Identification AAVS1-like locus from the porcine genome and site-specific integration of recombinase-mediated cassette exchange using CRISPR/Cas9.

Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

Targeted PMP22 TATA-box editing by CRISPR/Cas9 reduces demyelinating neuropathy of Charcot-Marie-Tooth disease type 1A in mice.

Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted TATA-box of human PMP22 promoter to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multiple copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a proof-of-concept that CRISPR/Cas9-mediated targeting of TATA-box can be utilized to treat CMT1A.

CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection.

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes (i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery.

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells.

Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

Mediator links epigenetic silencing of neuronal gene expression with x-linked mental retardation.

Mediator occupies a central role in RNA polymerase II transcription as a sensor, integrator, and processor of regulatory signals that converge on protein-coding gene promoters. Compared to its role in gene activation, little is known regarding the molecular mechanisms and biological implications of Mediator as a transducer of repressive signals. Here we describe a protein interaction network required for extraneuronal gene silencing comprising Mediator, G9a histone methyltransferase, and the RE1 silencing transcription factor (REST; also known as neuron restrictive silencer factor, NRSF). We show that the MED12 interface in Mediator links REST with G9a-dependent histone H3K9 dimethylation to suppress neuronal genes in nonneuronal cells. Notably, missense mutations in MED12 causing the X-linked mental retardation (XLMR) disorders FG syndrome and Lujan syndrome disrupt its REST corepressor function. These findings implicate Mediator in epigenetic restriction of neuronal gene expression to the nervous system and suggest a pathologic basis for MED12-associated XLMR involving impaired REST-dependent neuronal gene regulation.

Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases.

Despite the recent discoveries of and interest in numerous structural variations (SVs)--which include duplications and inversions--in the human and other higher eukaryotic genomes, little is known about the etiology and biology of these SVs, partly due to the lack of molecular tools with which to create individual SVs in cultured cells and model organisms. Here, we present a novel method of inducing duplications and inversions in a targeted manner without pre-manipulation of the genome. We found that zinc finger nucleases (ZFNs) designed to target two different sites in a human chromosome could introduce two concurrent double-strand breaks, whose repair via non-homologous end-joining (NHEJ) gives rise to targeted duplications and inversions of the genomic segments of up to a mega base pair (bp) in length between the two sites. Furthermore, we demonstrated that a ZFN pair could induce the inversion of a 140-kbp chromosomal segment that contains a portion of the blood coagulation factor VIII gene to mimic the inversion genotype that is associated with some cases of severe hemophilia A. This same ZFN pair could be used, in theory, to revert the inverted region to restore genomic integrity in these hemophilia A patients. We propose that ZFNs can be employed as molecular tools to study mechanisms of chromosomal rearrangements and to create SVs in a predetermined manner so as to study their biological roles. In addition, our method raises the possibility of correcting genetic defects caused by chromosomal rearrangements and holds new promise in gene and cell therapy.

Generation of knockout mice using engineered nucleases.

The use of engineered nucleases in one-cell stage mouse embryos is emerging as an efficient alternative to conventional gene targeting in mouse embryonic stem (ES) cells. These nucleases are designed or reprogrammed to specifically induce double strand breaks (DSBs) at a desired genomic locus, and efficiently introduce mutations by both error-prone and error-free DNA repair mechanisms. Since these mutations frequently result in the loss or alteration of gene function by inserting, deleting, or substituting nucleotide sequences, engineered nucleases are enabling us to efficiently generate gene knockout and knockin mice. Three kinds of engineered endonucleases have been developed and successfully applied to the generation of mutant mice: zinc-finger nuclease (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs). Based on recent advances, here we provide experimentally validated, detailed guidelines for generating non-homologous end-joining (NHEJ)-mediated mutant mice by microinjecting TALENs and RGENs into the cytoplasm or the pronucleus of one-cell stage mouse embryos.

Disruption of exogenous eGFP gene using RNA-guided endonuclease in bovine transgenic somatic cells.

Genome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis. CRISPR/Cas9 plasmids specifically targeting the eGFP gene were transfected into the eGFP fibroblasts by electroporation. After 10 days of culture, more than 40% of the cells had lost eGFP expression in fluorescence activated cell sorting (FACS) analysis. Targeted sequences of the transfected cells were analyzed, and various small indel mutations (6-203 bp deletions) in the target sequence were found. The fibroblasts mutated with the CRISPR/Cas9 system were applied for somatic cell nuclear transfer, and the reconstructed embryos were successfully developed into the blastocyst stage. In conclusion, the CRISPR/Cas9 system was successfully utilized in bovine cells and cloned embryos. This will be a useful technique to develop livestock transgenesis for agricultural science.

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Targeted dual base editing with Campylobacter jejuni Cas9 by single AAV-mediated delivery.

Various CRISPR‒Cas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.

Statins suppress cell-to-cell propagation of α-synuclein by lowering cholesterol.

Cell-to-cell propagation of protein aggregates has been implicated in the progression of neurodegenerative diseases. However, the underlying mechanism and modulators of this process are not fully understood. Here, we screened a small-molecule library in a search for agents that suppress the propagation of α-synuclein and mutant huntingtin (mHtt). These screens yielded several molecules, some of which were effective against both α-synuclein and mHtt. Among these molecules, we focused on simvastatin and pravastatin. Simvastatin administration in a transgenic model of synucleinopathy effectively ameliorated behavioral deficits and α-synuclein accumulation, whereas pravastatin had no effect. Because only simvastatin enters the brain effectively, these results suggest that inhibition of brain cholesterol synthesis is important in simvastatin effects. In cultured cells, accumulation of intracellular cholesterol, induced by genetic ablation of the NPC1 gene or by pharmacological treatment with U18666A, increased α-synuclein aggregation and secretion. In contrast, lowering cholesterol using methyl-ß-cyclodextrin or statins reversed α-synuclein aggregation and secretion in NPC1-knockout cells. Consistent with these observations, feeding a high-fat diet aggravated α-synuclein pathology and behavioral deficits in the preformed fibril-injected mouse model, an effect that was also reversed by simvastatin administration. These results suggest that statins suppress propagation of protein aggregates by lowering cholesterol in the brain.

In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector.

Recent therapeutic strategies for hemophilia include long-term therapeutic gene expression using adeno-associated virus (AAV) and rebalancing therapy via the downregulation of anticoagulant pathways. However, these approaches have limitations in immune responses or insufficiency to control acute bleeding. Thus, we developed a therapeutic strategy for hemophilia B by a combined rebalancing and human factor 9 (hF9) gene knockin (KI) using a lipid nanoparticle (LNP) and AAV. Antithrombin (AT; Serpin Family C Member 1 [Serpinc1]) was selected as the target anticoagulation pathway for the gene KI. First, the combined use of LNP-clustered regularly interspaced short palindromic repeats (CRISPR) and AAV donor resulted in 20% insertions or deletions (indels) in Serpinc1 and 67% reduction of blood mouse AT concentration. Second, hF9 coding sequences were integrated into approximately 3% of the target locus. hF9 KI yielded approximately 1,000 ng/mL human factor IX (hFIX) and restored coagulation activity to a normal level. LNP-CRISPR injection caused sustained AT downregulation and hFIX production up to 63 weeks. AT inhibition and hFIX protein-production ability could be maintained by the proliferation of genetically edited hepatocytes in the case of partial hepatectomy. The co-administration of AAV and LNP showed no severe side effects except random integrations. Our results demonstrate hemophilia B therapy by a combination of rebalancing and hF9 KI using LNP and AAV.

SUPT4H1-edited stem cell therapy rescues neuronal dysfunction in a mouse model for Huntington's disease.

Huntington's disease (HD) is a severe inherited neurological disorder caused by a CAG repeat expansion in the huntingtin gene (HTT), leading to the accumulation of mutant huntingtin with polyglutamine repeats. Despite its severity, there is no cure for this debilitating disease. HTT lowering strategies, including antisense oligonucleotides (ASO) showed promising results very recently. Attempts to develop stem cell-based therapeutics have shown efficacy in preclinical HD models. Using an HD patient's autologous cells, which have genetic defects, may hamper therapeutic efficacy due to mutant HTT. Pretreating these cells to reduce mutant HTT expression and transcription may improve the transplanted cells' therapeutic efficacy. To investigate this, we targeted the SUPT4H1 gene that selectively supports the transcription of long trinucleotide repeats. Transplanting SUPT4H1-edited HD-induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) into the YAC128 HD transgenic mouse model improved motor function compared to unedited HD iPSC-NPCs. Immunohistochemical analysis revealed reduced mutant HTT expression without compensating wild-type HTT expression. Further, SUPT4H1 editing increased neuronal and decreased reactive astrocyte differentiation in HD iPSC-NPCs compared to the unedited HD iPSC-NPCs. This suggests that ex vivo editing of SUPT4H1 can reduce mutant HTT expression and provide a therapeutic gene editing strategy for autologous stem cell transplantation in HD.

Genome editing-mediated knock-in of therapeutic genes ameliorates the disease phenotype in a model of hemophilia.

Recently, clinical trials of adeno-associated virus-mediated replacement therapy have suggested long-term therapeutic effects for several genetic diseases of the liver, including hemophilia. However, there remain concerns regarding decreased therapeutic effects when the liver is regenerated or when physiological proliferation occurs. Although genome editing using the clustered regularly interspaced short palindromic repeats/Cas9 system provides an opportunity to solve this problem, low knock-in efficiency may limit its application for therapeutically relevant expression. Here, we identified a novel gene, APOC3, in which a strong promoter facilitated the expression of knocked-in genes in hepatocytes. We also investigated the effects of APOC3 editing using a small Cas9 protein derived from Campylobacter jejuni (CjCas9) in a hemophilic model. We demonstrated that adeno-associated virus-mediated delivery of CjCas9 and donor led to moderate levels of human factor 9 expression in APOC3-humanized mice. Moreover, knock-in-driven expression induced substantial recovery of clotting function in mice with hemophilia B. There was no evidence of off-target editing in vitro or in vivo. Collectively, our findings demonstrated therapeutically relevant expression using a precise and efficient APOC3-editing platform, providing insights into the development of further long-term therapeutics for diverse monogenic liver diseases.

In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy.

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

Efficient and specific generation of knockout mice using Campylobacter jejuni CRISPR/Cas9 system.

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) system is now widely utilized to generate genome engineered mice; however, some studies raised issues related to off-target mutations with this system. Herein, we utilized the Campylobacter jejuni Cas9 (CjCas9) system to generate knockout mice. We designed sgRNAs targeting mouse Tyr or Foxn1 and microinjected into zygotes along with CjCas9 mRNA. We obtained newborn mice from the microinjected embryos and confirmed that 50% (Tyr) and 38.5% (Foxn1) of the newborn mice have biallelic mutation on the intended target sequences, indicating efficient genome targeting by CjCas9. In addition, we analyzed off-target mutations in founder mutant mice by targeted deep sequencing and whole genome sequencing. Both analyses revealed no off-target mutations at potential off-target sites predicted in silico and no unexpected random mutations in analyzed founder animals. In conclusion, the CjCas9 system can be utilized to generate genome edited mice in a precise manner.

Mediator modulates Gli3-dependent Sonic hedgehog signaling.

The physiological and pathological manifestations of Sonic hedgehog (Shh) signaling arise from the specification of unique transcriptional programs dependent upon key nuclear effectors of the Ci/Gli family of transcription factors. However, the underlying mechanism by which Gli proteins regulate target gene transcription in the nucleus remains poorly understood. Here, we identify and characterize a physical and functional interaction between Gli3 and the MED12 subunit within the RNA polymerase II transcriptional Mediator. We show that Gli3 binds to MED12 and intact Mediator both in vitro and in vivo through a Gli3 transactivation domain (MBD; MED12/Mediator-binding domain) whose activity derives from concerted functional interactions with both Mediator and the histone acetyltransferase CBP. Analysis of MBD truncation mutants revealed an excellent correlation between the in vivo activation strength of an MBD derivative and its ability to bind MED12 and intact Mediator in vitro, indicative of a critical functional interaction between the Gli3 MBD and the MED12 interface in Mediator. Disruption of the Gli3-MED12 interaction through dominant-negative interference inhibited, while RNA interference-mediated MED12 depletion enhanced, both MBD transactivation function and Gli3 target gene induction in response to Shh signaling. We propose that activated Gli3 physically targets the MED12 interface within Mediator in order to functionally reverse Mediator-dependent suppression of Shh target gene transcription. These findings thus link MED12 to the modulation of Gli3-dependent Shh signaling and further implicate Mediator in a broad range of developmental and pathological processes driven by Shh signal transduction.

Programmable Nuclease-Based Integration into Novel Extragenic Genomic Safe Harbor Identified from Korean Population-Based CNV Analysis.

Here, we found two genomic safe harbor (GSH) candidates from chromosomes 3 and 8, based on large-scale population-based cohort data from 4,694 Koreans by CNV analysis. Furthermore, estimated genotype of these CNVRs was validated by quantitative real-time PCR, and epidemiological data examined no significant genetic association between diseases or traits and two CNVRs. After screening the GSH candidates by in silico approaches, we designed TALEN pairs to integrate EGFP expression cassette into human cell lines in order to confirm the functionality of GSH candidates in an in vitro setting. As a result, transgene insertion into one of the two loci using TALEN showed robust transgene expression comparable to that with an AAVS1 site without significantly perturbing neighboring genes. Changing the promoter or cell type did not noticeably disturb this trend. Thus, we could validate two CNVRs as a site for effective and safe transgene insertion in human cells.

CRISPR-Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9-mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.