T7 Endonuclease Assay Research Articles

CRISPR/Cas9 genome editing in wheat.

Genome editing has been a long-term challenge for molecular biology research, particularly for plants possess complex genome. The recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile tool for genome editing which enables editing of multiple genes based on the guidance of small RNAs. Even though the efficiency of CRISPR/Cas9 system has been shown with several studies from diploid plants, its application remains a challenge for plants with polyploid and complex genome. Here, we applied CRISPR/Cas9 genome editing system in wheat protoplast to conduct the targeted editing of stress-responsive transcription factor genes, wheat dehydration responsive element binding protein 2 (TaDREB2) and wheat ethylene responsive factor 3 (TaERF3). Targeted genome editing of TaDREB2 and TaERF3 was achieved with transient expression of small guide RNA and Cas9 protein in wheat protoplast. The effectiveness of mutagenesis in wheat protoplast was confirmed with restriction enzyme digestion assay, T7 endonuclease assay, and sequencing. Furthermore, several off-target regions for designed sgRNAs were analyzed, and the specificity of genome editing was confirmed with amplicon sequencing. Overall results suggested that CRISPR/Cas9 genome editing system can easily be established on wheat protoplast and it has a huge potentiality for targeted manipulation of wheat genome for crop improvement purposes.

Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the “safe harbor” AAVS1 locus

We report the generation-characterization of a fetal liver (FL) B-cell progenitor (BCP)-derived human induced pluripotent stem cell (hiPSC) line CRISPR/Cas9-edited to carry/express a single copy of doxycycline-inducible Cas9 gene in the “safe locus” AAVS1 (iCas9-FL-BCP-hiPSC). Gene-edited iPSCs remained pluripotent after CRISPR/Cas9 genome-edition. Correct genomic integration of a unique copy of Cas9 was confirmed by PCR and Southern blot. Cas9 was robustly and specifically expressed on doxycycline exposure. T7-endonuclease assay demonstrated that iCas9 induces robust gene-edition when gRNAs against hematopoietic transcription factors were tested. This iCas9-FL-BCP-hiPSC will facilitate gene-editing approaches for studies on developmental biology, drug screening and disease modeling.

CRISPR/Cas9‐Mediated Genome Editing of the Murine Pulmonary Circulation

CRISPR/Cas9 is increasingly being tested for in vivo genome editing and we describe here the optimization, pharmacokinetic properties, and efficiency of this system to target the pulmonary vasculature in mice. We used cationic liposomes to deliver a PX458 vector expressing Cas9, eGFP, and gRNA to the pulmonary circulation and studied both its bio‐distribution and time needed to knock down proteins.Different amounts of liposomes were injected containing 25, 50, 75, and 100μg of plasmid DNA. After 4 days, mice were sacrificed and several organs were studied for eGFP expression. We found a dose‐dependent increase in pulmonary expression. The only other organ with detectable expression was the liver, but expression was at least 7‐fold higher in the lungs compared to the liver even at the highest dose tested. We found no eGFP expression in kidney, heart, brain, or skeletal muscle (n=4 animals/dose). Within the lung, we used immunohistochemistry to show that endothelial cells and to a lesser degree pulmonary artery smooth muscle cells were selectively targeted. We did not detect any expression in airway epithelial cells or airway macrophages. Lung expression was very temporary, with expression peaking at 4 days and returning to baseline after 28 days (n=4 mice/group), minimizing the chances of side effects, more specifically non‐specific alterations in the DNA that could result from continued expression of the endonuclease Cas9. Using mT mice that ubiquitously express tandem dimer Tomato fluorescent protein, were able to show a downregulation of fluorescence specifically in the majority of the pulmonary blood vessels after just 7 days (n=3 mice/group). Genomic alterations were confirmed by the T7 endonuclease assay and Next‐Generation Sequencing. Importantly, we did not observe any thrombosis or other adverse side‐effects that might be associated with expression of Cas9 in the pulmonary endothelium.In conclusion, we used a non‐viral delivery method to specifically deliver CRISPR/Cas9 to the pulmonary vasculature. The temporary expression of CRISPR/Cas9 led to a pronounced downregulation of the targeted fluorescent protein within 7 days, but could easily be adapted to target almost any gene. Currently available animal models do not allow to knock out a gene specifically in the pulmonary endothelial cells and effects on the pulmonary circulation might be compounded by effects in the systemic circulation. Therefore, our work provides a novel tool to specifically target and study genes in the pulmonary circulation.Support or Funding InformationThis work was supported in part by a Parker B. Francis fellowship and American Heart Scientist Development grant 15SDG23250002 (GM), NIH‐R01‐GM094220 (JR) and NIH‐R01‐HL045638, R01‐HL090152, R01‐HL118068, P01‐HL060678, P01‐HL077806 (ABM).

132. Targeting the BTK Locus in Primary Human Hematopoietic Cells with TALENs and AAV Donor Template

X-linked agammaglobulinemia (XLA), a rare immunodeficiency, is caused by mutations in the Bruton's Tyrosine Kinase (BTK) gene. BTK is required for B-cell maturation, activation and BCR-mediated signaling. Patients lacking functional BTK have predominantly immature B-cells with near absence of mature B cells, minimal antibody production and are prone to recurrent and life-threatening bacterial infections. Available treatments are limited to intravenous immunoglobulin therapy, which lessens the severity of these infections and judicious use of antibiotic therapy. BTK is an ideal target for gene editing as BTK-expressing B cells exhibit a marked selective advantage in vivo, and even a small number of corrected HSC or B cell progenitors rescues B cell development and function in murine models and is expected to rescue the B cell compartment in XLA patients. Here we outline a strategy to edit the endogenous BTK locus in primary human cells, targeting a codon-optimized BTK cDNA into the first coding exon of BTK using BTK specific DNA nucleases and donor template. Four pairs of transcription activator-like effector nucleases (TALENs) cleaving within introns 1 or 2 of BTK were constructed and their cleavage efficiency in primary human T cells evaluated. Using the T7 Endonuclease assay, we determined that disruption (indel) rates of ~75% were obtained using the TALENs. Next, utilizing co-delivery of TALEN mRNA and an AAV donor template (consisting of an MND promoter-GFP cDNA expression cassette flanked by 1.0 kb BTK homology arms), we achieved up to 25% homology directed repair (HDR) in primary T cells. The translation of this gene editing strategy to human mobilized peripheral CD34+ cells is currently being optimized. If successful, this targeting approach will be used to test expression levels of the targeted cDNA as regulated by the endogenous BTK promoter.

569. Precision Editing of the WAS Locus via Homologous Recombination in Primary Human Hematopoietic Cells Mediated by Either TALEN or CRISPR/Cas Nucleases

Wiskott-Aldrich Syndrome (WAS) is an inherited primary immunodeficiency caused by mutations in the WAS gene which encodes a protein (WASp) that regulates the actin cytoskeleton in multiple hematopoietic cell lineages. Currently, allogeneic stem cell transplantation constitutes the only available cure for WAS, although phase I/II clinical trials using lentiviral gene therapy of autologous stem cells are currently underway and have shown improvement in immune system defects. Although effective, the gene therapy approach carries a risk of insertional mutagenesis, and unpredictable expression due to promoter choice as well as influences of the random integration site, including epigenetic status and the presence of neighboring transcription regulatory elements. As a refinement, our goal is to develop gene editing methodologies that would allow the specific targeting of a WASp cDNA to a position within the WAS locus allowing transcriptional regulation by the endogenous WAS promoter. To this end, we first designed and tested the cleavage efficiency of several guide RNAs (delivered as self-complementary AAV along with Cas9, delivered as mRNA), as well as candidate TALEN pairs (delivered as mRNAs), in primary human T cells. Using the T7 endonuclease assay, we identified candidate nucleases from both platforms (CRISPR/Cas9 and TALEN) that achieved a high Indel frequency: 73 and 85%, respectively. We next created a synthetic AAV6 donor template for homology-directed repair (HDR) that contained an MND promoter driven GFP cDNA with 1kb of WAS homology arms flanking it. When delivered with the TALEN or CRISPR/Cas9 nucleases, we observed stable integration of the GFP reporter within >25% of primary human T cells. Subsequently, we utilzed the identical reagents to target integration of the reporter cDNA into the WAS locus in adult human mobilized CD34+ cells, albeit at lower efficiencies. The off-target cleavage sites for TALENs identified using the Prognos software were amplified and sequenced, with no evidence of off-target cleavage observed at any of the predicted loci. Thus, we have generated genome editing tools that possess a high degree of specificity, providing the foundation for site-specific modification of the WAS locus as a therapeutic option.

116. MegaTAL Nucleases Outperform TALENs in Promoting Homology-Directed-Gene Modification in Primary Human T Cells

Advances in rare cleaving nuclease technologies have dramatically increased the potential for seamless manipulation of virtually any gene (i.e. gene editing). Among these platforms, TALENs and Cas9-based nucleases have become broadly applied owing to ease in customizing their DNA recognition properties. In contrast, LAGLIDADG homing endonucleases (LHE) are more challenging to engineer, yet exhibit superior physical attributes, DNA recognition and hydrolysis properties. Recent advances have overcome hurdles in LHE engineering leading to the ability to more rapidly customize LHEs as therapeutic nucleases. In addition, LHEs can be fused to TAL effector arrays to create chimeric proteins [referred to here as megaTALs (MTAL)] that exhibit further enhancements in activity. We previously developed a CCR5-specific MTAL as a potential HIV therapeutic. In parallel, we generated high-activity TALEN pairs targeting the identical region in CCR5. In the current study, we directly compared the ability of these nuclease platforms to induce homology directed repair (HDR) in primary human T-cells. We first verified that both reagents facilitated efficient disruption of the CCR5 locus. We directly compared activity in primary CD4 T cells using mRNA nuclease delivery. Indel frequency was estimated using both the T7 endonuclease assay and a re-cleavage assay (RCA; where genomic DNA surrounding the cleavage site is amplified and digested in vitro with the original LHE used to create the MTAL). NHEJ rates were 65-82% for the TALEN vs. 44-61% (6 experiments) for the MTAL using these respective assays. Next, we coupled mRNA-mediated nuclease expression with AAV delivery of a donor template. We co-delivered mRNA with a range of doses of an AAV-CCR5-GFP donor (containing 1.3kb CCR5 homology arms and an internal MND-GFP expression cassette). Cell viability was not significantly different; and control experiments using AAV BFP (lacking homology arms) demonstrated that infection and expression rates were equivalent. Strikingly, the proportion of cells with sustained GFP expression (at day 16) was up to 5-fold higher in MTAL treated cells (across a range of AAV doses; n=6, independent donors); with the greatest differences observed when access to donor template was limiting. Molecular analysis verified HDR in both test groups. While the mechanism for these differences remains incompletely defined, preliminary comparisons of HR:NHEJ ratios using single cell analysis suggest that alternative DNA repair pathways may be preferentially utilized by the 3’ vs 5’ overhangs generated by LHE vs Fok1 nucleases, respectively. Taken together, our findings suggest that the MTAL platform and the co-delivery method described here may provide significant clinical utility in future editing applications in human hematopoietic cells.

56. AAV Vector-Mediated CRISPR Attacks on Proviral HIV-1 DNA for Purging of Cellular Reservoirs

HIV, the human immunodeficiency virus, is highly successful as a viral pathogen due to its ability to persist in the infected host as an integrated proviral DNA within cellular reservoirs, particularly in resting CD4+ T cells and macrophages. Current antiretroviral combination therapy can control viral spread and viremia but cannot eradicate latent HIV genomes, raising an urgent need for alternative strategies for long-term remission or cure. We combined two powerful technologies, synthetic AAV vectors and CRISPR-mediated gene engineering, with the ultimate aim to eliminate integrated HIV from latently infected human cells. Therefore, we first screened a library of peptide display mutants based on 12 different AAV serotypes and identified novel capsids that mediate potent transduction of relevant HIV target cells, including primary human T-lymphocytes and macrophages. Our lead candidates – two peptide-modified variants of AAV9 or AAVrh.10 – were capable of transducing >65% of primary humanCD4+ T cells and were superior to all 12 wildtype AAV isolates. Concurrently, we created AAV vector genomes that express – alone or in combination – the two essential CRISPR components, i.e., the Cas9 protein and a g(uide)RNA directed against either the HIV-1 long terminal repeats (LTRs), or against viral structural genes. AAV/CRISPR vector efficiency and specificity were validated using a T7 endonuclease assay, showing successful Cas9-mediated induction of double-stranded DNA breaks at the specific HIV-1 target regions and subsequent error-prone repair in HeLaP4 cells with a stably integrated HIV-1 genome. To further potentiate the approach, we exploited a highly customizable self-complementary AAV vector backbone recently developed in our lab that permits multiplexing up to three gRNAs under control of the U6, H1 and 7SK promoters in a single vector particle. We engineered this template to co-express our best gRNAs against the two HIV-1 LTRs and the viral gag gene, and compared its potency with our different first-generation single gRNA vectors. In reporter cells carrying a GFP-encoding HIV-1 genome, simultaneous cleavage was indeed detected at three different sites (5’LTR, 3’LTR and gag), reaching an impressive 63.5% cutting efficiency (versus 36.6-50% for the single gRNA vectors) and demonstrating the power of these vectors to cleave HIV-1 proviral DNA. Finally, we will present results from our ongoing validation of proviral cleavage in human T-cell lines that are latently infected with HIV-1 as a physiologically relevant model of HIV-1 latency. The described experiments prove the possibility to target, cut and destroy integrated HIV-1 proviral DNA with AAV/CRISPR vectors in human cells, and therefore support the further development of our tools and approach into a novel clinical modality for purging of latent HIV-1 reservoirs.

569. Development of CRISPR Toolkit for Knock-In Transgene Targeting to the Rat Rosa26 Locus

The advent of CRISPR-based genome modification has lowered the investment cost required for the creation of highly-customized model organisms. Here we describe the development of a Cas9-based genetic toolkit for the rat, which streamlines the targeted insertion of transgenes into the Rosa26 locus. We have constructed a set of plasmids which express the Cas9(D10A) nickase (Cas9n) and a pair of rat Rosa26-targeting guide RNAs (gRNAs) along with a selectable marker. When transfected into rat adrenal gland pheochromocytoma (PC-12) cells, these plasmids induce the formation of double-strand breaks and thereby lead to the accumulation of small insertion/deletions (InDels) between the gRNA binding sites. These plasmids also increase the efficiency of homology-directed insertion of a transgene (donor template) when delivered by cotransfection. The verified Rosa26-targeting gRNAs were transcribed and purified in vitro, and microinjected alongside Cas9n mRNA into Long-Evans rat embryos. InDel frequency at the Rosa26 locus in the resulting pups was assessed using a T7 endonuclease assay. For three rounds of injections, the frequency of InDels detected was (2 out of 16), (7 out of 12), and (10 out of 15). The ability to enhance homology-directed insertions in vivo was also validated by coinjecting these gRNAs with single-stranded oligonucleotides (ssODN) containing a novel loxP site flanked on each side by 72 basepairs of Rosa26 sequence. A single round of injections produced 15 pups, 4 of which carried the loxP allele. Next, a single round of injections employing a plasmid-based donor template with 1 kilobase homologous arms produced 7 pups, 1 of which carried the transgene. The use of this rat Rosa26 CRISPR toolkit will simplify the creation of “defined” transgenic rat models and should reduce the inconsistencies attributed to copy number- and position-effects when dealing with randomly integrated transgenes.

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases