Epigenetic control of multiple genes with a lentiviral vector encoding transcriptional repressors fused to compact zinc finger arrays.

Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here, we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells.

Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells.

Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddA, that can act upon double-stranded DNA.

Inducible CRISPR-targeted "knockdown" of human gut in gnotobiotic mice discloses glycan utilization strategies.

Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic species.

Improving CRISPR-Cas9 Genome Editing Efficiency by Fusion with Chromatin-Modulating Peptides.

Bacterial-derived CRISPR-Cas9 nucleases have become a common tool in genome engineering. However, the editing efficiency by even the best-crafted Cas9 nucleases varies considerably with different genomic sites, and efforts to explore the vast natural Cas9 diversity have often met with mixed or little success. Here, we show that modification of the widely used Cas9 by fusion with chromatin-modulating peptides (CMPs), derived from high mobility group proteins HMGN1 and HMGB1, histone H1, and chromatin remodeling complexes, improves its activity by up to several fold, particularly on refractory target sites.

Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells ( Jinek , 2012 ; Cong , 2013 ; Mali , 2013 ; Zetsche , 2015 ). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved.

Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome.

Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.

Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner.

Gene editing using ssODNs with engineered endonucleases.

Gene editing using engineered endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases, requires the creation of a targeted, chromosomal DNA double-stranded break (DSB). In mammalian cells, these DSBs are typically repaired by one of the two major DNA repair pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone repair process that can result in a wide range of end-joining events that leads to somewhat random mutations at the site of DSB.

Donor plasmid design for codon and single base genome editing using zinc finger nucleases.

In recent years, CompoZr zinc finger nuclease (ZFN) technology has matured to the point that a user-defined double strand break (DSB) can be placed at virtually any location in the human genome within 50 bp of a desired site. Such high resolution ZFN engineering is well within the conversion tract limitations demarcated by the mammalian DNA repair machinery, resulting in a nearly universal ability to create point mutations throughout the human genome. Additionally, new architectures for targeted nuclease engineering have been rapidly developed, namely transcription activator like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems, further expanding options for placement of DSBs.

Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin.

High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs.

Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates.

Derivation and genetic modification of embryonic stem cells from disease-model inbred rat strains.

The lack of rat embryonic stem cells (ESCs) and approaches for manipulation of their genomes have restricted the ability to create new genetic models and to explore the function of a single gene in complex diseases in the laboratory rat. The recent breakthrough in isolating germline-competent ESCs from rat and subsequent demonstration of gene knockout has propelled the field forward, but such tools do not yet exist for many disease-model rat strains. Here we derive new ESCs from several commonly used rat models including the Dahl Salt Sensitive (SS), the sequenced Brown Norway (BN), and Fischer (F344) rat and establish the first germline-competent ESCs from a hypertension disease model strain, the Fawn Hooded Hypertensive (FHH) rat.

High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.

Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.

Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells.

Knockout rats via embryo microinjection of zinc-finger nucleases.

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline.