Lau, A. Y., Wyatt, M. D., Glassner, B. J., Samson, L. D. & Ellenberger, T. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG. Proc. Natl Acad. Sci. USA 97, 13573–13578 (2000). (PMID: 111063951761710.1073/pnas.97.25.13573) Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013). (PMID: 23873081396985810.1038/nbt.2647) Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014). (PMID: 24752079414567210.1038/nbt.2889) Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013). (PMID: 23792628377302310.1038/nbt.2623) Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013). (PMID: 23934178378261110.1038/nbt.2673) Grünewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019). This article demonstrates that DNA base editors have transcriptome-wide RNA off-targets. (PMID: 30995674665734310.1038/s41586-019-1161-z) Li, S., Liu, L., Sun, W., Zhou, X. & Zhou, H. A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants. Genome Biol. 23, 51 (2022). (PMID: 35139891882665410.1186/s13059-022-02618-w) 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). (PMID: 3239388910.1038/s41556-020-0518-8) Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019). (PMID: 31332326672821010.1038/s41587-019-0193-0) Schmidheini, L. et al. Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility. Nat. Chem. Biol. https://doi.org/10.1038/s41589-023-01427-x (2023). Huang, T. P. et al. High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs. Nat. Biotechnol. 41, 96–107 (2022). (PMID: 36076084984914010.1038/s41587-022-01410-2) 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). (PMID: 28191901538857410.1038/nbt.3803) 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). (PMID: 32433547735782110.1038/s41587-020-0453-z) 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). (PMID: 31110355655127610.1038/s41587-019-0134-y) 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). (PMID: 30683865634762510.1038/s41467-018-08034-8) 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). (PMID: 32005820699448510.1038/s41467-020-14465-z) 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). (PMID: 31637015679983210.1038/s41392-019-0072-7) Jiang, W. et al. BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity. Cell Res. 28, 855–861 (2018). (PMID: 29875396608291410.1038/s41422-018-0052-4) Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016). (PMID: 27798611555728810.1038/nmeth.4038) McGrath, E. et al. Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing. Nat. Commun. 10, 5353 (2019). (PMID: 31767844687763910.1038/s41467-019-13342-8) Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016). (PMID: 2772375410.1038/nmeth.4027) Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017). (PMID: 2839834510.1038/nbt.3852) Liang, P. et al. Genome-wide profiling of adenine base editor specificity by EndoV-seq. Nat. Commun. 10, 67 (2019). (PMID: 30622278632512610.1038/s41467-018-07988-z) 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). (PMID: 3083365810.1038/s41587-019-0050-1) 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). (PMID: 32042165733542410.1038/s41587-020-0414-6) Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019). (PMID: 30819928730130810.1126/science.aav9973) Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019). (PMID: 3081993110.1126/science.aaw7166) Gehrke, J. M. et al. An APOBEC3A–Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018). (PMID: 30059493618177010.1038/nbt.4199) Liu, Z. et al. Precise base editing with CC context-specificity using engineered human APOBEC3G–nCas9 fusions. BMC Biol. 18, 111 (2020). (PMID: 32867757746134410.1186/s12915-020-00849-6) 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). (PMID: 3107126710.1096/fj.201900476RR) Liu, L. D. et al. Intrinsic nucleotide preference of diversifying base editors guides antibody ex vivo affinity maturation. Cell Rep. 25, 884–892.e3 (2018). (PMID: 3035549510.1016/j.celrep.2018.09.090) 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). (PMID: 32345976718938210.1038/s41467-020-15887-5) Lee, S. et al. Single C-to-T substitution using engineered APOBEC3G–nCas9 base editors with minimum genome- and transcriptome-wide off-target effects. Sci. Adv. 6, eaba1773 (2020). (PMID: 32832622743935910.1126/sciadv.aba1773) Liu, Z. et al. Efficient base editing with high precision in rabbits using YFE-BE4max. Cell Death Dis. 11, 36 (2020). (PMID: 31959743697125010.1038/s41419-020-2244-3) Lam, D. K. et al. Improved cytosine base editors generated from TadA variants. Nat. Biotechnol. 41, 686–697 (2023). (PMID: 366241491018836710.1038/s41587-022-01611-9) Neugebauer, M. E. et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat. Biotechnol. 41, 673–685 (2022). This article presents a mitochondrial, CRISPR-free, base editor. (PMID: 363577191018836610.1038/s41587-022-01533-6) 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). (PMID: 31086823650623710.1126/sciadv.aax5717) Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892–900 (2020). (PMID: 3228458610.1038/s41587-020-0491-6) Rothgangl, T. et al. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nat. Biotechnol. 39, 949–957 (2021). Together with ref. 148, this work shows the applicability of ABEs in non-human primates. (PMID: 34012094835278110.1038/s41587-021-00933-4) Musunuru, K. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429–434 (2021). Together with ref. 147, this work shows the applicability of ABEs in non-human primates. (PMID: 3401208210.1038/s41586-021-03534-y) Chen, P. J. & Liu, D. R. Prime editing for precise and highly versatile genome manipulation. Nat. Rev. Genet. 24, 161–177 (2023). (PMID: 3634474910.1038/s41576-022-00541-1) Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652.e29 (2021). (PMID: 34653350858403410.1016/j.cell.2021.09.018) Kweon, J. et al. Engineered prime editors with PAM flexibility. Mol. Ther. 29, 2001–2007 (2021). (PMID: 33636398817845610.1016/j.ymthe.2021.02.022) Böck, D. et al. In vivo prime editing of a metabolic liver disease in mice. Sci. Transl. Med. 14, eabl9238 (2022). (PMID: 35294257761413410.1126/scitranslmed.abl9238) Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 2121 (2021). (PMID: 33837189803519010.1038/s41467-021-22295-w) Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2021). (PMID: 34608327893041810.1038/s41587-021-01039-7) Zhang, G. et al. Enhancement of prime editing via xrRNA motif-joined pegRNA. Nat. Commun. 13, 1856 (2022). (PMID: 35387980898680410.1038/s41467-022-29507-x) Li, X. et al. Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes. J. Mol. Cell Biol. 14, mjac022 (2022). (PMID: 35411929934530910.1093/jmcb/mjac022) Liu, Y. et al. Enhancing prime editing by Csy4-mediated processing of pegRNA. Cell Res. 31, 1134–1136 (2021). (PMID: 34103663848685910.1038/s41422-021-00520-x) Li, X. et al. Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure. Nat. Commun. 13, 1669 (2022). (PMID: 35351879896472510.1038/s41467-022-29339-9) Liu, B. et al. A split prime editor with untethered reverse transcriptase and circular RNA template. Nat. Biotechnol. 40, 1388–1393 (2022). (PMID: 3537996210.1038/s41587-022-01255-9) Bhagwat, A. M. et al. MultiCRISPR: gRNA design for prime editing and parallel targeting of thousands of targets. Life Sci. Alliance 3, e202000757 (2020). (PMID: 32907859749481410.26508/lsa.202000757) Chow, R. D., Chen, J. S., Shen, J. & Chen, S. A web tool for the design of prime-editing guide RNAs. Nat. Biomed. Eng. 5, 190–194 (2021). (PMID: 3298928410.1038/s41551-020-00622-8) Hwang, G.-H. et al. PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing. Nucleic Acids Res. 49, W499–W504 (2021). (PMID: 33939828826510210.1093/nar/gkab319) Li, Y., Chen, J., Tsai, S. Q. & Cheng, Y. Easy-prime: a machine learning-based prime editor design tool. Genome Biol. 22, 235 (2021). (PMID: 34412673837785810.1186/s13059-021-02458-0) Standage-Beier, K., Tekel, S. J., Brafman, D. A. & Wang, X. Prime editing guide RNA design automation using PINE-CONE. ACS Synth. Biol. 10, 422–427 (2021). (PMID: 33464043790101710.1021/acssynbio.0c00445) Siegner, S. M., Karasu, M. E., Schröder, M. S., Kontarakis, Z. & Corn, J. E. PnB designer: a web application to design prime and base editor guide RNAs for animals and plants. BMC Bioinformatics 22, 101 (2021). (PMID: 33653259792353810.1186/s12859-021-04034-6) Hsu, J. Y. et al. PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat. Commun. 12, 1034 (2021). (PMID: 33589617788477910.1038/s41467-021-21337-7) Kim, H. K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat. Biotechnol. 39, 198–206 (2021). (PMID: 3295895710.1038/s41587-020-0677-y) Yu, G. et al. Prediction of efficiencies for diverse prime editing systems in multiple cell types. Cell 186, 2256–2272.e23 (2023). (PMID: 3711981210.1016/j.cell.2023.03.034) Mathis, N. et al. Predicting prime editing efficiency and product purity by deep learning. Nat. Biotechnol. 41, 1151–1159 (2023). (PMID: 3664693310.1038/s41587-022-01613-7) Song, M. et al. Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain. Nat. Commun. 12, 5617 (2021). (PMID: 34556671846072610.1038/s41467-021-25928-2) Park, S.-J. et al. Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor. Genome Biol. 22, 170 (2021). (PMID: 34082781817382010.1186/s13059-021-02389-w) Velimirovic, M. et al. Peptide fusion improves prime editing efficiency. Nat. Commun. 13, 3512 (2022). (PMID: 35717416920666010.1038/s41467-022-31270-y) Zong, Y. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat. Biotechnol. 40, 1394–1402 (2022). (PMID: 3533234110.1038/s41587-022-01254-w) Xu, W. et al. A design optimized prime editor with expanded scope and capability in plants. Nat. Plants 8, 45–52 (2022). (PMID: 3494980210.1038/s41477-021-01043-4) Zheng, C. et al. A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver. Mol. Ther. 30, 1343–1351 (2022). (PMID: 34998953889960210.1016/j.ymthe.2022.01.005) Gao, Z. et al. A truncated reverse transcriptase enhances prime editing by split AAV vectors. Mol. Ther. 30, 2942–2951 (2022). (PMID: 35808824948198610.1016/j.ymthe.2022.07.001) Davis, J. R. et al. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01758-z (2023). (PMID: 10.1038/s41587-023-01758-z3779841611098749) Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002.e26 (2023). (PMID: 376574191048298210.1016/j.cell.2023.07.039) Ferreira da Silva, J. et al. Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair. Nat. Commun. 13, 760 (2022). (PMID: 35140211882878410.1038/s41467-022-28442-1) Roberts, J. D., Bebenek, K. & Kunkel, T. A. The accuracy of reverse transcriptase from HIV-1. Science 242, 1171–1173 (1988). (PMID: 246092510.1126/science.2460925) Jaganathan, D., Ramasamy, K., Sellamuthu, G., Jayabalan, S. & Venkataraman, G. CRISPR for crop improvement: an update review. Front. Plant Sci. 9, 985 (2018). (PMID: 30065734605666610.3389/fpls.2018.00985) Horwitz, A. A. et al. Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas. Cell Syst. 1, 88–96 (2015). (PMID: 2713568810.1016/j.cels.2015.02.001) Dunbar, C. E. et al. Gene therapy comes of age. Science 359, eaan4672 (2018). (PMID: 2932624410.1126/science.aan4672) Landrum, M. J. et al. ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 46, D1062–D1067 (2018). (PMID: 2916566910.1093/nar/gkx1153) Chaikind, B., Bessen, J. L., Thompson, D. B., Hu, J. H. & Liu, D. R. A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Res. 44, 9758–9770 (2016). (PMID: 275155115175349) Pallarès-Masmitjà, M. et al. Find and cut-and-transfer (FiCAT) mammalian genome engineering. Nat. Commun. 12, 7071 (2021). (PMID: 34862378864241910.1038/s41467-021-27183-x) Rezazade Bazaz, M., Ghahramani Seno, M. M. & Dehghani, H. Transposase-CRISPR mediated targeted integration (TransCRISTI) in the human genome. Sci. Rep. 12, 3390 (2022). (PMID: 35232993888862610.1038/s41598-022-07158-8) Kovač, A. et al. RNA-guided retargeting of S transposition in human cells. eLife 9, e53868 (2020). (PMID: 32142408707798010.7554/eLife.53868) Wang, C. et al. dCas9-based gene editing for cleavage-free genomic knock-in of long sequences. Nat. Cell Biol. 24, 268–278 (2022). (PMID: 35145221884381310.1038/s41556-021-00836-1) Peters, J. E., Makarova, K. S., Shmakov, S. & Koonin, E. V. Recruitment of CRISPR-Cas systems by Tn7-like transposons. Proc. Natl Acad. Sci. USA 114, E7358–E7366 (2017). (PMID: 28811374558445510.1073/pnas.1709035114) Faure, G. et al. CRISPR–Cas in mobile genetic elements: counter-defence and beyond. Nat. Rev. Microbiol. 17, 513–525 (2019). (PMID: 3116578110.1038/s41579-019-0204-7) Vo, P. L. H. et al. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat. Biotechnol. 39, 480–489 (2021). (PMID: 3323029310.1038/s41587-020-00745-y) Rubin, B. E. et al. Species- and site-specific genome editing in complex bacterial communities. Nat. Microbiol. 7, 34–47 (2022). (PMID: 3487329210.1038/s41564-021-01014-7) George, J. T. et al. Mechanism of target site selection by type V-K CRISPR-associated transposases. Science 382, eadj8543 (2023). (PMID: 379721611077133910.1126/science.adj8543) Tou, C. J., Orr, B. & Kleinstiver, B. P. Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases. Nat. Biotechnol. 41, 968–979 (2023). (PMID: 3659341310.1038/s41587-022-01574-x) Park, J.-U. et al. Structures of the holo CRISPR RNA-guided transposon integration complex. Nature 613, 775–782 (2022). (PMID: 36442503987679710.1038/s41586-022-05573-5) Schmitz, M., Querques, I., Oberli, S., Chanez, C. & Jinek, M. Structural basis for the assembly of the type V CRISPR-associated transposon complex. Cell 185, 4999–5010.e17 (2022). This article identifies ribosomal S15 as an additional bona fide component of type V CAST complex. (PMID: 36435179979883110.1016/j.cell.2022.11.009) Querques, I., Schmitz, M., Oberli, S., Chanez, C. & Jinek, M. Target site selection and remodelling by type V CRISPR–transposon systems. Nature 599, 497–502 (2021). (PMID: 34759315761340110.1038/s41586-021-04030-z) Halpin-Healy, T. S., Klompe, S. E., Sternberg, S. H. & Fernández, I. S. Structural basis of DNA targeting by a transposon-encoded CRISPR–Cas system. Nature 577, 271–274 (2019). (PMID: 3185306510.1038/s41586-019-1849-0) Park, J.-U. et al. Structural basis for target site selection in RNA-guided DNA transposition systems. Science 373, 768–774 (2021). (PMID: 34385391908005910.1126/science.abi8976) Park, J.-U., Tsai, A. W.-L., Chen, T. H., Peters, J. E. & Kellogg, E. H. Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM. Proc. Natl Acad. Sci. USA 119, e2202590119 (2022). (PMID: 35914146937166510.1073/pnas.2202590119) Jiang, T., Zhang, X. O., Weng, Z. & Xue, W. Deletion and replacement of long genomic sequences using prime editing. Nat. Biotechnol. 40, 227–234 (2022). (PMID: 3465027010.1038/s41587-021-01026-y) Choi, J. et al. Precise genomic deletions using paired prime editing. Nat. Biotechnol. 40, 218–226 (2021). (PMID: 34650269884732710.1038/s41587-021-01025-z) Tao, R. et al. Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells. Nucleic Acids Res. 50, 6423–6434 (2022). (PMID: 35687127922652910.1093/nar/gkac506) Wang, J. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat. Methods 19, 331–340 (2022). (PMID: 3522872610.1038/s41592-022-01399-1) Sun, C. et al. Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01769-w (2023). 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). (PMID: 30010673639093810.1038/nbt.4192) Adikusuma, F. et al. Large deletions induced by Cas9 cleavage. Nature 560, E8–E9 (2018). (PMID: 3008992210.1038/s41586-018-0380-z) Zhuang, Y. et al. Increasing the efficiency and precision of prime editing with guide RNA pairs. Nat. Chem. Biol. 18, 29–37 (2022). (PMID: 3471198110.1038/s41589-021-00889-1) Tao, R. et al. WT-PE: prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing. Signal Transduct. Target. Ther. 7, 108 (2022). (PMID: 35440051901873410.1038/s41392-022-00936-w) Adikusuma, F. et al. Optimized nickase- and nuclease-based prime editing in human and mouse cells. Nucleic Acids Res. 49, 10785–10795 (2021). (PMID: 34534334850194810.1093/nar/gkab792) Fujiwara, H. Site-specific non-LTR retrotransposons. Microbiol. Spectr. 3, MDNA3-0001-2014 (2015). (PMID: 2610470010.1128/microbiolspec.MDNA3-0001-2014) Eickbush, T. H. & Eickbush, D. G. Integration, regulation, and long-term stability of R2 retrotransposons. Microbiol. Spectr. 3, MDNA3-0011-2014 (2015). (PMID: 2610470310.1128/microbiolspec.MDNA3-0011-2014) Luan, D. D., Korman, M. H., Jakubczak, J. L. & Eickbush, T. H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72, 595–605 (1993). (PMID: 767995410.1016/0092-8674(93)90078-5) Yang, J., Malik, H. S. & Eickbush, T. H. Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements. Proc. Natl Acad. Sci. USA 96, 7847–7852 (1999). (PMID: 103939102215010.1073/pnas.96.14.7847) Wilkinson, M. E., Frangieh, C. J., Macrae, R. K. & Zhang, F. Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription. Science 380, 301–308 (2023). This study resolves the R2 retrotransposon structure. (PMID: 370231711049905010.1126/science.adg7883) Deng, P. et al. Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon. Cell 186, 2865–2879.e20 (2023). (PMID: 3730119610.1016/j.cell.2023.05.032) Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015). (PMID: 25730490439388310.1038/nmeth.3312) Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016). (PMID: 27214048492735610.1038/nmeth.3871) Dominguez, A. A. et al. CRISPR-mediated synergistic epigenetic and transcriptional control. CRISPR J. 5, 264–275 (2022). (PMID: 35271371920648210.1089/crispr.2021.0099) Zhou, H. et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR–dCas9-activator transgenic mice. Nat. Neurosci. 21, 440–446 (2018). (PMID: 2933560310.1038/s41593-017-0060-6) Tycko, J. et al. Development of compact transcriptional effectors using high-throughput measurements in diverse contexts. Preprint at bioRxiv https://doi.org/10.1101/2023.05.12.540558 (2023). Alerasool, N., Leng, H., Lin, Z.-Y., Gingras, A.-C. & Taipale, M. Identification and functional characterization of transcriptional activators in human cells. Mol. Cell 82, 677–695.e7 (2022). (PMID: 3501603510.1016/j.molcel.2021.12.008) Arnold, C. D. et al. A high-throughput method to identify trans-activation domains within transcription factor sequences. EMBO J. 37, e98896 (2018). (PMID: 30006452609262110.15252/embj.201798896) DelRosso, N. et al. Large-scale mapping and mutagenesis of human transcriptional effector domains. Nature 616, 365–372 (2023). (PMID: 370200221048423310.1038/s41586-023-05906-y) Sanborn, A. L. et al. Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to mediator. eLife 10, e68068 (2021). (PMID: 33904398813714310.7554/eLife.68068) Tycko, J. et al. High-throughput discovery and characterization of human transcriptional effectors. Cell 183, 2020–2035.e16 (2020). (PMID: 33326746817879710.1016/j.cell.2020.11.024) Alerasool, N., Segal, D., Lee, H. & Taipale, M. An efficient KRAB domain for CRISPRi applications in human cells. Nat. Methods 17, 1093–1096 (2020). (PMID: 3302065510.1038/s41592-020-0966-x) Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat. Methods 15, 611–616 (2018). (PMID: 30013045612939910.1038/s41592-018-0048-5) Carleton, J. B., Berrett, K. C. & Gertz, J. Multiplex enhancer interference reveals collaborative control of gene regulation by estrogen receptor α-bound enhancers. Cell Syst. 5, 333–344.e5 (2017). (PMID: 28964699567935310.1016/j.cels.2017.08.011) Replogle, J. M. et al. Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors. eLife 11, e81856 (2022). (PMID: 36576240982940910.7554/eLife.81856) Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016). (PMID: 27662090503911110.1016/j.cell.2016.09.006) Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019). (PMID: 31534142675119710.1038/s41467-019-12166-w) Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018). (PMID: 29426832580736510.1038/s41467-017-02708-5) Hofacker, D. et al. Engineering of effector domains for targeted DNA methylation with reduced off-target effects. Int. J. Mol. Sci. 21, 502 (2020). (PMID: 31941101701345810.3390/ijms21020502) Li, J. et al. Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase. Nat. Commun. 12, 896 (2021). (PMID: 33563994787327710.1038/s41467-021-21188-2) Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015). (PMID: 25849900443040010.1038/nbt.3199) Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60, 385–397 (2015). (PMID: 26593719466026910.1016/j.molcel.2015.10.008) Tong, H. et al. High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects. Nat. Biotechnol. 41, 108–119 (2022). (PMID: 3595367310.1038/s41587-022-01419-7) Wang, Q. et al. The CRISPR-Cas13a gene-editing system induces collateral cleavage of RNA in glioma cells. Adv. Sci. 6, 1901299 (2019). (PMID: 10.1002/advs.201901299) Ai, Y., Liang, D. & Wilusz, J. E. CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells. Nucleic Acids Res. 50, e65 (2022). (PMID: 35244715922654310.1093/nar/gkac159) Kelley, C. P., Haerle, M. C. & Wang, E. T. Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells. Cell Rep. 40, 111226 (2022). (PMID: 35977479980906210.1016/j.celrep.2022.111226) Powell, J. E. et al. Targeted gene silencing in the nervous system with CRISPR-Cas13. Sci. Adv. 8, eabk2485 (2022). (PMID: 35044815876954510.1126/sciadv.abk2485) Li, Y. et al. The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model. Genome Biol. 24, 20 (2023). (PMID: 36726140989354710.1186/s13059-023-02860-w) Shi, P. et al. Collateral activity of the CRISPR/RfxCas13d system in human cells. Commun. Biol. 6, 334 (2023). (PMID: 369779231004999810.1038/s42003-023-04708-2) Xu, C. et al. Programmable RNA editing with compact CRISPR–Cas13 systems from uncultivated microbes. Nat. Methods 18, 499–506 (2021). (PMID: 3394193510.1038/s41592-021-01124-4) Wessels, H.-H. et al. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat. Biotechnol. 38, 722–727 (2020). (PMID: 32518401729499610.1038/s41587-020-0456-9) Méndez-Mancilla, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells. Cell Chem. Biol. 29, 321–327.e4 (2022). (PMID: 3434348410.1016/j.chembiol.2021.07.011) Guo, X. et al. Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens. Cell Genom. 1, 100001 (2021). (PMID: 35664829916447510.1016/j.xgen.2021.100001) Bartolomé, R. A. et al. IL13 receptor α2 signaling requires a scaffold protein, FAM120A, to activate the FAK and PI3K pathways in colon cancer metastasis. Cancer Res. 75, 2434–2444 (2015). (PMID: 2589632710.1158/0008-5472.CAN-14-3650) Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2019). (PMID: 31857715890552510.1038/s41579-019-0299-x) Kato, K. et al. Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex. Cell 185, 2324–2337.e16 (2022). Together with refs. 254 and 255, this study identifies RNA-guided peptidases. (PMID: 3564308310.1016/j.cell.2022.05.003) Strecker, J. et al. RNA-activated protein cleavage with a CRISPR-associated endopeptidase. Science 378, 874–881 (2022). Together with refs. 253 and 255, this study identified RNA-guided peptidases. (PMID: 364232761002873110.1126/science.add7450) Hu, C. et al. Craspase is a CRISPR RNA-guided, RNA-activated protease. Science 377, 1278–1285 (2022). Together with refs. 253 and 254, this study identified RNA-guided peptidases. (PMID: 360070611004182010.1126/science.add5064) Kato, K. et al. RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease. Science 378, 882–889 (2022). (PMID: 3642330410.1126/science.add7347) Rouillon, C. et al. Antiviral signalling by a cyclic nucleotide activated CRISPR protease. Nature 614, 168–174 (2023). (PMID: 3642365710.1038/s41586-022-05571-7) Jiang, K. et al. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR. Nat. Biotechnol. 41, 698–707 (2022). (PMID: 3630298810.1038/s41587-022-01534-5) Kaseniit, K. E. et al. Modular, programmable RNA sensing using ADAR editing in living cells. Nat. Biotechnol. 41, 482–487 (2022). (PMID: 3619877210.1038/s41587-022-01493-x) Qian, Y. et al. Programmable RNA sensing for cell monitoring and manipulation. Nature 610, 713–721 (2022). (PMID: 361988031034834310.1038/s41586-022-05280-1) Fricke, T. et al. Targeted RNA knockdown by a type III CRISPR-Cas complex in zebrafish. CRISPR J. 3, 299–313 (2020). (PMID: 32833532746970110.1089/crispr.2020.0032) Woodside, W. T. et al. Type III-A CRISPR systems as a versatile gene knockdown technology. RNA 28, 1074–1088 (2022). (PMID: 35618430929784110.1261/rna.079206.122) Colognori, D., Trinidad, M. & Doudna, J. A. Precise transcript targeting by CRISPR-Csm complexes. Nat. Biotechnol. 41, 1256–1264 (2023). (PMID: 366907621049741010.1038/s41587-022-01649-9) Abudayyeh, O. O. et al. A cytosine deaminase for programmable single-base RNA editing. Science 365, 382–386 (2019). This article reports a laboratory-evolved C-to-U deaminase fused to dCas13. (PMID: 31296651695656510.1126/science.aax7063) Kapoor, U. et al. ADAR-deficiency perturbs the global splicing landscape in mouse tissues. Genome Res. 30, 1107–1118 (2020). (PMID: 32727871746207910.1101/gr.256933.119) Blencowe, B. J. Alternative splicing: new insights from global analyses. Cell 126, 37–47 (2006). (PMID: 1683987510.1016/j.cell.2006.06.023) Di Giammartino, D. C., Nishida, K. & Manley, J. L. Mechanisms and consequences of alternative polyadenylation. Mol. Cell 43, 853–866 (2011). (PMID: 21925375319400510.1016/j.molcel.2011.08.017) Vandivier, L. E., Anderson, Z. D. & Gregory, B. D. HAMR: high-throughput annotation of modified ribonucleotides. Methods Mol. Biol. 1870, 51–67 (2019). (PMID: 3053954610.1007/978-1-4939-8808-2_4) Vogel, P. et al. Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs. Nat. Methods 15, 535–538 (2018). (PMID: 29967493632265010.1038/s41592-018-0017-z) Stafforst, T. & Schneider, M. F. An RNA-deaminase conjugate selectively repairs point mutations. Angew. Chem. Int. Ed. Engl. 51, 11166–11169 (2012). (PMID: 2303840210.1002/anie.201206489) Katrekar, D. et al. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nat. Methods 16, 239–242 (2019). (PMID: 30737497639552010.1038/s41592-019-0323-0) Montiel-González, M. F., Vallecillo-Viejo, I. C. & Rosenthal, J. J. C. An efficient system for selectively altering genetic information within mRNAs. Nucleic Acids Res. 44, e157 (2016). (PMID: 275577105137428) Merkle, T. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat. Biotechnol. 37, 133–138 (2019). (PMID: 3069269410.1038/s41587-019-0013-6) Reautschnig, P. et al. CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo. Nat. Biotechnol. 40, 759–768 (2022). (PMID: 3498091310.1038/s41587-021-01105-0) Fukuda, M. et al. Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing. Sci. Rep. 7, 41478 (2017). (PMID: 28148949528865610.1038/srep41478) Wettengel, J., Reautschnig, P., Geisler, S., Kahle, P. J. & Stafforst, T. Harnessing human ADAR2 for RNA repair – recoding a PINK1 mutation rescues mitophagy. Nucleic Acids Res. 45, 2797–2808 (2017). (PMID: 27907896) Qu, L. et al. Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nat. Biotechnol. 37, 1380 (2019). (PMID: 3155494010.1038/s41587-019-0292-y) Yi, Z. et al. Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo. Nat. Biotechnol. 40, 946–955 (2022). (PMID: 3514531310.1038/s41587-021-01180-3) Katrekar, D. et al. Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs. Nat. Biotechnol. 40, 938–945 (2022). (PMID: 35145312923283910.1038/s41587-021-01171-4) Wang, X. et al. Develop a compact RNA base editor by fusing ADAR with engineered EcCas6e. Adv. Sci. 10, e2206813 (2023). (PMID: 10.1002/advs.202206813) Huang, X. et al. Programmable C-to-U RNA editing using the human APOBEC3A deaminase. EMBO J. 40, e108209 (2021). (PMID: 33938012809082810.15252/embj.2021108209) Collantes, J. C. et al. Development and characterization of a modular CRISPR and RNA aptamer mediated base editing system. CRISPR J. 4, 58–68 (2021). (PMID: 33616445789845910.1089/crispr.2020.0035) Pecori, R., Di Giorgio, S., Paulo Lorenzo, J. & Nina Papavasiliou, F. Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination. Nat. Rev. Genet. 23, 505–518 (2022). (PMID: 35256818890047310.1038/s41576-022-00459-8) Helm, M. & Motorin, Y. Detecting RNA modifications in the epitranscriptome: predict and validate. Nat. Rev. Genet. 18, 275–291 (2017). (PMID: 2821663410.1038/nrg.2016.169) Roundtree, I. A., Evans, M. E., Pan, T. & He, C. Dynamic RNA modifications in gene expression regulation. Cell 169, 1187–1200 (2017). (PMID: 28622506565724710.1016/j.cell.2017.05.045) Wilson, C., Chen, P. J., Miao, Z. & Liu, D. R. Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase. Nat. Biotechnol. 38, 1431–1440 (2020). (PMID: 32601430771842710.1038/s41587-020-0572-6) Xie, S. et al. Programmable RNA N1 -methyladenosine demethylation by a Cas13d-directed demethylase. Angew. Chem. Int. Ed. Engl. 60, 19592–19597 (2021). (PMID: 3408182710.1002/anie.202105253) Xia, Z. et al. Epitranscriptomic editing of the RNA N6-methyladenosine modification by dCasRx conjugated methyltransferase and demethylase. Nucleic Acids Res. 49, 7361–7374 (2021). (PMID: 34181729828792010.1093/nar/gkab517) Chang, C., Ma, G., Cheung, E. & Hutchins, A. P. A programmable system to methylate and demethylate N6-methyladenosine (m6A) on specific RNA transcripts in mammalian cells. J. Biol. Chem. 298, 102525 (2022). (PMID: 36162509959789210.1016/j.jbc.2022.102525) Li, J. et al. Targeted mRNA demethylation using an engineered dCas13b–ALKBH5 fusion protein. Nucleic Acids Res. 48, 5684–5694 (2020). (PMID: 32356894726118910.1093/nar/gkaa269) Cheng, X. et al. Modeling CRISPR–Cas13d on-target and off-target effects using machine learning approaches. Nat. Commun. 14, 752 (2023). (PMID: 36765063991224410.1038/s41467-023-36316-3) Deep learning and CRISPR-Cas13d ortholog discovery for optimized RNA targeting. Cell Systems 14, 1087–1102.e13 (2023). Kushawah, G. et al. CRISPR-Cas13d induces efficient mRNA knockdown in animal embryos. Dev. Cell 54, 805–817.e7 (2020). (PMID: 3276842110.1016/j.devcel.2020.07.013) Blanchard, E. L. et al. Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents. Nat. Biotechnol. 39, 717–726 (2021). (PMID: 3353662910.1038/s41587-021-00822-w) Abbott, T. R. et al. Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and influenza. Cell 181, 865–876.e12 (2020). (PMID: 32353252718986210.1016/j.cell.2020.04.020) Cui, Z. et al. Cas13d knockdown of lung protease Ctsl prevents and treats SARS-CoV-2 infection. Nat. Chem. Biol. 18, 1056–1064 (2022). (PMID: 358795451008299310.1038/s41589-022-01094-4) Guo, Y. et al. Specific knockdown of Htra2 by CRISPR-CasRx prevents acquired sensorineural hearing loss in mice. Mol. Ther. Nucleic Acids 28, 643–655 (2022). (PMID: 35615000911205310.1016/j.omtn.2022.04.014) Zhao, X. et al. A CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment. Cancer Lett. 431, 171–181 (2018). (PMID: 2987077410.1016/j.canlet.2018.05.042) Kannan, S. et al. Compact RNA editors with small Cas13 proteins. Nat. Biotechnol. 40, 194–197 (2022). (PMID: 3446258710.1038/s41587-021-01030-2) Nakagawa, R. et al. Structure and engineering of the minimal type VI CRISPR-Cas13bt3. Mol. Cell 82, 3178–3192.e5 (2022). (PMID: 36027912761369610.1016/j.molcel.2022.08.001) US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05398029 (2023). Xiao, Q. et al. Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor. Sci. Transl. Med. 14, eabn0449 (2022). (PMID: 3585782410.1126/scitranslmed.abn0449) Li, G. et al. Mini-dCas13X-mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy. J. Clin. Invest. 133, e162809 (2023). (PMID: 36512423988837710.1172/JCI162809) Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020). (PMID: 3202968710.1126/science.aba7365) Xu, L. et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N. Engl. J. Med. 381, 1240–1247 (2019). (PMID: 3150966710.1056/NEJMoa1817426) Chiesa, R. et al. Base-edited CAR7 T cells for relapsed T-cell acute lymphoblastic leukemia. N. Engl. J. Med. 389, 899–910 (2023). (PMID: 3731435410.1056/NEJMoa2300709) McGuirk, J. P. et al. CTX110 allogeneic CRISPR-Cas9-engineered CAR T cells in patients (Pts) with relapsed or refractory (R/R) large B-cell lymphoma (LBCL): results from the phase 1 dose escalation carbon study. Blood 140, 10303–10306 (2022). (PMID: 10.1182/blood-2022-166432) Uchida, N. et al. Fertility-preserving myeloablative conditioning using single-dose CD117 antibody–drug conjugate in a rhesus gene therapy model. Nat. Commun. 14, 6291 (2023). (PMID: 378280211057033510.1038/s41467-023-41153-5) Frangoul, H. et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N. Engl. J. Med. 384, 252–260 (2021). (PMID: 3328398910.1056/NEJMoa2031054) US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05444894 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04774536 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05565248 (2023). Gillmore, J. D. et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 385, 493–502 (2021). (PMID: 3421502410.1056/NEJMoa2107454) US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05120830 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT03872479 (2022). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05477563 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05144386 (2023). Lek, A. et al. Death after high-dose rAAV9 gene therapy in a patient with Duchenne’s muscular dystrophy. N. Engl. J. Med. 389, 1203–1210 (2023). (PMID: 3775428510.1056/NEJMoa2307798) Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014). (PMID: 2433656910.1126/science.1246981) Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014). (PMID: 2433657110.1126/science.1247005) US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT03399448 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT03655678 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT03745287 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04925206 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04035434 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05356195 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05329649 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04819841 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05066165 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04557436 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04849910 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05951205 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05662904 (2022). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05397184 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05643742 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04244656 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04502446 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04438083 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05795595 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04637763 (2024). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05722418 (2024). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04426669 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05566223 (2022). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04601051 (2023). US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05514249 (2022). Kenjo, E. et al. Low immunogenicity of LNP allows repeated administrations of CRISPR–Cas9 mRNA into skeletal muscle in mice. Nat. Commun. 12, 7101 (2021). (PMID: 34880218865481910.1038/s41467-021-26714-w) Wei, T. et al. Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models. Nat. Commun. 14, 7322 (2023). (PMID: 379519481064056310.1038/s41467-023-42948-2) Li, B. et al. Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing. Nat. Biotechnol. 41, 1410–1415 (2023). (PMID: 369976801054467610.1038/s41587-023-01679-x) Villiger, L. et al. In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA. Nat. Biomed. Eng. 5, 179–189 (2021). (PMID: 33495639761098110.1038/s41551-020-00671-z) Finn, J. D. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227–2235 (2018). (PMID: 2949026210.1016/j.celrep.2018.02.014) Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179–1187 (2017). Demonstration of efficient, lipid nanoparticle-mediated genome editing in the mouse liver. (PMID: 29131148590166810.1038/nbt.4005) Raguram, A., Banskota, S. & Liu, D. R. Therapeutic in vivo delivery of gene editing agents. Cell 185, 2806–2827 (2022). Comprehensive review on in vivo delivery of genome editors. (PMID: 35798006945433710.1016/j.cell.2022.03.045) 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). (PMID: 31937940698078310.1038/s41551-019-0501-5) Davis, J. R. et al. Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors. Nat. Biomed. Eng. 6, 1272–1283 (2022). (PMID: 35902773965215310.1038/s41551-022-00911-4) 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). (PMID: 3029790410.1038/s41591-018-0209-1) 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). (PMID: 2970263710.1038/nbt.4148) Koblan, L. W. et al. In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice. Nature 589, 608–614 (2021). (PMID: 33408413787220010.1038/s41586-020-03086-7) Arbab, M. et al. Base editing rescue of spinal muscular atrophy in cells and in mice. Science 380, eadg6518 (2023). (PMID: 369961701027000310.1126/science.adg6518) Srivastava, A. In vivo tissue-tropism of adeno-associated viral vectors. Curr. Opin. Virol. 21, 75–80 (2016). (PMID: 27596608513812510.1016/j.coviro.2016.08.003) Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34, 204–209 (2016). (PMID: 26829320508805210.1038/nbt.3440) Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Mol. Ther. 18, 80–86 (2010). (PMID: 1990423410.1038/mt.2009.255) Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019). (PMID: 3118156710.1038/s41586-019-1314-0) Banskota, S. et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 185, 250–265.e16 (2022). This article reports on engineering of virus-like particles for in vivo base editor delivery. (PMID: 35021064880925010.1016/j.cell.2021.12.021) Campbell, L. A. et al. Gesicle-mediated delivery of CRISPR/Cas9 ribonucleoprotein complex for inactivating the HIV provirus. Mol. Ther. 27, 151–163 (2019). (PMID: 3038935510.1016/j.ymthe.2018.10.002) Choi, J. G. et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther. 23, 627–633 (2016). (PMID: 2705280310.1038/gt.2016.27) Hamilton, J. R. et al. Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell Rep. 35, 109207 (2021). (PMID: 34077734823621610.1016/j.celrep.2021.109207) Zhuo, C. et al. Spatiotemporal control of CRISPR/Cas9 gene editing. Signal Transduct. Target. Ther. 6, 238 (2021). (PMID: 34148061821462710.1038/s41392-021-00645-w) Parnas, O. et al. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162, 675–686 (2015). (PMID: 26189680452237010.1016/j.cell.2015.06.059) Jain, I. H. et al. Hypoxia as a therapy for mitochondrial disease. Science 352, 54–61 (2016). (PMID: 26917594486074210.1126/science.aad9642) Marceau, C. D. et al. Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens. Nature 535, 159–163 (2016). (PMID: 27383987496479810.1038/nature18631) Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017). (PMID: 28854172567571610.1038/nature23875) Sanjana, N. E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016). (PMID: 27708104514410210.1126/science.aaf7613) Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, aah7111 (2017). (PMID: 2798008610.1126/science.aah7111) Joung, J. et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature 548, 343–346 (2017). (PMID: 28792927570665710.1038/nature23451) Replogle, J. M. et al. Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell 185, 2559–2575.e28 (2022). (PMID: 35688146938047110.1016/j.cell.2022.05.013) Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882.e21 (2016). (PMID: 27984733531557110.1016/j.cell.2016.11.048) Dixit, A. et al. Perturb-seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens. Cell 167, 1853–1866.e17 (2016). (PMID: 27984732518111510.1016/j.cell.2016.11.038) Xu, P. et al. Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs. Nat. Biotechnol. 39, 1403–1413 (2021). (PMID: 3415540710.1038/s41587-021-00944-1) Hanna, R. E. et al. Massively parallel assessment of human variants with base editor screens. Cell 184, 1064–1080.e20 (2021). (PMID: 3360697710.1016/j.cell.2021.01.012) Cuella-Martin, R. et al. Functional interrogation of DNA damage response variants with base editing screens. Cell 184, 1081–1097.e19 (2021). (PMID: 33606978801828110.1016/j.cell.2021.01.041) Perner, F. et al. MEN1 mutations mediate clinical resistance to menin inhibition. Nature 615, 913–919 (2023). (PMID: 369225891015789610.1038/s41586-023-05755-9) Morris, J. A. et al. Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens. Science 380, eadh7699 (2023). (PMID: 371413131051823810.1126/science.adh7699) Martin-Rufino, J. D. et al. Massively parallel base editing to map variant effects in human hematopoiesis. Cell 186, 2456–2474.e24 (2023). (PMID: 3713730510.1016/j.cell.2023.03.035) Li, S. et al. Screening for functional circular RNAs using the CRISPR–Cas13 system. Nat. Methods 18, 51–59 (2020). (PMID: 3328896010.1038/s41592-020-01011-4) Freije, C. A. et al. Programmable inhibition and detection of RNA viruses using Cas13. Mol. Cell 76, 826–837.e11 (2019). (PMID: 31607545742262710.1016/j.molcel.2019.09.013) Wessels, H.-H. et al. Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq. Nat. Methods 20, 86–94 (2023). (PMID: 3655027710.1038/s41592-022-01705-x) |
تواريخ الأحداث: | Date Created: 20240202 Date Completed: 20240528 Latest Revision: 20240529 |
رمز التحديث: | 20240530 |
DOI: | 10.1038/s41580-023-00697-6 |
PMID: | 38308006 |
قاعدة البيانات: | MEDLINE |
تدمد: | 1471-0080 |
---|---|
DOI: | 10.1038/s41580-023-00697-6 |