Jennifer A. Doudna
Howard Hughes Medical Institute Investigator, Li Ka Shing Chancellor's Chair in Biomedical and Health Sciences and Professor of Biochemistry, Biophysics and Structural BiologyLab Homepage: http://rna.berkeley.edu/
RNA molecules are uniquely capable of encoding and controlling the expression of genetic information, often as a consequence of their three-dimensional structures. We are interested in understanding and harnessing RNA-mediated control of the genome, including CRISPR-Cas bacterial adaptive immunity and related systems.
The CRISPR bacterial adaptive immune system
Prokaryotes have evolved a nucleic acid-based immune system that shares some functional similarities with RNA interference in eukaryotes. Central to this system are DNA repeats called CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPRs are genetic elements containing direct repeats separated by unique spacers, many of which are identical to sequences found in phage and other foreign genetic elements. Recent work has demonstrated the role of CRISPRs in adaptive immunity and shown that small RNAs derived from CRISPRs (crRNAs) are implemented as homing oligos for the targeted interference of foreign DNA.
Phylogenetic analysis of CRISPR-associated (Cas) proteins suggests there are at least seven distinct versions of this immune system. These systems can be extremely divergent mechanistically and provide a rich area to research RNA:protein interactions, including novel protein folds. To explore this diversity, we have determined the structures of diverse CRISPR-associated proteins, including the large E. coli CASCADE silencing complex. This seahorse-shaped assembly shows how the CRISPR RNA is cradled by six repeating subunits and presented for DNA inspection. Further, we have solved the structure of the CasA subunit by X-ray crystallography, which revealed that it is poised to have a role in discriminating between “nonself” (foreign DNA) or “self” (host DNA) prior to targeting. This step is critical, as reckless silencing could prove lethal to the host.
RNA-guided DNA cleavage with the Type-II CRISPR enzyme Cas9
Type II CRISPR-Cas systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9-mediated cleavage is strictly dependent on the presence of a protospacer adjacent motif (PAM) in the target DNA. The ability to program Cas9 for DNA cleavage at specific sites defined by guide RNAs has led to its adoption as a versatile platform for genome engineering and gene regulation. To compare the architectures and domain organization of diverse Cas9 proteins, we have solved the atomic structures of Cas9 from Streptococcus pyogenes (SpyCas9) and Actinomyces naeslundii (AnaCas9), revealing the structural core shared by all Cas9 family members, and the structurally divergent regions, including the PAM recognition loops, are likely responsible for distinct guide RNA and PAM specificities. Our EM analysis further shows that by triggering a conformational rearrangement in Cas9, the guide RNA acts as a critical determinant of target DNA binding (in collaboration with Eva Nogales, UC Berkeley, HHMI).
Adaptive immunity in bacteria and the genome engineering technologies derived from it employ RNA-guided cleavage of double-stranded DNA targets using CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts. In Type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to an 85-nucleotide single-guide RNA reveal a conformation distinct from both the apo and DNA-bound forms of the protein, in which the 10-nucleotide RNA “seed” sequence required for initial DNA interrogation is pre-ordered in an A-form helical conformation. Biochemical experiments show this segment of the guide RNA to be essential for Cas9 to form a DNA recognition-competent structure that is poised to engage double-stranded DNA target sequences. Together, these results suggest convergent evolution of a “seed” mechanism reminiscent of that employed by Argonaute proteins during RNA interference in eukaryotes. Furthermore, our structural and biochemical data show that Cas9 is subject to multi-layered regulation during its activation. The pre-ordered RNA seed sequence and protein PAM-interacting cleft enable the Cas9-sgRNA complex to interact productively with potential DNA sequences for target sampling. The inactive conformation of apo Cas9, as well as the additional conformational changes required for the complex to reach its ultimate catalytically-active state, could help avoid spurious DNA cleavage within the host genome and hence minimize off-target effects in Cas9-based genome editing.
RNA-guided RNA cleavage by the Csm complex
In a collaboration with John van der Oost’s laboratory, we are studying the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). Recently, we showed that multiple Cas proteins and a crRNA guide assemble to recognize and cleave invader RNAs at multiple sites. Our negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes, suggesting a model for cleavage of the target RNA at periodic intervals (in collaboration with Eva Nogales, UC Berkeley, HHMI).
RNA-guided DNA cleavage with CASCADE
Cascade is composed of Cse1, Cse2, Cas7, Cas5e, and Cas6e subunits and one crRNA, forming a structure that binds and unwinds dsDNA to form an R-loop in which the target strand of the DNA base pairs with the 32-nt crRNA guide sequence. Recently, we used single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 to reveal that Cascade positions the PAM-proximal end of the DNA duplex at the Cse1 subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with Cse1 implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of Cse1, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the Cse1 subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage (in collaboration with Eva Nogales, UC Berkeley).
Tunable expression of the human transcriptome
Eukaryotic cells exert control over gene expression at multiple layers. Control at the step of protein production, or translation, is frequently used to effect tight spatiotemporal regulation of gene expression. High-throughput methods such as ribosome profiling have revolutionized the study of translational control in cells. However, many eukaryotes frequently generate multiple transcript isoforms from a gene through the combined action of alternative transcription initiation, splicing, and polyadenylation, which are inaccessible to ribosome profiling. For example, in humans there is a median of five transcript isoforms per gene, each of which may have a distinct set of regulatory features. We developed a new method termed Transcript Isoforms in Polysomes sequencing, or TrIP-seq, to directly measure how well each individual human transcript isoform is translated in human cells. We found that thousands of human genes express multiple transcript isoforms that are differentially translated. Furthermore, we showed that regulatory regions from transcript isoforms are sufficient to control translation of an orthogonal gene in a manner predicted by TrIP-seq. Translational control conferred by transcript 5’ leader sequences is robust across cell types, while 3’ UTRs can exhibit cell-type specific expression. All told, our work has uncovered an underappreciated layer in gene regulation due to differential translation of transcript isoforms that broadly impacts human gene expression, and may be part of the reason mRNA and protein levels are poorly correlated.
RNA-mediated signaling in eukaryotic innate immunity
In collaboration with the Berger lab (Johns Hopkins Medical School) and Vance lab (UC-Berkeley), we are investigating a mammalian signaling network where small RNA oligonucleotide second messengers are enzymatically synthesized in response to pathogen infection. The human enzyme cyclic GMP–AMP synthase (cGAS) is responsible for detection of cytosolic DNA and synthesis of a cyclic GMP–AMP dinucleotide containing one canonical 3ʹ–5ʹ and one unique 2ʹ–5ʹ phosphodiester bond (2ʹ,3ʹ cGAMP). Previously, we determined the X-ray crystal structure of human cGAS revealing a bi-lobed enzyme that utilizes a zinc-ribbon modified cleft to couple DNA recognition and enzymatic activity. Through analysis of a distantly related bacterial enzyme encoded by Vibrio cholerae, we went on to delineate the molecular rules for mammalian-specific 2ʹ–5ʹ linkage formation and rationally reprogrammed human cGAS to produce an exclusively 3ʹ,3ʹ linked cGAMP product. These results reveal unexpected mechanistic homology between bacterial signaling and mammalian innate immunity, illustrating active site configurations that may underlie distinct 2ʹ,3ʹ and 3ʹ,3ʹ cGAMP signaling in the human population. Currently, we are using a paleo-biochemistry approach to investigate the unique potency of human 2ʹ,3ʹ cGAMP signaling.
Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Schumann K, Lin S, Boyer E, Simeonov DR, Subramaniam M, Gate RE, Haliburton GE, Ye CJ, Bluestone JA, Doudna JA, Marson A Proc Natl Acad Sci U S A 2015 Jul 27
A Cas9-guide RNA complex preorganized for target DNA recognition. Jiang F, Zhou K, Ma L, Gressel S, Doudna JA Science 2015 Jun 26;348(6242):1477-81
Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning. Taylor DW, Zhu Y, Staals RH, Kornfeld JE, Shinkai A, van der Oost J, Nogales E, Doudna JA Science 2015 May 1;348(6234):581-5
Rational design of a split-Cas9 enzyme complex. Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld JE, Doudna JA Proc Natl Acad Sci U S A 2015 Mar 10;112(10):2984-9
Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nuñez JK, Lee AS, Engelman A, Doudna JA Nature 2015 Mar 12;519(7542):193-8
Dicer-TRBP complex formation ensures accurate mammalian microRNA biogenesis. Wilson RC, Tambe A, Kidwell MA, Noland CL, Schneider CP, Doudna JA Mol Cell 2015 Feb 5;57(3):397-407
Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Lin S, Staahl BT, Alla RK, Doudna JA Elife 2014;3:e04766
RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Staals RH, Zhu Y, Taylor DW, Kornfeld JE, Sharma K, Barendregt A, Koehorst JJ, Vlot M, Neupane N, Varossieau K, Sakamoto K, Suzuki T, Dohmae N, Yokoyama S, Schaap PJ, Urlaub H, Heck AJ, Nogales E, Doudna JA, Shinkai A, van der Oost J Mol Cell 2014 Nov 20;56(4):518-30
Programmable RNA recognition and cleavage by CRISPR/Cas9. O'Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA Nature 2014 Dec 11;516(7530):263-6
RNA-guided assembly of Rev-RRE nuclear export complexes. Bai Y, Tambe A, Zhou K, Doudna JA Elife 2014;3:e03656
Evolutionarily conserved roles of the dicer helicase domain in regulating RNA interference processing. Kidwell MA, Chan JM, Doudna JA J Biol Chem 2014 Oct 10;289(41):28352-62
Structure-guided reprogramming of human cGAS dinucleotide linkage specificity. Kranzusch PJ, Lee AS, Wilson SC, Solovykh MS, Vance RE, Berger JM, Doudna JA Cell 2014 Aug 28;158(5):1011-21
Insights into RNA structure and function from genome-wide studies. Mortimer SA, Kidwell MA, Doudna JA Nat Rev Genet 2014 Jul;15(7):469-79
Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity. Nuñez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA Nat Struct Mol Biol 2014 Jun;21(6):528-34
CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference. Hochstrasser ML, Taylor DW, Bhat P, Guegler CK, Sternberg SH, Nogales E, Doudna JA Proc Natl Acad Sci U S A 2014 May 6;111(18):6618-23
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA Nature 2014 Mar 6;507(7490):62-7
Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases. Niewoehner O, Jinek M, Doudna JA Nucleic Acids Res 2014 Jan;42(2):1341-53
Photo credit: Keegan Houser.
Last Updated 2015-09-17