Abstract
Small molecule drugs known as modulators can treat ~90% of people with cystic fibrosis (CF), but do not work for premature termination codon variants such as W1282X (c.3846G>A). Here we ...evaluated two gene editing strategies, Adenine Base Editing (ABE) to correct W1282X, and Homology-Independent Targeted Integration (HITI) of a CFTR superexon comprising exons 23–27 (SE23–27) to enable expression of a CFTR mRNA without W1282X. In Flp-In-293 cells stably expressing a CFTR expression minigene bearing W1282X, ABE corrected 24% of W1282X alleles, rescued CFTR mRNA from nonsense mediated decay and restored protein expression. However, bystander editing at the adjacent adenine (c.3847A>G), caused an amino acid change (R1283G) that affects CFTR maturation and ablates ion channel activity. In primary human nasal epithelial cells homozygous for W1282X, ABE corrected 27% of alleles, but with a notably lower level of bystander editing, and CFTR channel function was restored to 16% of wild-type levels. Using the HITI approach, correct integration of a SE23–27 in intron 22 of the CFTR locus in 16HBEge W1282X cells was detected in 5.8% of alleles, resulting in 7.8% of CFTR transcripts containing the SE23–27 sequence. Analysis of a clonal line homozygous for the HITI-SE23–27 produced full-length mature protein and restored CFTR anion channel activity to 10% of wild-type levels, which could be increased three-fold upon treatment with the triple combination of CF modulators. Overall, these data demonstrate two different editing strategies can successfully correct W1282X, the second most common class I variant, with a concomitant restoration of CFTR function.
•Traditional stone spouts can supplement the water demand in the Kathmandu valley if sufficiently conserved.•Rapid unplanned constructions and excessive groundwater extractions are affecting the ...recharge and discharge of spout water.•Nitrate and bacteria are affecting the spout water quality and call for effective urban planning and policy interventions for ensuring sound health of the community.
The speedy unplanned urbanization in the Kathmandu Valley is creating an increasing demand for water. In 2021, the water demand was 470 MLD (million liters per day), and the supply was 106 MLD during the wet season and 80 MLD during the dry season. In such scarce conditions, traditional stone spouts have the potential to fulfill water demands of certain populations. However, these spouts themselves are degrading due to extensive pumping of groundwater, unstoppable constructions, and contamination from sewage, septic tanks, and industrial waste. This review paper essentially focuses on the status of stone spouts in the Kathmandu Valley in terms of quantity and quality. It also explores the reasons that affected the quantity and quality of spout water and its impact upon communities that depend on spout water. In 2019, the Kathmandu Water Supply Development Board (KVWSMB) identified 573 spouts in the Valley, out of which 224 were functional at the time of survey and 94 were completely lost. These functional spouts were the primary source of drinking water for low-income households and had total discharge of 2.4 MLD. Spout water is perceived as “clean” drinking water but it is easily contaminated due to being mostly supplied by shallow groundwater. The observed samples exceeded WHO standards (sometimes national standards too) for total coliform, E-coli and nitrate, and the quality was heavily affected during monsoon. This indicates potential impacts on community health if not treated properly. These culturally significant spouts are to be conserved for sustainable water supply. Policy interventions regarding haphazard constructions, over-extraction of groundwater, waste and sewage management, source protections, renovation, and restorations can help to protect these traditional structures and fulfill water needs.
CRISPR/Cas9 genome-editing system is a powerful, fairly accurate, and efficient tool for modifying genomic DNA. Despite obvious advantages, it is not devoid of certain drawbacks, such as propensity ...for introduction of additional nonspecific DNA breaks, insufficient activity against aneuploid genomes, and relative difficulty in delivering its components to cells. In this review, we focus on the difficulties that can limit the use of CRISPR/Cas9 and suggest a number of practical recommendations and information sources that will make it easier for the beginners to work with this outstanding technological achievement of the XXI century.
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•Developed a CRISPR/Cas9-mediated EGT system for F. oxysporum.•First instance of a HITI strategy to insert a large fragment into the fungal genome.•FoChs5 showed polarized location at ...the hyphal tip.•Used the HDRI strategy to tag FoSSO1.•FoSso1 and FoSso2 are localized to different fungal structures.
Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, we developed a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The HITI strategy was able to facilitate integration of a large DNA fragment, ∼8 kb in length, into the genome of F. oxysporum at the sgRNA cleavage site, and was used to insert a C-terminal 3×sGFP tag to the FoCHS5 gene and a N-terminal mCherry tag to the FoSSO2 gene. The HDRI strategy was used to tag the paralogous gene, FoSSO1, with a C-terminal mCherry marker. FoChs5-3×sGFP localized to conidia, some septa, and fungal tips. A majority of the FoSso1-mCherry was distributed in the conidia, septum, and hyphae that were distal from the fungal tips. While FoSso1-mCherry showed a very weak fluorescent signal at the fungal tips, mCherry-FoSso2 accumulated in the plasma membrane of conidia, germlings, fungal tips, hyphae, and phialides, suggesting FoSSO1 and FoSSO2 are regulated differently during fungal development. These results indicate this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.
Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, ...access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA).
We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach.
Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 10
-3.6 × 10
CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity.
Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.
A genome editing tool targeting the high-risk human papillomavirus (HPV) oncogene is a promising therapeutic strategy to treat HPV-related cervical cancer. To improve gene knockout efficiency, we ...developed a gene knockout chain reaction (GKCR) method for continually generating mutagenic disruptions and used this method to disrupt the HPV18 E6 and E7 genes. We verified that the GKCR Cas9/guide RNA (gRNA) cassettes could integrated into the targeted loci via homology-independent targeted insertion (HITI). The qPCR results revealed that the GKCR method enabled a relatively higher Cas9/gRNA cassette insertion rate than a control method (the common CRISPR-Cas9 strategy). Tracking of Indels by DEcomposition (TIDE) assay results showed that the GKCR method produced a significantly higher percentage of insertions or deletions (indels) in the HPV18 E6 and E7 genes. Furthermore, by targeting the HPV18 E6/E7 oncogenes, we found that the GKCR method significantly upregulated the P53/RB proteins and inhibited the proliferation and motility of HeLa cells. The GKCR method significantly improved the gene knockout efficiency of the HPV18 E6/E7 oncogenes, which might provide new insights into treatment of HPV infection and related cervical cancer.
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We developed a gene knockout chain reaction (GKCR) method by inserting CRISPR-Cas9 into the edited allele and generating sustained disruptions. Compared with the traditional CRISPR-Cas9 strategy, the GKCR method could result in higher disruption of the HPV18 E6/E7 oncogenes, which provides new insights into treatment of HPV infection and related cervical cancer.
Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to ...provide sustained transgene expression following neonatal treatment. We show that targeting the 3′ end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.
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•AAV-HITI provides sustained transgene expression from newborn and adult liver•Liver-directed AAV-HITI is effective in mouse models of inherited diseases•AAV-HITI results in undetectable off-targets and large gene rearrangements•AAV-HITI is safe at the doses tested up to 1 year after AAV-HITI delivery
Esposito et al. explore the therapeutic potential of liver-directed adeno-associated viral vector-mediated homology-independent targeted integration (AAV-HITI) through CRISPR-Cas9 at the mouse albumin locus. Effective integration yields stable therapeutic protein levels in mouse models of inherited diseases without adverse effects, supporting AAV-HITI as a safe and efficient liver-directed knockin strategy.
Ex vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) has emerged as a promising therapeutic approach for treatment of inherited human blood disorders. Use of engineered ...nucleases to target therapeutic transgenes to their endogenous genetic loci addresses many of the limitations associated with viral vector-based gene replacement strategies, such as insertional mutagenesis, variable gene dosage, and ectopic expression. Common methods of nuclease-mediated site-specific integration utilize the homology-directed repair (HDR) pathway. However, these approaches are inefficient in HSPCs, where non-homologous end joining (NHEJ) is the primary DNA repair mechanism. Recently, a novel NHEJ-based approach to CRISPR-Cas9-mediated transgene knockin, known as homology-independent targeted integration (HITI), has demonstrated improved site-specific integration frequencies in non-dividing cells. Here we utilize a HITI-based approach to achieve robust site-specific transgene integration in human mobilized peripheral blood CD34+ HSPCs. As proof of concept, a reporter gene was targeted to a clinically relevant genetic locus using a recombinant adeno-associated virus serotype 6 vector and single guide RNA/Cas9 ribonucleoprotein complexes. We demonstrate high levels of stable HITI-mediated genome editing (∼21%) in repopulating HSPCs after transplantation into immunodeficient mice. Our study demonstrates that HITI-mediated genome editing provides an effective alternative to HDR-based transgene integration in CD34+ HSPCs.
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Common methods of genome editing utilize the homology-directed repair (HDR) pathway. However, these approaches are inefficient in human hematopoietic stem and progenitor cells (HSPCs), where non-homologous end joining (NHEJ) is the primary DNA repair mechanism. Bloomer et al. demonstrate stable high-level genome editing (∼21%) in human repopulating HSPCs using a novel NHEJ-based approach known as homology-independent targeted integration (HITI).
CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, ...especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.
Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between ...in vitro and in vivo gene integration rates. Homology-directed repair occurs primarily in the S/G2 phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton’s tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G1 resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors.
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There are alternative methods for nuclease-mediated targeted gene integration, including homology-independent targeted integration (HITI) and precise integration into target chromosome (PITCh), which utilize non-homologous end joining and microhomology-mediated end joining, respectively. These DNA repair pathways can be utilized in both cell lines and primary hematopoietic stem cells.
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