Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development.

Resolution of hepatic fibrosis after ZFN-mediated gene editing in the PiZ mouse model of human α1-antitrypsin deficiency.

Background: α1-antitrypsin deficiency is most commonly caused by a mutation in exon-7 of SERPINA1 (SA1-ATZ), resulting in hepatocellular accumulation of a misfolded variant (ATZ). Human SA1-ATZ-transgenic (PiZ) mice exhibit hepatocellular ATZ accumulation and liver fibrosis. We hypothesized that disrupting the SA1-ATZ transgene in PiZ mice by in vivo genome editing would confer a proliferative advantage to the genome-edited hepatocytes, enabling them to repopulate the liver.

Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes.

Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa).

Unveiling the Role of the Most Impactful Cardiovascular Risk Locus through Haplotype Editing.

The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease (CAD), accounting for ∼10%-15% of disease in non-African populations. The ∼60 kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function.

Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells.

This unit describes the protocol for the delivery of reagents for targeted genome editing to CD34(+) hematopoietic stem/progenitor cells (HSPCs). Specifically, this unit focuses on the process of thawing and pre-stimulating CD34(+) HSPCs, as well as the details of their electroporation with in vitro-transcribed mRNA-encoding site-specific nucleases [in this case zinc-finger nucleases (ZFNs)]. In addition, discussed is delivery of a gene editing donor template in the form of an oligonucleotide or integrase-defective lentiviral vector (IDLV).

Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification.

Genome editing-enabled HTS assays expand drug target pathways for Charcot-Marie-tooth disease.

Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot-Marie-Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay.

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation.

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models.

Human intestinal tissue with adult stem cell properties derived from pluripotent stem cells.

Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine.

Translating dosage compensation to trisomy 21.

Down's syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down's syndrome pluripotent stem cells.

In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration.

Targeted DNA integration is commonly used to eliminate position effects on transgene expression. Integration can be targeted to specific sites in the genome via both homology-based and homology-independent processes. Both pathways start the integration process with a site-specific break in the chromosome, typically from a zinc-finger nuclease (ZFN).

Genetic engineering of human pluripotent cells using TALE nucleases.

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci.

Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene.

Dissection of splicing regulation at an endogenous locus by zinc-finger nuclease-mediated gene editing.

Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells.

A TALE nuclease architecture for efficient genome editing.

Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%.

Characterization of immunoglobulin heavy chain knockout rats.

The rat is a species frequently used in immunological studies but, until now, there were no models with introduced gene-specific mutations. In a recent study, we described for the first time the generation of novel rat lines with targeted mutations using zinc-finger nucleases. In this study, we compare immune development in two Ig heavy-chain KO lines; one with truncated Cμ and a new line with removed JH segments.

Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies.

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily alpha1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a alpha1,6-fucosyltransferase. FUT8(-/-) CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8(-/-) cells as hosts for antibody production.

Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.

We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two approximately 750 bp regions of chromosomal sequence required for homology-directed DNA repair.

Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases.

Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem.

Generation of gene-specific mutated rats using zinc-finger nucleases.

The genetic dissection of physiological and pathological traits in laboratory model organisms is accelerated by the ability to engineer loss-of-function mutations at investigator-specified loci. This chapter describes the use of zinc-finger nucleases (ZFNs) for the targeted disruption of endogenous rat genes directly in the embryo. ZFNs can specifically disrupt target genes in cultured rat cells and in embryos from inbred and outbred strains, leading to permanently genetically modified animals.

BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells.

Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis.

Retrotransposon overdose and genome integrity.

Yeast and mammalian genomes are replete with nearly identical copies of long dispersed repeats in the form of retrotransposons. Mechanisms clearly exist to maintain genome structure in the face of potential rearrangement between the dispersed repeats, but the nature of this machinery is poorly understood. Here we describe a series of distinct "retrotransposon overdose" (RO) lineages in which the number of Ty1 elements in the Saccharomyces cerevisiae genome has been increased by as much as 10 fold.