CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.United States. National Institutes of Health (DK085713)National Cancer Institute (U.S.) (P30-CA14051)National Institute of Environmental Health Sciences (P30-ES002109)United States. National Institutes of Health (1K08DK101754

Characterization and application of type VI-B RNA-targeting CRISPR systems

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.Cataloged from PDF version of thesis.Includes bibliographical references.The ability to modify nucleic acids is critical for establishing the role of genetic and transcribed elements in mediating biological phenotypes. Manipulating endogenous DNA sequences in eukaryotic genomes has been greatly aided by the advent of genome editing technologies that utilize programmable nucleases. DNA nucleases derived from class 2 CRISPR systems, which provide adaptive immunity in prokaryotes through cleavage of nucleic acids using a single, multi-domain, RNA-guided endonuclease, have been particularly useful in this regard because they enable targeting of new sites through simple Watson-Crick base pairing rules. Recent computational studies have uncovered the existence of predicted RNA-targeting class 2 CRISPR systems, suggesting that the power of genome editing techniques might be extended to the level of transcripts. In this thesis, I present work describing the discovery and characterization of a new RNA-targeting class 2 CRISPR system: type VI-B. Using a combination of biochemistry and bacterial genetics, we demonstrated that the predicted nuclease of the VI-B system, Casl3b, is an RNA-guided RNase, whose activity can be modulated by the csx genes that often appear in genetic proximity to casl3b. Next, we characterized the behavior of Casl3b and the related enzymes Casl3a and Casl3c in mammalian cells, identifying orthologs of Casl3a and Casl3b with specific RNA interference activity in mammalian cells. Finally, we showed that catalytically inactive versions of a Casl3b ortholog can direct adenosine-to-inosine deaminase activity to transcripts in human cells when fused to the catalytic domain of ADAR2. Using structure-guided mutagenesis, we created a high-specificity version of this system that can be utilized in research or potentially therapeutic contexts. The description of a Casl3b ortholog that can be used to knockdown or recruit RNA-modifying domains to transcripts in mammalian cells suggests the utility of this technology to interrogate and modify transcript function in diverse contexts.by David Benjamin Turitz Cox.Ph. D

RNA editing with CRISPR-Cas13

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.National Institute of Allergy and Infectious Diseases (U.S.) (Grant R01AI117043)United States. Air Force. Office of Scientific Research (Grant FA9550-14-1-0060)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

Engineered Cpf1 variants with altered PAM specificities

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ~11 bp.National Institute of General Medical Sciences (U.S.) (Grant T32GM007753)National Institute of General Medical Sciences (U.S.) (Grant T32GM007753)National Institutes of Health (U.S.) (Grant 2T32GM7287-41)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.National Institute of General Medical Sciences (U.S.) (Award T32GM007753)National Institute of Mental Health (U.S.) (Award 5DP1-MH100706)National Institute of Mental Health (U.S.) (Award 1R01-MH110049

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

The clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the class 2 type VI CRISPR-Cas effector C2c2 and demonstrate its RNA-guided ribonuclease function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis shows that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave single-stranded RNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, mutations of which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.United States. Dept. of Energy (Computational Science Graduate Fellowship)Massachusetts Institute of Technology. Simons Center for the Social BrainNational Institute of General Medical Sciences (U.S.) (Award T32GM007753)National Institutes of Health (U.S.) (National Institute of Mental Health (U.S.) Grants 5DP1-MH100706 and 1R01-MH110049)National Science Foundation (U.S.)New York Stem Cell FoundationSimons FoundationPaul G. Allen Family FoundationVallee FoundationPoitras FoundationRobert MetcalfeDavid R. Chen

Therapeutic genome editing: prospects and challenges

Available in PMC 2015 July 06Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.Robert MetcalfeSimons FoundationMerkin Family Foundation for Stem Cell ResearchNational Science Foundation (U.S.) (NSF Waterman Award)National Science Foundation (U.S.) (NSF Graduate Research Fellowship, grant number 1122374)National Institute of General Medical Sciences (U.S.) (Award number T32GM007753)National Institutes of Health (U.S.) ((NIH) Director’s Pioneer Award (DP1-MH100706))W. M. Keck FoundationNational Institute of Neurological Disorders and Stroke (U.S.) (NIH Transformative R01 grant (R01-NS 07312401))Damon Runyon Cancer Research FoundationSearle Scholars ProgramEsther A. & Joseph Klingenstein Fund, Inc.Vallee Foundatio