Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.National Institutes of Health (U.S.) (Grant 5DP1-MH100706

Optical Control of Mammalian Endogenous Transcription and Epigenetic States

The dynamic nature of gene expression enables cellular programming, homeostasis, and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high precision spatiotemporal control of many cellular functions1-11. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here, we describe the development of Light-Inducible Transcriptional Effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain12-14 with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical co-factors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility3,4,6,7,15. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of awake mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states

Optical control of mammalian endogenous transcription and epigenetic states

A theoretical underpinning of the standard model of fundamental particles and interactions is CPT invariance, which requires that the laws of physics be invariant under the combined discrete operations of charge conjugation, parity and time reversal. Antimatter, the existence of which was predicted by Dirac, can be used to test the CPT theorem—experimental investigations involving comparisons of particles with antiparticles are numerous. Cold atoms and anti-atoms, such as hydrogen and antihydrogen, could form the basis of a new precise test, as CPT invariance implies that they must have the same spectrum. Observations of antihydrogen in small quantities and at high energies have been reported at the European Organization for Nuclear Research (CERN) and at Fermilab, but these experiments were not suited to precision comparison measurements. Here we demonstrate the production of antihydrogen atoms at very low energy by mixing trapped antiprotons and positrons in a cryogenic environment. The neutral anti-atoms have been detected directly when they escape the trap and annihilate, producing a characteristic signature in an imaging particle detector

Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems

Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant 5R01DK097768-03)National Institutes of Health (U.S.) (Grant GM10407

Optical Control of Mammalian Endogenous Transcription and Epigenetic States

The dynamic nature of gene expression enables cellular programming, homeostasis, and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high precision spatiotemporal control of many cellular functions1-11. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here, we describe the development of Light-Inducible Transcriptional Effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain12-14 with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical co-factors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility3,4,6,7,15. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of awake mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.National Institutes of Health (U.S.) (Grant RO1-GM34277)National Institutes of Health (U.S.) (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant PO1-CA42063)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Director's Pioneer Award 1DP1-MH100706)Damon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramSimons Foundatio

Interrogation and control of mammalian transcription

Thesis: Ph. D. in Neuroscience, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, 2016.Cataloged from PDF version of thesis.Includes bibliographical references (pages 119-128).Gene expression is dynamic in living systems, enabling environmental adaptation and homeostasis. Transcript levels may change temporarily during distinct phases of biological processes, while longer lasting modifications to their regulatory machinery can lead to specific cell states or disease phenotypes. However, versatile and robust methods to investigate causal relationships between gene expression states and biological phenotypes remain elusive. My thesis work - divided into two main parts - has focused on the development of technologies to enable efficient, generalizable, and precise perturbation of mammalian gene expression. The first part of my research focused on the development of light-inducible transcriptional effectors (LITEs) to mediate positive and negative regulation of endogenous mammalian gene expression (Konermann et al. Nature 2013). Optical stimulation enables precise spatiotemporal control to closely match endogenous transcriptional dynamics. I engineered the LITE system based on the programmable TALE DNA binding proteins from plant pathogens in combination with the light-inducible dimer cryptochrome 2 - cibi from Arabidopsis thaliana. Light enables fast and reversible recruitment of transcriptional effector domains to the TALE bound to the endogenous target promoter through dimerization of cryptochrome 2 - cibi. I applied LITEs to control gene expression in primary neurons as well as in the mouse brain in vivo, demonstrating their potential to dissect genetic contributions to dynamic behaviors such as learning. Epigenetic regulation of transcriptional state is an additional layer of endogenous control exerted by the cell to store more permanent states such as memories. To interrogate epigenetic in addition to transcriptional dynamics, I next developed TALE-mediated targeting of 32 repressive histone effectors to alter epigenetic states in a locus-specific manner. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states. One major limiting aspect of TALE-based transcriptional activators is the costly and labor-intensive construction of their repetitive DNA binding domains. As a result, the utility of TALEs for higher-throughput gene targeting experiments remains limited. The CRISPR nuclease Cas9, however, can be easily programmed using a short guide RNA homologous to the target genomic DNA of interest. Additionally, Cas9 can be easily converted into an RNA-guided DNA binding protein (dCas9) via inactivation of its two catalytic domains. The ease and scalability of the CRISPR-Cas9 system potentially enables systematic, genome-scale perturbation, but the magnitude of transcriptional upregulation achieved by the current generation of Cas9 transcriptional activators typically ranges from low to ineffective. In order to achieve a system where the majority of Cas9 activators are highly functional, I undertook structure-guided engineering to generate a potent, sjynergistic Cas9 activation complex (SAM) capable of mediating robust upregulation with a single sgRNA (Konermann et al., Nature, 2015) which outperforms current systems by more than two orders of magnitude. I demonstrated that these transcriptional effectors are capable of activating up to 10 genes simultaneously, allowing for understanding of complex genetic and regulatory networks. Genome-scale GOF screening approaches have largely remained limited to the use of cDNA library systems, which are costly and challenging to use in a pooled format. To overcome this limitation, I designed a genome-scale sgRNA library targeting every coding isoform from the RefSeq database (23,430 isoforms) for a final library of 70,290 guides. I next aimed to identify gain-of-function changes that can lead to the development of BRAF inhibitor resistance in BRAFV 600 mutant melanoma cells. The screen results highlighted a number of gene candidates that both confirm known BRAF inhibitor-resistance pathways and suggest novel mechanisms of action. SAM activators present a highly reliable and generalizable tool for genome-wide interrogation of gene function and interaction in diverse biological processes. Recently, we have extended the utility of the SAM system to enable bimodal control through the use of modified, truncated deadRNAs (dRNAs) (Dahlman, et al., Nature Biotechnology 2015). These dRNAs prevent nucleolytic activity of an active Cas9 nuclease and transform the wildtype enzyme into an efficient transcriptional activator when combined with the SAM activator-components. This system enables simultaneous knock-out of gene A and activation of gene B in the same cell population, enabling bidirectional interrogation of gene interaction and regulatory networks.by Silvana Konermann.Ph. D. in Neuroscienc