المؤلفون: Cofsky, Joshua C, Karandur, Deepti, Huang, Carolyn J, Witte, Isaac P, Kuriyan, John, Doudna, Jennifer A

المساهمون: Howard Hughes Medical Institute, National Science Foundation

المصدر: eLife ; volume 9 ; ISSN 2050-084X

الوصف: Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3′ side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

الإتاحة: https://doi.org/10.7554/elife.55143Test
https://cdn.elifesciences.org/articles/55143/elife-55143-v2.pdfTest
https://cdn.elifesciences.org/articles/55143/elife-55143-v2.xmlTest
https://elifesciences.org/articles/55143Test

المؤلفون: Knott, Gavin J, Cress, Brady F, Liu, Jun-Jie, Thornton, Brittney W, Lew, Rachel J, Al-Shayeb, Basem, Rosenberg, Daniel J, Hammel, Michal, Adler, Benjamin A, Lobba, Marco J, Xu, Michael, Arkin, Adam P, Fellmann, Christof, Doudna, Jennifer A

المساهمون: Defense Advanced Research Projects Agency, Paul G. Allen Frontiers Group, National Science Foundation, Howard Hughes Medical Institute, National Institutes of Health, National Institute of General Medical Sciences, W. M. Keck Foundation

المصدر: eLife ; volume 8 ; ISSN 2050-084X

الوصف: CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

الإتاحة: https://doi.org/10.7554/elife.49110Test
https://cdn.elifesciences.org/articles/49110/elife-49110-v2.pdfTest
https://cdn.elifesciences.org/articles/49110/elife-49110-v2.xmlTest
https://elifesciences.org/articles/49110Test

المؤلفون: Strutt, Steven C, Torrez, Rachel M, Kaya, Emine, Negrete, Oscar A, Doudna, Jennifer A

المساهمون: National Science Foundation, Howard Hughes Medical Institute, German Academic Exchange Program, Laboratory Directed Research and Development, Paul Allen Frontiers Science Program

المصدر: eLife ; volume 7 ; ISSN 2050-084X

الوصف: Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.

الإتاحة: https://doi.org/10.7554/elife.32724Test
https://cdn.elifesciences.org/articles/32724/elife-32724-v2.pdfTest
https://cdn.elifesciences.org/articles/32724/elife-32724-v2.xmlTest
https://elifesciences.org/articles/32724Test

المؤلفون: Schulze-Gahmen, Ursula, Echeverria, Ignacia, Stjepanovic, Goran, Bai, Yun, Lu, Huasong, Schneidman-Duhovny, Dina, Doudna, Jennifer A, Zhou, Qiang, Sali, Andrej, Hurley, James H

المساهمون: National Institutes of Health, National Institute of Allergy and Infectious Diseases

المصدر: eLife ; volume 5 ; ISSN 2050-084X

الوصف: HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM.

الإتاحة: https://doi.org/10.7554/elife.15910Test
https://cdn.elifesciences.org/articles/15910/elife-15910-v3.pdfTest
https://cdn.elifesciences.org/articles/15910/elife-15910-v3.xmlTest
https://elifesciences.org/articles/15910Test

المؤلفون: Floor, Stephen N, Doudna, Jennifer A

المساهمون: Howard Hughes Medical Institute, Helen Hay Whitney Foundation

المصدر: eLife ; volume 5 ; ISSN 2050-084X

الوصف: Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.

الإتاحة: https://doi.org/10.7554/elife.10921Test
https://cdn.elifesciences.org/articles/10921/elife-10921-v2.pdfTest
https://cdn.elifesciences.org/articles/10921/elife-10921-v2.xmlTest
https://elifesciences.org/articles/10921Test

المؤلفون: Carlson, Lars-Anders, Bai, Yun, Keane, Sarah C, Doudna, Jennifer A, Hurley, James H

المساهمون: National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences

المصدر: eLife ; volume 5 ; ISSN 2050-084X

الوصف: HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5’ untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another.

الإتاحة: https://doi.org/10.7554/elife.14663Test
https://cdn.elifesciences.org/articles/14663/elife-14663-v2.pdfTest
https://cdn.elifesciences.org/articles/14663/elife-14663-v2.xmlTest
https://elifesciences.org/articles/14663Test

المؤلفون: Isaac, R Stefan, Jiang, Fuguo, Doudna, Jennifer A, Lim, Wendell A, Narlikar, Geeta J, Almeida, Ricardo

المساهمون: Merck Fellow of the Damon Runyon Cancer Research Foundation, National Science Foundation, National Institutes of Health, Howard Hughes Medical Institute

المصدر: eLife ; volume 5 ; ISSN 2050-084X

الوصف: The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.

الإتاحة: https://doi.org/10.7554/elife.13450Test
https://cdn.elifesciences.org/articles/13450/elife-13450-v2.pdfTest
https://cdn.elifesciences.org/articles/13450/elife-13450-v2.xmlTest
https://elifesciences.org/articles/13450Test

المؤلفون: Bai, Yun, Tambe, Akshay, Zhou, Kaihong, Doudna, Jennifer A

المساهمون: National Institute of General Medical Sciences, Howard Hughes Medical Institute

المصدر: eLife ; volume 3 ; ISSN 2050-084X

الوصف: HIV replication requires nuclear export of unspliced and singly spliced viral transcripts. Although a unique RNA structure has been proposed for the Rev-response element (RRE) responsible for viral mRNA export, how it recruits multiple HIV Rev proteins to form an export complex has been unclear. We show here that initial binding of Rev to the RRE triggers RNA tertiary structural changes, enabling further Rev binding and the rapid formation of a viral export complex. Analysis of the Rev-RRE assembly pathway using SHAPE-Seq and small-angle X-ray scattering (SAXS) reveals two major steps of Rev-RRE complex formation, beginning with rapid Rev binding to a pre-organized region presenting multiple Rev binding sites. This step induces long-range remodeling of the RNA to expose a cryptic Rev binding site, enabling rapid assembly of additional Rev proteins into the RNA export complex. This kinetic pathway may help maintain the balance between viral replication and maturation.

الإتاحة: https://doi.org/10.7554/elife.03656Test
https://cdn.elifesciences.org/articles/03656/elife-03656-v1.pdfTest
https://cdn.elifesciences.org/articles/03656/elife-03656-v1.xmlTest
https://elifesciences.org/articles/03656Test

المؤلفون: Lin, Steven, Staahl, Brett T, Alla, Ravi K, Doudna, Jennifer A

المساهمون: Damon Runyon Cancer Research Foundation (Damon Runyon), Roche

المصدر: eLife ; volume 3 ; ISSN 2050-084X

الوصف: The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

الإتاحة: https://doi.org/10.7554/elife.04766Test
https://cdn.elifesciences.org/articles/04766/elife-04766-v2.pdfTest
https://cdn.elifesciences.org/articles/04766/elife-04766-v2.xmlTest
https://elifesciences.org/articles/04766Test

المؤلفون: Jinek, Martin, East, Alexandra, Cheng, Aaron, Lin, Steven, Ma, Enbo, Doudna, Jennifer

المساهمون: Howard Hughes Medical Institute, National Institutes of Health

المصدر: eLife ; volume 2 ; ISSN 2050-084X

الوصف: Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.

الإتاحة: https://doi.org/10.7554/elife.00471Test
https://cdn.elifesciences.org/articles/00471/elife-00471-v1.pdfTest
https://cdn.elifesciences.org/articles/00471/elife-00471-v1.xmlTest
https://elifesciences.org/articles/00471Test