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Smith, J., Rodriguez, T., Mou, H., Kwan, S-Y., Pratt, H., Zhang, X-O., Cao, Y., Liang, S., Ozata, D. M., Yu, T., Yin, Q., Hazeltine, M., Weng, Z., Sontheimer, E. J., Xue, W. (2020). YAP1 withdrawal in hepatoblastoma drives therapeutic differentiation of tumor cells to functional hepatocyte-like cells. Hepatology, in press. (PMID: 32452550)

Chatterjee, P., Lee, J., Nip, L., Koseki, S. R. T., Tysinger, E., Sontheimer, E. J., Jacobson, J. M. and Jakimo, N. (2020). A Cas9 with PAM recognition for adenine dinucleotides.  Nature Communications 11, 2474(PMID: 32424114)

Chatterjee, P., Jakimo, N., Lee, J., Amrani, N., Rodriguez, T., Koseki, S. R. T., Tysinger, E., Qing, R., Sontheimer, E. J. and Jacobson, J. (2020) An engineered ScCas9 with broad PAM range and high specificity and activity.  Nature Biotechnology, in press(PMID: 32393822)

Iyer, S., Mir, A., Vega-Badillo, J., Roscoe, B. P., Ibraheim, R., Zhu, L. J., Lee, J., Liu, P., Luk, K., Mintzer, E., Soares de Brito, J., Zamore, P. D., Sontheimer, E. J. and Wolfe, S. A. (2019) Efficient homology-directed repair with circular ssDNA donors.  Manuscript in revision. 

Ghanta, K. S., Dokshin, G. A., Mir, A., Shin, M., Medha, P., Edraki, A., Gneid, H., Lawson, N., Watts, J., Sontheimer, E. J., and Mello, C. C. (2019) 5’-Modified DNA donors are highly potent at low concentrations and improve efficiency of homology-directed genome editing. Manuscript in revision. 

Davidson, A. R., Lu, W.-T., Stanley, S.Y., Wang, J., Mejdani, M., Trost, C. N., Hicks, B.T., Lee, J. and Sontheimer, E. J.  (2020) Anti-CRISPRs: Protein inhibitors of CRISPR-Cas systems.  Annual Review of Biochemistry 89, 309-332. (PMID: 32186918)

Barrangou, R. and Sontheimer, E. J. (2020) CRISPR shields: Fending off diverse Cas nucleases with nucleus-like structures.  Fending off diverse Cas nucleases with nucleus-like structures. Molecular Cell 77, 934-935. (PMID: 32142691)

Sun, W., Yang, J., Cheng, Z., Amrani, N., Liu, C., Wang, K., Ibraheim, R., Edraki, A., Huang, X., Wang, M., Wang, J., Liu, L., Sheng, G., Yang, Y., Liu, J., Sontheimer, E. J., and Wang, Y. (2019) Structures of Neisseria meningitidis Cas9 complexes in catalytically poised and anti-CRISPR-inhibited states. Molecular Cell 76, 938-952. (PMID: 31668930)

Garcia, B., Lee, J., Edraki, A., Hidalgo-Reyes, Y., Erwood, S., Mir, A., Trost, C., Seroussi, U., Stanley, S. Y., Cohn, R. D., Claycomb, J. M., Sontheimer, E. J., Maxwell, K. L. and Davidson, A. R. (2019) Anti-CRISPR AcrIIA5 potently inhibits all Cas9 homologs used for genome editing. Cell Reports 29, 1739-1746.  (PMID: 31722192)

Lee, J., Mou, H., Ibraheim, R., Liang, S.-Q., Xue, W., and Sontheimer, E. J. (2019) Tissue-specific genome editing in vivo by microRNA-repressible anti-CRISPR proteins. RNA 25, 1421-1431. (PMID: 31439808)

Sontheimer, E. J. (2019) X-Tracting a new CRISPR-Cas genome-editing platform from metagenomic data sets. The CRISPR Journal 2, 148-150.  (PMID: 31225753)

Thavalingam, A., Cheng, Z., Garcia, B., Huang, X., Shah, M., Sun, W., Wang, M., Harrington, L., Hwang, S., Hidalgo-Reyes, Y., Sontheimer, E. J., Doudna, J. A., Davidson, A. R., Moraes, T. F., Wang, Y., and Maxwell, K. L. (2019) Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anit-CRISPR AcrIIC2 Nature Communications 10, 2806. (PMID: 31243272)

Edraki, A., Mir, A., Ibraheim, R., Gainetdinov, I., Yoon, Y., Gallant, J., Song, C.-Q., Cao, Y., Xue, W., Rivera-Pérez, J. A., and Sontheimer, E. J. (2019) A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing. Molecular Cell 73, 714-726. (PMID: 30581144)

Gao, X. D., Rodriguez, T., and Sontheimer, E. J. (2018) Adapting dCas9-APEX2 for subnuclear proteomic profiling. Methods in Enzymology 616, 365-383. (PMID: 30691651)

Amrani, N., Gao, X. D., Liu, P., Edraki, A., Mir, A., Ibraheim, R., Gupta, A., Sasaki, K. E., Wu, T., Donohoue, P. D., Settle, A. H., Lied, A. M., McGovern, K., Fuller, C. K., Cameron, P., Fazzio, T. G., Zhu, L. J., Wolfe, S. A., and Sontheimer, E. J. (2018) NmeCas9 is an intrinsically high-fidelity genome editing platform. Genome Biology 19, 214. (PMID: 30518407)

Lee, J., Mir, A., Edraki, A., Garcia, B., Amrani, N., Lou, H. E., Gainetdinov, I., Pawluk, A., Ibraheim, R., Gao, X. D., Liu, P., Davidson, A. R., Maxwell, K. L., and Sontheimer, E. J. (2018) Potent Cas9 inhibition in bacterial and human cells by AcrIIC4 and AcrIIC5 anti-CRISPR proteins. mBio 9, e02321-18. (PMID: 30514786)

Bolukbasi, M. F., Liu, P., Luk, K., Kwok, S. F., Gupta, A., Amrani, N., Sontheimer, E. J., Zhu, L. J., and Wolfe, S. A. (2018) Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing. Nature Communications 9, 4856. (PMID: 30531933)

Bondy-Denomy, J., Davidson, A. R., Doudna, J. A., Fineran, P. C., Maxwell, K. L., Moineau, S., Peng, X., Sontheimer, E. J., and Weidenheft, B. (2018) A unified resource for tracking anti-CRISPR names. The CRISPR Journal 1, 304-305. (PMID: 31021273)

Ibraheim, R., Song, C.-Q., Mir, A., Amrani, N., Xue, W., and Sontheimer, E. J. (2018) All-in-one Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo. Genome Biology 19, 137. (PMID: 30231914)

Mir, A., Alterman, J. A., Hassler, M. R., Debacker, A. J., Hudgens, E., Echevarria, D., Brodsky, M. H., Khvorova, A., Watts, J. K., and Sontheimer, E. J. (2018) Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing. Nature Communications 9, 2641. (PMID: 29980686)

Stone, N., Hilbert, B. J., Hidalgo, D., Halloran, K. T., Lee, J., Sontheimer, E. J., and Kelch, B. A. (2018) A hyperthermophilic phage decoration protein suggests common evolutionary origin with herpesvirus triplex proteins and an anti-CRISPR protein. Structure 26, 936-947. (PMID: 29779790)

Gao, X. D., Tu, L.-C., Mir, A., Rodriguez, T., Ding, Y., Leszyk, J., Dekker, J., Shaffer, S. A., Zhu, L. J., Wolfe, S. A., and Sontheimer, E. J. (2018) C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2. Nature Methods 15, 433-436. (PMID: 29735996)

Edraki, A., and Sontheimer, E. J. (2018) CRISPRs from scratch. Nature Microbiology 3, 261-262. (PMID: 29463924)

Mir, A., Edraki, A., Lee, J., and Sontheimer, E. J. (2018) Type II-C CRISPR-Cas9 biology, mechanism and application. ACS Chemical Biology 13, 357-365. (PMID: 29202216)

Harrington, L. B., Doxzen, K. W., Ma, E., Liu, J.-J., Knott, G. J., Edraki, A., Amrani, N., Chen, J. S., Cofsky, J. C., Kranzusch, P. J., Sontheimer, E. J., Davidson, A. R., Maxwell, K., and Doudna, J. A. (2017). A broad-spectrum inhibitor of CRISPR-Cas9. Cell 170, 1224-1233. (PMID: 28844692)

Sontheimer, E. J. and Davidson, A. R. (2017) Inhibition of CRISPR-Cas systems by mobile genetic elements. Current Opinion in Microbiology 37, 120-127. (PMID: 28668720)

Mou, H., Smith, J. L., Yin, H., Peng, L., Moore, J., Zhang, X.-O., Song, C.-Q., Sheel, A., Wu, Q., Ozata, D. M., Li, Y., Anderson, D. G., Emerson, C. P., Sontheimer, E. J., Moore, M. J., Weng, Z., and Xue, W. (2017) CRISPR-mediated genome editing induces exon skipping by alternative splicing or exon deletion. Genome Biology 18, 108. (PMID: 28615073)

Pawluk, A., Amrani, N., Zhang, Y., Garcia, B., Hidalgo-Reyes, Y., Lee, J., Edraki, A., Shah, M., Sontheimer, E. J., Maxwell, K., and Davidson, A. R. (2016) Naturally occurring off-switches for CRISPR-Cas9. Cell 167, 1829-1838. (PMID: 27984730)

Sontheimer, E. J. and Marraffini, L. A. (2016) CRISPR goes retro. Science 351, 920-921. (PMID:26917756

Sontheimer, E. J. and Wolfe, S. A. (2015) Cas9 gets a classmate. Nature Biotechnology 33, 1240-1241. (PMID: 26650011)

Zhang, Y., Rajan, R., Seifert, H. S., Mondragón, A. and Sontheimer, E. J. (2015) DNase H activity of Neisseria meningitidis Cas9. Molecular Cell 60, 242-255. (PMID: 26474066)

Wakefield, N., Rajan, R. and Sontheimer, E. J. (2015) Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis. FEBS Letters 589, 3197-3204. (PMID: 26364721)

Wang, D., Mou, H., Li, S., Li, Y., Hough, S., Tran, K., Li, J., Yin, H., Anderson, D. G., Sontheimer, E. J., Weng, Z., Gao, G. and Xue, W. (2015) Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Human Gene Therapy 26, 432-442. (PMID: 26086867)

Sontheimer, E. J. and Barrangou, R. (2015) The bacterial origins of the CRISPR genome editing revolution. Human Gene Therapy 26, 413-424. (PMID: 26078042)

Doudna, J. A. and Sontheimer, E. J., Editors (2014) The use of CRISPR-Cas9, ZFNs, and TALENs in generating site-specific genome alterations. Methods in Enzymology, Volume 516. (PMID: 25398356)

Zhang, Y. and Sontheimer, E.J. (2014) Cascading into focus. Science 345, 1452-1453. (PMID: 25237089)

Hurtado, S. B., Kim Guisbert, K. S. and Sontheimer, E. J. (2014) SPO24 is a transcriptionally dynamic, small ORF-encoding locus required for efficient sporulation in S. cerevisiae. PLoS ONE 9(8), e105058. (PMID: 25127041)

Hou, Z., Zhang, Y., Propson, N. E., Howden, S. E., Chu, L.-F., Sontheimer, E. J. and Thomson, J. A. (2013) Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proceedings of the National Academy of Sciences U.S.A. 110, 15644-15649. (PMID: 23940360)

Zhang, Y., Heidrich, N., Joseph, B., Gunderson, C. Seifert, H. S., Schoen, C., Vogel, J. and Sontheimer, E. J. (2013) Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Molecular Cell 50, 488-503. (PMID: 23706818)

Kim Guisbert, K. S. and Sontheimer, E. J. (2013) Quit stalling or you’ll be silenced. Cell 152, 938-939. (PMID: 23452843)

Sontheimer, E. J. (2012) Small RNAs of opposite sign…but same absolute value. Cell 151, 1157-1158. (PMID: 23217700)

Kim Guisbert, K. S., Zhang, Y., Flatow, J., Hurtado, S., Staley, J. P., Lin, S. and Sontheimer, E. J. (2012) Meiosis-induced alterations in transcript architecture and noncoding RNA expression in S. cerevisiae. RNA 18, 1142-1153. (PMID: 22539527)

Gerbasi, V. R., Preall, J. B., Golden, D. E., Powell, D. W., Cummins, T. D. and Sontheimer, E. J. (2011) Blanks, a nuclear siRNA/dsRNA binding complex component, is required for Drosophila spermiogenesis. Proceedings of the National Academy of Sciences U.S.A. 108, 3204-3209. (PMID: 21300896)

Sontheimer, E. J. and Marraffini, L. A. (2010) Microbiology: Slicer for DNA. Nature 468, 45-46. (PMID: 21048757)

Gerbasi, V. R., Golden, D. E., Hurtado, S. B. and Sontheimer, E. J. (2010) Proteomic identification of Drosophila siRNA-associated factors. Molecular & Cellular Proteomics 9, 1866-1872. (PMID: 20472918)

Marraffini, L. A. and Sontheimer, E. J. (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature Reviews Genetics 11, 181-190. (PMID: 20125085)

Marraffini, L. A. and Sontheimer, E. J. (2010) Self vs. non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568-571. (PMID: 20072129)

Marraffini, L. A. and Sontheimer, E. J. (2009) Invasive DNA, chopped and in the CRISPR. Structure 17, 786-787. (PMID: 19523896)

Lee, Y. S., Pressman, S., Andress, A. P., Kim, K., White, J. L., Cassidy, J. J., Li, X., Lubell, K., Lim, D. H., Cho, I. S., Nakahara, K., Preall, J. B., Bellare, P., Sontheimer, E. J. and Carthew, R. W. (2009) Silencing by small RNAs is linked to endosome trafficking. Nature Cell Biology 11, 1150-1156. (PMID: 19684574)

Carthew, R. W., and Sontheimer, E. J. (2009) Origins and mechanisms of siRNAs and miRNAs. Cell 136, 642-655. (PMID: 19239886)

Marraffini, L. A. and Sontheimer, E. J. (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843-1845. (PMID: 19095942)

Bellare, P., Small, E. C., Huang, X., Wohlschlegel, J. A., Staley, J. P. and Sontheimer, E. J. (2008) A role for ubiquitin in the spliceosome assembly pathway. Nature Structural & Molecular Biology 15, 444-451. (PMID: 18425143)

Golden, D. E., Gerbasi, V. R., and Sontheimer, E. J. (2008) An inside job for siRNAs. Molecular Cell 31, 309-312. (PMID: 18691963)

Bellare, P., and Sontheimer, E. J. (2007) A fork in the road for microRNAs. Nature Structural & Molecular Biology 14, 684-686. (PMID: 17676029)

Preall, J. B., He, Z., Gorra, J. and Sontheimer, E. J. (2006). Short interfering RNA strand selection is independent of double-stranded RNA processing polarity during RNA interference in Drosophila. Current Biology 16, 530-535. (PMID: 16527750)

Bellare, P., Kutach, A. K., Rines, A., Guthrie, C. and Sontheimer, E. J. (2006). Ubiquitin binding by a variant Jab1/MPN domain in the essential pre-mRNA splicing factor Prp8p. RNA 12, 292-302. (PMID: 16428608)

Pham, J. W. and Sontheimer, E. J. (2005). Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway. Journal of Biological Chemistry 280, 39278-39283. (PMID: 16179342)

Preall, J. B., and Sontheimer, E. J. (2005). RNAi: RISC gets loaded. Cell 123, 543-545. (PMID: 16286001)

Sontheimer, E. J., and Carthew, R. W. (2005). Silence from within: Endogenous siRNAs and miRNAs. Cell 122, 9-12. (PMID: 16009127)

Pham, J. W., and Sontheimer, E. J. (2005). Separation of Drosophila RNA silencing complexes by native gel electrophoresis. In “Methods in Molecular Biology, vol. 309: RNA Silencing: Methods and Protocols” (ed. G. G. Carmichael), pp. 11-16. Humana Press, Totowa, New Jersey. (PMID: 15990394)

Sontheimer, E. J. (2005). Assembly and function of RNA silencing complexes. Nature Reviews Molecular Cell Biology 6, 127-138. (PMID: 15654322)

Pellino, J. L., Jaskiewicz, L., Filipowicz, W. and Sontheimer, E. J. (2005). ATP modulates siRNA interactions with an endogenous human Dicer complex. RNA 11, 1719-1724. (PMID: 16177131)

Pham, J. W., Radhakrishnan, I. and Sontheimer, E. J. (2004). Thermodynamic and structural characterization of 2’-nitrogen-modified RNA duplexes. Nucleic Acids Research 32, 3446-3455. (PMID: 15247335)

Sontheimer, E. J., and Carthew, R. W. (2004). Argonaute journeys into the heart of RISC. Science 305, 1409-1410. (PMID: 15353786)

Pham, J. W., and Sontheimer, E. J. (2004). The making of an siRNA. Molecular Cell 15, 163-164. (PMID: 15260964)

He, Z., and Sontheimer, E. J. (2004). “siRNAs and miRNAs”: A meeting review on RNA silencing. RNA 10, 1165-1173. (PMID: 15272116)

Pham, J. W., Pellino, J. L., Lee, Y. S., Carthew, R. W. and Sontheimer, E. J. (2004). A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117, 83-94. (PMID: 15066284)

Lee, Y. S., Nakahara, K., Pham, J. W., Kim, K., He, Z., Sontheimer, E. J. and Carthew, R. W. (2004). Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69-81. (PMID: 15066283)

Pellino, J. L. and Sontheimer, E. J. (2003). R2D2 leads the silencing trigger to mRNA's death star. Cell 115, 132-133. (PMID: 14567910)

Sontheimer, E. J. (2001). The spliceosome shows its metal. Nature Structural Biology 8, 11-13. (PMID: 11135658)

Gordon, P. M., Sontheimer, E. J. and Piccirilli, J. A. (2000). Kinetic characterization of the second step of group II intron splicing: Role of metal ions and the cleavage site 2’-OH in catalysis. Biochemistry 39, 12939-12952. (PMID: 11041859)

Gordon, P. M., Sontheimer, E. J. and Piccirilli, J. A. (2000). Metal ion catalysis during the exon ligation step of nuclear pre-mRNA splicing: Extending the parallels between the spliceosome and group II introns. RNA 6, 199-205. (PMID: 10688359)

Warnecke, J. M., Sontheimer, E. J., Piccirilli, J. A. and Hartmann, R. K. (2000). Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: Analysis of precursor tRNAs with a single 3’-S-phosphorothiolate internucleotide linkage. Nucleic Acids Research 28, 720-727. (PMID: 10637323)

Sontheimer, E. J., Gordon, P. M. and Piccirilli, J. A. (1999). Metal ion catalysis during group II intron self-splicing: Parallels with the spliceosome. Genes and Development 13, 1729-1741. (PMID: 10398685)

Sontheimer, E. J. (1999). Bridging sulfur substitutions in the analysis of pre-mRNA splicing. Methods 18, 29-37. (PMID: 10208814)

Sontheimer, E. J., Sun, S. and Piccirilli, J. A. (1997). Metal ion catalysis during splicing of premessenger RNA. Nature 388, 801-805. (PMID: 9285595)

Sontheimer, E. J.  (1994). Site-specific RNA crosslinking with 4-thiouridine. Molecular Biology Reports 20, 35-44. (PMID: 7531281)

Sontheimer, E. J. and Steitz, J. A. (1993). The U5 and U6 small nuclear RNAs as active site components of the spliceosome. Science 262, 1989-1996. (PMID: 8266094)

Cortes, J. J., Sontheimer, E. J., Seiwert, S. D. and Steitz, J. A. (1993). Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5' splice sites in vivo. EMBO Journal 12, 5181-5189. (PMID: 8262061)

Wyatt, J. R., Sontheimer, E. J. and Steitz, J. A. (1992). Site-specific crosslinking of mammalian U5 snRNP to the 5' splice site prior to the first step of premessenger RNA splicing. Genes and Development 6, 2542-2553. (PMID: 1340469)

Gelpi, C., Sontheimer, E. J. and Rodriguez-Sanchez, J. L. (1992). Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion. Proceedings of the National Academy of Sciences U.S.A. 89, 9739-9743. (PMID: 1409691

Sontheimer, E. J. and Steitz, J. A. (1992). Three novel functional variants of human U5 small nuclear RNA. Molecular and Cell Biology12, 734-746. (PMID: 1310151)

 
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  • Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

    Author(s): Pengpeng Liu,Shun-Qing Liang,Chunwei Zheng,Esther Mintzer,Yan G Zhao,Karthikeyan Ponnienselvan,Aamir Mir,Erik J Sontheimer,Guangping Gao,Terence R Flotte,Scot A Wolfe,Wen Xue
    Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both...
  • The NIH Somatic Cell Genome Editing program

    Author(s): Krishanu Saha,Erik J Sontheimer,P J Brooks,Melinda R Dwinell,Charles A Gersbach,David R Liu,Stephen A Murray,Shengdar Q Tsai,Ross C Wilson,Daniel G Anderson,Aravind Asokan,Jillian F Banfield,Krystof S Bankiewicz,Gang Bao,Jeff W M Bulte,Nenad Bursac,Jarryd M Campbell,Daniel F Carlson,Elliot L Chaikof,Zheng-Yi Chen,R Holland Cheng,Karl J Clark,David T Curiel,James E Dahlman,Benjamin E Deverman,Mary E Dickinson,Jennifer A Doudna,Stephen C Ekker,Marina E Emborg,Guoping Feng,Benjamin S Freedman,David M Gamm,Guangping Gao,Ionita C Ghiran,Peter M Glazer,Shaoqin Gong,Jason D Heaney,Jon D Hennebold,John T Hinson,Anastasia Khvorova,Samira Kiani,William R Lagor,Kit S Lam,Kam W Leong,Jon E Levine,Jennifer A Lewis,Cathleen M Lutz,Danith H Ly,Samantha Maragh,Paul B McCray,Todd C McDevitt,Oleg Mirochnitchenko,Ryuji Morizane,Niren Murthy,Randall S Prather,John A Ronald,Subhojit Roy,Sushmita Roy,Venkata Sabbisetti,W Mark Saltzman,Philip J Santangelo,David J Segal,Mary Shimoyama,Melissa C Skala,Alice F Tarantal,John C Tilton,George A Truskey,Moriel Vandsburger,Jonathan K Watts,Kevin D Wells,Scot A Wolfe,Qiaobing Xu,Wen Xue,Guohua Yi,Jiangbing Zhou,SCGE Consortium
    The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome...
  • Shutting down RNA-targeting CRISPR

    Author(s): Rodolphe Barrangou,Erik J Sontheimer
    No abstract
  • Publisher Correction: An engineered ScCas9 with broad PAM range and high specificity and activity

    Author(s): Pranam Chatterjee,Noah Jakimo,Jooyoung Lee,Nadia Amrani,Tomás Rodríguez,Sabrina R T Koseki,Emma Tysinger,Rui Qing,Shilei Hao,Erik J Sontheimer,Joseph Jacobson
    An amendment to this paper has been published and can be accessed via a link at the top of the paper.
  • YAP1 Withdrawal in Hepatoblastoma Drives Therapeutic Differentiation of Tumor Cells to Functional Hepatocyte-Like Cells

    Author(s): Jordan L Smith,Tomás C Rodríguez,Haiwei Mou,Suet-Yan Kwan,Henry Pratt,Xiao-Ou Zhang,Yueying Cao,Shunqing Liang,Deniz M Ozata,Tianxiong Yu,Qiangzong Yin,Max Hazeltine,Zhiping Weng,Erik J Sontheimer,Wen Xue
    CONCLUSIONS: YAP1^(S127A) withdrawal, without silencing oncogenic β-catenin, significantly regresses hepatoblastoma, providing in vivo data to support YAP1 as a therapeutic target for HB. YAP1^(S127A) withdrawal alone sufficiently drives long-term regression in HB, as it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.
  • A Cas9 with PAM recognition for adenine dinucleotides

    Author(s): Pranam Chatterjee,Jooyoung Lee,Lisa Nip,Sabrina R T Koseki,Emma Tysinger,Erik J Sontheimer,Joseph M Jacobson,Noah Jakimo
    CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5[Formula: see text]-NAAN-3[Formula: see text] PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the...
  • An engineered ScCas9 with broad PAM range and high specificity and activity

    Author(s): Pranam Chatterjee,Noah Jakimo,Jooyoung Lee,Nadia Amrani,Tomás Rodríguez,Sabrina R T Koseki,Emma Tysinger,Rui Qing,Shilei Hao,Erik J Sontheimer,Joseph Jacobson
    CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9-Sc^(++) and a higher-fidelity mutant HiFi-Sc^(++)-that have simultaneously broad 5'-NNG-3' PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc^(++) and HiFi-Sc^(++) extend the use of CRISPR editing for...
  • Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems

    Author(s): Alan R Davidson,Wang-Ting Lu,Sabrina Y Stanley,Jingrui Wang,Marios Mejdani,Chantel N Trost,Brian T Hicks,Jooyoung Lee,Erik J Sontheimer
    Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The...
  • CRISPR Shields: Fending Off Diverse Cas Nucleases with Nucleus-like Structures

    Author(s): Rodolphe Barrangou,Erik J Sontheimer
    Two recent studies have uncovered a novel means by which bacteriophages thwart host immunity. Mendoza et al. (2020) and Malone et al. (2020) demonstrate that a nucleus-like proteinaceous structure shields phage DNA from CRISPR-associated nucleases encompassing Cascade-Cas3, Cas9, and Cas12.
  • Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing

    Author(s): Bianca Garcia,Jooyoung Lee,Alireza Edraki,Yurima Hidalgo-Reyes,Steven Erwood,Aamir Mir,Chantel N Trost,Uri Seroussi,Sabrina Y Stanley,Ronald D Cohn,Julie M Claycomb,Erik J Sontheimer,Karen L Maxwell,Alan R Davidson
    CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory...
  • Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States

    Author(s): Wei Sun,Jing Yang,Zhi Cheng,Nadia Amrani,Chao Liu,Kangkang Wang,Raed Ibraheim,Alireza Edraki,Xue Huang,Min Wang,Jiuyu Wang,Liang Liu,Gang Sheng,Yanhua Yang,Jizhong Lou,Erik J Sontheimer,Yanli Wang
    High-resolution Cas9 structures have yet to reveal catalytic conformations due to HNH nuclease domain positioning away from the cleavage site. Nme1Cas9 and Nme2Cas9 are compact nucleases for in vivo genome editing. Here, we report structures of meningococcal Cas9 homologs in complex with sgRNA, dsDNA, or the AcrIIC3 anti-CRISPR protein. DNA-bound structures represent an early step of target recognition, a later HNH pre-catalytic state, the HNH catalytic state, and a cleaved-target-DNA-bound...
  • Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins

    Author(s): Jooyoung Lee,Haiwei Mou,Raed Ibraheim,Shun-Qing Liang,Pengpeng Liu,Wen Xue,Erik J Sontheimer
    CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from editing activity in unintended cell types or tissues upon in vivo delivery (e.g., by adeno-associated virus (AAV) vectors). Although tissue-specific promoters and serotypes with tissue tropisms...
  • Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2

    Author(s): Annoj Thavalingam,Zhi Cheng,Bianca Garcia,Xue Huang,Megha Shah,Wei Sun,Min Wang,Lucas Harrington,Sungwon Hwang,Yurima Hidalgo-Reyes,Erik J Sontheimer,Jennifer Doudna,Alan R Davidson,Trevor F Moraes,Yanli Wang,Karen L Maxwell
    CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2(Nme) alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2(Nme) inhibits Cas9 through interactions with the positively charged bridge...
  • X-Tracting a New CRISPR-Cas Genome-Editing Platform from Metagenomic Data Sets

    Author(s): Erik J Sontheimer
    No abstract
  • A Unified Resource for Tracking Anti-CRISPR Names

    Author(s): Joseph Bondy-Denomy,Alan R Davidson,Jennifer A Doudna,Peter C Fineran,Karen L Maxwell,Sylvain Moineau,Xu Peng,Eric J Sontheimer,Blake Wiedenheft
    No abstract
  • Adapting dCas9-APEX2 for subnuclear proteomic profiling

    Author(s): Xin D Gao,Tomás C Rodríguez,Erik J Sontheimer
    Genome organization and subnuclear protein localization are essential for normal cellular function and have been implicated in the control of gene expression, DNA replication, and genomic stability. The coupling of chromatin conformation capture (3C), chromatin immunoprecipitation and sequencing, and related techniques have continuously improved our understanding of genome architecture. To profile site-specifically DNA-associated proteins in a high-throughput and unbiased manner, the...
  • A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing

    Author(s): Alireza Edraki,Aamir Mir,Raed Ibraheim,Ildar Gainetdinov,Yeonsoo Yoon,Chun-Qing Song,Yueying Cao,Judith Gallant,Wen Xue,Jaime A Rivera-Pérez,Erik J Sontheimer
    CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria...
  • Publisher Correction: Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing

    Author(s): Mehmet Fatih Bolukbasi,Pengpeng Liu,Kevin Luk,Samantha F Kwok,Ankit Gupta,Nadia Amrani,Erik J Sontheimer,Lihua Julie Zhu,Scot A Wolfe
    The original version of this Article contained errors in the author affiliations. Mehmet Fatih Bolukbasi was incorrectly associated with Bluebird Bio., Cambridge, MA, USA and Ankit Gupta was incorrectly associated with Exonics Therapeutics, Watertown, MA, USA. This has now been corrected in the HTML version of the Article. The PDF version of the Article was correct at the time of publication.
  • NmeCas9 is an intrinsically high-fidelity genome-editing platform

    Author(s): Nadia Amrani,Xin D Gao,Pengpeng Liu,Alireza Edraki,Aamir Mir,Raed Ibraheim,Ankit Gupta,Kanae E Sasaki,Tong Wu,Paul D Donohoue,Alexander H Settle,Alexandra M Lied,Kyle McGovern,Chris K Fuller,Peter Cameron,Thomas G Fazzio,Lihua Julie Zhu,Scot A Wolfe,Erik J Sontheimer
    CONCLUSIONS: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.
  • Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins

    Author(s): Jooyoung Lee,Aamir Mir,Alireza Edraki,Bianca Garcia,Nadia Amrani,Hannah E Lou,Ildar Gainetdinov,April Pawluk,Raed Ibraheim,Xin D Gao,Pengpeng Liu,Alan R Davidson,Karen L Maxwell,Erik J Sontheimer
    In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell...
  • Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing

    Author(s): Mehmet Fatih Bolukbasi,Pengpeng Liu,Kevin Luk,Samantha F Kwok,Ankit Gupta,Nadia Amrani,Erik J Sontheimer,Lihua Julie Zhu,Scot A Wolfe
    The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site...
  • All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo

    Author(s): Raed Ibraheim,Chun-Qing Song,Aamir Mir,Nadia Amrani,Wen Xue,Erik J Sontheimer
    CONCLUSIONS: Our findings indicate that NmeCas9 can enable the editing of disease-causing loci in vivo, expanding the targeting scope of RNA-guided nucleases.
  • Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing

    Author(s): Aamir Mir,Julia F Alterman,Matthew R Hassler,Alexandre J Debacker,Edward Hudgens,Dimas Echeverria,Michael H Brodsky,Anastasia Khvorova,Jonathan K Watts,Erik J Sontheimer
    RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified...
  • A Hyperthermophilic Phage Decoration Protein Suggests Common Evolutionary Origin with Herpesvirus Triplex Proteins and an Anti-CRISPR Protein

    Author(s): Nicholas P Stone,Brendan J Hilbert,Daniel Hidalgo,Kevin T Halloran,Jooyoung Lee,Erik J Sontheimer,Brian A Kelch
    Virus capsids are protein shells that protect the viral genome from environmental assaults, while maintaining the high internal pressure of the tightly packaged genome. To elucidate how capsids maintain stability under harsh conditions, we investigated the capsid components of the hyperthermophilic phage P74-26. We determined the structure of capsid protein gp87 and show that it has the same fold as decoration proteins in many other phages, despite lacking significant sequence homology. We also...
  • C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2

    Author(s): Xin D Gao,Li-Chun Tu,Aamir Mir,Tomás Rodriguez,Yuehe Ding,John Leszyk,Job Dekker,Scott A Shaffer,Lihua Julie Zhu,Scot A Wolfe,Erik J Sontheimer
    Mapping proteomic composition at distinct genomic loci in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. C-BERST enables the high-throughput identification of proteins associated with specific sequences, thereby facilitating annotation of these factors and their roles.
  • CRISPRs from scratch

    Author(s): Alireza Edraki,Erik J Sontheimer
    No abstract
  • Type II-C CRISPR-Cas9 Biology, Mechanism, and Application

    Author(s): Aamir Mir,Alireza Edraki,Jooyoung Lee,Erik J Sontheimer
    Genome editing technologies have been revolutionized by the discovery of prokaryotic RNA-guided defense system called CRISPR-Cas. Cas9, a single effector protein found in type II CRISPR systems, has been at the heart of this genome editing revolution. Nearly half of the Cas9s discovered so far belong to the type II-C subtype but have not been explored extensively. Type II-C CRISPR-Cas systems are the simplest of the type II systems, employing only three Cas proteins. Cas9s are central players in...
  • A Broad-Spectrum Inhibitor of CRISPR-Cas9

    Author(s): Lucas B Harrington,Kevin W Doxzen,Enbo Ma,Jun-Jie Liu,Gavin J Knott,Alireza Edraki,Bianca Garcia,Nadia Amrani,Janice S Chen,Joshua C Cofsky,Philip J Kranzusch,Erik J Sontheimer,Alan R Davidson,Karen L Maxwell,Jennifer A Doudna
    CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs...
  • Inhibition of CRISPR-Cas systems by mobile genetic elements

    Author(s): Erik J Sontheimer,Alan R Davidson
    Clustered, regularly interspaced, short, palindromic repeats (CRISPR) loci, together with their CRISPR-associated (Cas) proteins, provide bacteria and archaea with adaptive immunity against invasion by bacteriophages, plasmids, and other mobile genetic elements. These host defenses impart selective pressure on phages and mobile elements to evolve countermeasures against CRISPR immunity. As a consequence of this pressure, phages and mobile elements have evolved 'anti-CRISPR' proteins that...
  • CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion

    Author(s): Haiwei Mou,Jordan L Smith,Lingtao Peng,Hao Yin,Jill Moore,Xiao-Ou Zhang,Chun-Qing Song,Ankur Sheel,Qiongqiong Wu,Deniz M Ozata,Yingxiang Li,Daniel G Anderson,Charles P Emerson,Erik J Sontheimer,Melissa J Moore,Zhiping Weng,Wen Xue
    CRISPR is widely used to disrupt gene function by inducing small insertions and deletions. Here, we show that some single-guide RNAs (sgRNAs) can induce exon skipping or large genomic deletions that delete exons. For example, CRISPR-mediated editing of β-catenin exon 3, which encodes an autoinhibitory domain, induces partial skipping of the in-frame exon and nuclear accumulation of β-catenin. A single sgRNA can induce small insertions or deletions that partially alter splicing or unexpected...
  • Naturally Occurring Off-Switches for CRISPR-Cas9

    Author(s): April Pawluk,Nadia Amrani,Yan Zhang,Bianca Garcia,Yurima Hidalgo-Reyes,Jooyoung Lee,Alireza Edraki,Megha Shah,Erik J Sontheimer,Karen L Maxwell,Alan R Davidson
    CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we...
  • RNA. CRISPR goes retro

    Author(s): Erik J Sontheimer,Luciano A Marraffini
    No abstract
  • Cas9 gets a classmate

    Author(s): Erik J Sontheimer,Scot A Wolfe
    No abstract
  • DNase H Activity of Neisseria meningitidis Cas9

    Author(s): Yan Zhang,Rakhi Rajan,H Steven Seifert,Alfonso Mondragón,Erik J Sontheimer
    Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate...
  • Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis

    Author(s): Noelle Wakefield,Rakhi Rajan,Erik J Sontheimer
    In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from...
  • Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses

    Author(s): Dan Wang,Haiwei Mou,Shaoyong Li,Yingxiang Li,Soren Hough,Karen Tran,Jia Li,Hao Yin,Daniel G Anderson,Erik J Sontheimer,Zhiping Weng,Guangping Gao,Wen Xue
    CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived...
  • The Bacterial Origins of the CRISPR Genome-Editing Revolution

    Author(s): Erik J Sontheimer,Rodolphe Barrangou
    Like most of the tools that enable modern life science research, the recent genome-editing revolution has its biological roots in the world of bacteria and archaea. Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci are found in the genomes of many bacteria and most archaea, and underlie an adaptive immune system that protects the host cell against invasive nucleic acids such as viral genomes. In recent years, engineered versions of these systems have enabled efficient DNA...
  • Methods in Enzymology. The use of CRISPR/Cas9, ZFNs, and TALENs in generating site-specific genome alterations. Preface

    Author(s): Jennifer A Doudna,Erik J Sontheimer
    No abstract
  • Structural biology. Cascading into focus

    Author(s): Yan Zhang,Erik J Sontheimer
    No abstract
  • SPO24 is a transcriptionally dynamic, small ORF-encoding locus required for efficient sporulation in Saccharomyces cerevisiae

    Author(s): Sara Hurtado,Karen S Kim Guisbert,Erik J Sontheimer
    In Saccharomyces cerevisiae, meiosis and sporulation are highly regulated responses that are driven in part by changes in RNA expression. Alternative mRNA forms with extended 5' UTRs are atypical in S. cerevisiae, and 5' extensions with upstream open reading frames (uORFs) are even more unusual. Here we characterize the gene YPR036W-A, now renamed SPO24, which encodes a very small (67-amino-acid) protein. This gene gives rise to two mRNA forms: a shorter form throughout meiosis and a longer,...
  • Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

    Author(s): Zhonggang Hou,Yan Zhang,Nicholas E Propson,Sara E Howden,Li-Fang Chu,Erik J Sontheimer,James A Thomson
    Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus), each with...
  • Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis

    Author(s): Yan Zhang,Nadja Heidrich,Biju Joseph Ampattu,Carl W Gunderson,H Steven Seifert,Christoph Schoen,Jörg Vogel,Erik J Sontheimer
    CRISPR interference confers adaptive, sequence-based immunity against viruses and plasmids and is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from spacer-repeat units. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here, our studies of crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp. reveal a unique crRNA maturation pathway in which crRNAs are transcribed from promoters that are embedded within...
  • Quit stalling or you'll be silenced

    Author(s): Karen S Kim Guisbert,Erik J Sontheimer
    The use of small RNAs to program gene regulation and genome defense necessitates ever-changing choices about the sequences used for small-RNA biogenesis. Dumesic et al. now reveal stalled spliceosomes as a trigger for small-RNA biogenesis in the pathogenic fungus Cryptococcus neoformans.
  • Small RNAs of opposite sign… but same absolute value

    Author(s): Erik J Sontheimer
    In ciliates, small RNAs have been shown to target foreign sequences for silencing via elimination from the somatic genome. Fang et al. now reveal a set of Piwi-interacting RNAs (piRNAs) in Oxytricha trifallax that likewise enable genomic self versus nonself discrimination, this time by specifying self sequences for genome retention.
  • Meiosis-induced alterations in transcript architecture and noncoding RNA expression in S. cerevisiae

    Author(s): Karen S Kim Guisbert,Yong Zhang,Jared Flatow,Sara Hurtado,Jonathan P Staley,Simon Lin,Erik J Sontheimer
    Changes in transcript architecture can have powerful effects on protein expression. Regulation of the transcriptome is often dramatically revealed during dynamic conditions such as development. To examine changes in transcript architecture we analyzed the expression and transcript boundaries of protein-coding and noncoding RNAs over the developmental process of meiosis in Saccharomyces cerevisiae. Custom-designed, high-resolution tiling arrays were used to define the time-resolved transcriptome...
  • Blanks, a nuclear siRNA/dsRNA-binding complex component, is required for Drosophila spermiogenesis

    Author(s): Vincent R Gerbasi,Jonathan B Preall,Daniel E Golden,David W Powell,Timothy D Cummins,Erik J Sontheimer
    Small RNAs and a diverse array of protein partners control gene expression in eukaryotes through a variety of mechanisms. By combining siRNA affinity chromatography and mass spectrometry, we have identified the double-stranded RNA-binding domain protein Blanks to be an siRNA- and dsRNA-binding protein from Drosophila S2 cells. We find that Blanks is a nuclear factor that contributes to the efficiency of RNAi. Biochemical fractionation of a Blanks-containing complex shows that the Blanks complex...
  • Microbiology: slicer for DNA

    Author(s): Erik J Sontheimer,Luciano A Marraffini
    Many bacteria and archaea protect themselves from viruses and other invasive genomes through a genetic interference pathway. The small RNAs that guide this defence specify the direct cleavage of foreign DNA.
  • Proteomics identification of Drosophila small interfering RNA-associated factors

    Author(s): Vincent R Gerbasi,Daniel E Golden,Sara B Hurtado,Erik J Sontheimer
    The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an approximately 80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we...
  • CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea

    Author(s): Luciano A Marraffini,Erik J Sontheimer
    Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids....
  • Self versus non-self discrimination during CRISPR RNA-directed immunity

    Author(s): Luciano A Marraffini,Erik J Sontheimer
    All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway. CRISPR loci are present in approximately 40% and approximately 90% of sequenced bacterial and archaeal genomes, respectively, and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral...
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