Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Targeting Hepatitis B Virus with Zinc Finger Nucleases

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy

Successful Targeting and Disruption of an Integrated Reporter Lentivirus Using the Engineered Homing Endonuclease Y2 I-AniI

Current antiviral therapy does not cure HIV-infected individuals because the virus establishes lifelong latent infection within long-lived memory T cells as integrated HIV proviral DNA. Here, we report a new therapeutic approach that aims to cure cells of latent HIV infection by rendering latent virus incapable of replication and pathogenesis via targeted cellular mutagenesis of essential viral genes. This is achieved by using a homing endonuclease to introduce DNA double-stranded breaks (dsb) within the integrated proviral DNA, which is followed by triggering of the cellular DNA damage response and error-prone repair. To evaluate this concept, we developed an in vitro culture model of viral latency, consisting of an integrated lentiviral vector with an easily evaluated reporter system to detect targeted mutagenesis events. Using this system, we demonstrate that homing endonucleases can efficiently and selectively target an integrated reporter lentivirus within the cellular genome, leading to mutation in the proviral DNA and loss of reporter gene expression. This new technology offers the possibility of selectively disabling integrated HIV provirus within latently infected cells

High-throughput mapping of regulatory DNA

Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9–based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ~40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell–specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair–resolution functional motifs of regulatory elements.National Institutes of Health (U.S.) (1U01HG007037

Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy

<p>Abstract</p> <p>Background</p> <p>Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that <it>ex-vivo </it>or even <it>in-vivo </it>abolition <it>or </it>disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger <it>arrays </it>and <it>nucleases </it>(ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype.</p> <p>Methods and Results</p> <p><it>First, </it>we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) <it>arrays </it>(ZFAs or Zif_HIV-pol) and (b) two zinc-finger <it>nucleases </it>(ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (Zif_HIV-polF_N). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (Zif_HIV-1F_N). <it>Second, </it>a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either Zif_HIV-polF_Nor Zif_HIV-1F_Nchimeric genes (termed <b>LV- 2xZif</b>_{<b>HIV-pol</b>}<b>F</b>_{<b>N </b>}and <b>LV- 2xZif</b>_<b>HIV-1</b><b>F</b>_{<b>N, </b>}respectively) is proposed. <it>Third, </it>two preclinical models for controlled testing of the safety and efficacy of either of these LVs are described using active HIV-infected TZM-bl reporter cells (HeLa-derived JC53-BL cells) and latent HIV-infected cell lines.</p> <p>Conclusion</p> <p><b>LV-2xZif</b>_{<b>HIV-pol</b>}<b>F</b>_{<b>N </b>}and <b>LV- 2xZif</b>_<b>HIV-1</b><b>F</b>_{<b>N </b>}may offer the <it>ex-vivo </it>or even <it>in-vivo </it>experimental opportunity to halt HIV replication functionally by directly abrogating HIV-pol-gene-action <it>or </it>disrupting/excising over 80% of the proviral HIV DNA from latently infected cells.</p

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

Validating the concept of mutational signatures with isogenic cell models.

The diversity of somatic mutations in human cancers can be decomposed into individual mutational signatures, patterns of mutagenesis that arise because of DNA damage and DNA repair processes that have occurred in cells as they evolved towards malignancy. Correlations between mutational signatures and environmental exposures, enzymatic activities and genetic defects have been described, but human cancers are not ideal experimental systems-the exposures to different mutational processes in a patient's lifetime are uncontrolled and any relationships observed can only be described as an association. Here, we demonstrate the proof-of-principle that it is possible to recreate cancer mutational signatures in vitro using CRISPR-Cas9-based gene-editing experiments in an isogenic human-cell system. We provide experimental and algorithmic methods to discover mutational signatures generated under highly experimentally-controlled conditions. Our in vitro findings strikingly recapitulate in vivo observations of cancer data, fundamentally validating the concept of (particularly) endogenously-arising mutational signatures

CPP-ZFN: A potential DNA-targeting anti-malarial drug

<p>Abstract</p> <p>Background</p> <p>Multidrug-resistant <it>Plasmodium </it>is of major concern today. Effective vaccines or successful applications of RNAi-based strategies for the treatment of malaria are currently unavailable. An unexplored area in the field of malaria research is the development of DNA-targeting drugs that can specifically interact with parasitic DNA and introduce deleterious changes, leading to loss of vital genome function and parasite death.</p> <p>Presentation of the hypothesis</p> <p>Advances in the development of zinc finger nuclease (ZFN) with engineered DNA recognition domains allow us to design and develop nuclease of high target sequence specificity with a mega recognition site that typically occurs only once in the genome. Moreover, cell-penetrating peptides (CPP) can cross the cell plasma membrane and deliver conjugated protein, nucleic acid, or any other cargo to the cytoplasm, nucleus, or mitochondria. This article proposes that a drug from the combination of the CPP and ZFN systems can effectively enter the intracellular parasite, introduce deleterious changes in its genome, and eliminate the parasite from the infected cells.</p> <p>Testing the hypothesis</p> <p>Availability of a DNA-binding motif for more than 45 triplets and its modular nature, with freedom to change number of fingers in a ZFN, makes development of customized ZFN against diverse target DNA sequence of any gene feasible. Since the <it>Plasmodium </it>genome is highly AT rich, there is considerable sequence site diversity even for the structurally and functionally conserved enzymes between <it>Plasmodium </it>and humans. CPP can be used to deliver ZFN to the intracellular nucleus of the parasite. Signal-peptide-based heterologous protein translocation to <it>Plasmodium</it>-infected RBCs (iRBCs) and different <it>Plasmodium </it>organelles have been achieved. With successful fusion of CPP with mitochondrial- and nuclear-targeting peptides, fusion of CPP with 1 more <it>Plasmodium </it>cell membrane translocation peptide seems achievable.</p> <p>Implications of the hypothesis</p> <p>Targeting of the <it>Plasmodium </it>genome using ZFN has great potential for the development of anti-malarial drugs. It allows the development of a single drug against all malarial infections, including multidrug-resistant strains. Availability of multiple ZFN target sites in a single gene will provide alternative drug target sites to combat the development of resistance in the future.</p