Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing (WGS) results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders.

Precision Genome and Base Editing by Regulating mRNA Nuclear Export Using Selective Inhibitors of Nuclear Export (SINEs)

CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. In the present study, we screened for irreversible small molecule off-switches of CRISPR-Cas9 and discovered that selective inhibitors of nuclear export (SINEs) could inhibit the cellular activity of CRISPR-Cas9 by interfering with the nuclear export of Cas9 mRNA. We subsequently found that SINEs, including an FDA-approved anticancer drug KPT330, could improve the specificities of CRISPR-Cas9-based genome and base editing tools in human cells.

Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.

Highly Efficient Gene Editing of Human Hematopoietic Stem Cells to Treat X-Linked Sideroblastic Anemia

X-linked sideroblastic anemia (XLSA) is an anemic disease caused by mutations in the gene encoding 5-aminolevulinate synthase 2 (ALAS2), which catalyzes the rate-limiting step of heme biosynthesis. With current interventions including pyridoxine treatment and allogeneic hematopoietic stem cell transplantation, severe unmet needs remain for patients with XLSA. Here, we used CRISPR/Cas9 technology to efficiently and functionally correct the pathogenic mutation in intron 1 of ALAS2 in CD34+ hematopoietic stem cell and progenitor cells (HSPCs) from two patients. Intriguingly, we found that the gene-editing efficiency of CD34+HSPCs from the elder patient was much lower than that from the younger patient, consistent with the poor hematopoiesis of older XLSA patients observed in clinical practice. Furthermore, we performed single cell RNA-sequencing (scRNA-seq) to investigate the causes of those phenomenon in-depth. Previously we and others identified the A>G mutation in the GATA1 binding region of intron 1 of ALAS2 in particular XLSA families and have demonstrated the key role of this site in regulating ALAS expression. Thus, we first designed a series of sgRNAs, along with single-stranded DNA oligonucleotide donors (ssODN). After co-electroporating with Cas9 mRNA into patient-derived hiPSCs, sgRNA-1 and ssODN were selected for further experiments based on optimal correction rate (43.93±3.43% via HDR). Next, using a well-established erythroid protocol, CD34+ HSPCs of control and gene-edited groups were differentiated into erythroid cells in vitro. Surprisingly, heme biosynthesis examined by benzidine staining showed that compared with the mock cells, the gene-corrected group significantly increased the frequency of benzidine-positive cells. To examine the multilineage differentiation potential of gene-corrected CD34+ HSCPs, we performed colony-forming unit (CFU) assays to quantify various types of colonies. Compared with mock cells, gene-edited group significantly enhanced the generation of total, CFU-GM and BFU-E colonies, suggesting higher clonogenic potential. Next, gene-corrected CD34+ HSPCs were transplanted into nonobese diabetic (NOD)/Prkdcscid/IL-2Rγnull (NPG) mice to evaluate the repopulating potential. All transplanted mice displayed engraftment in multiple organs at 10-16 weeks post transplantation, and the gene-corrected cells showed greater engraftment potential than mock group. In addition, hematopoietic reconstitution analysis indicated that the gene-corrected cells maintained normal lineage distribution, while the B cell development of mock group was impaired. Moreover, gene-editing efficiency analysis of bone marrow samples 16 weeks after transplantation exhibited high editing rate (34±7.18% via HDR), comparable to the in vitro efficiency. The specificity of the Cas9 mRNA-based gene editing system was examined using unbiased Digenome-Seq. In total, 32 potential off-target sites were identified and deeply interrogated via targeted PCR and NGS analysis of XLSA iPSCs electroporated with Cas9 mRNA and sgRNA. No off-target cleavage events were detected at these sites, suggesting a lack of detectable off-target events. Finally, scRNA-seq of CD34+ HSPCs from healthy donor and XLSA patients revealed more HSC/LMPP and erythroid progenitor cells in older XLSA patient. Further analysis showed that cell cycle and gene expression in older HSC/LMPP cells were significantly different from that from healthy donors and younger patients. Hence, we speculated that the compensatory differentiation of HSCs caused by long-term functional red blood cell deficiency caused the abnormal expansion of HSCs, which led to the poor hematopoiesis in elderly patients. Our study firstly uses CRISPR/Cas9 gene-editing technology to correct the disease mutation in patient's CD34+ HSPCs and rescues ALAS2 expression and heme biosynthesis, directly confirming that this mutation is the pathogenic factor for XLSA. In addition, we dissect the transcriptional profile of CD34+ HSCPs from XLSA patients at single cell resolution for the first time, shedding light on mechanistic insights into the XLSA pathogenesis. The robust gene-correction rates and significant function rescue in patient's CD34+ HSPCs further suggest a curable option of gene-edited HSC transplantation for the treatment of the patients with XLSA. Disclosures Fang: EdiGene Inc.: Current Employment. Yuan:EdiGene Inc.: Current Employment. Yang:EdiGene Inc.: Current Employment. Yu:EdiGene Inc.: Current Employment. Zhang:EdiGene Inc.: Current Employment. Shi:EdiGene Guangzhou Inc.: Current Employment. Qi:Novogene Co, Ltd: Current Employment. Wei:EdiGene Inc.: Current Employment.

Evaluation of Mosaicism and Off Target Mutations in CRISPR-Mediated Genome Edited Bovine Embryos

ABSTRACTThe CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrates that the use of Cas9 protein, rather than Cas9 mRNA, results in a higher editing efficiency in bovine embryos while lowering the level of mosaicism. However, further optimization must be carried out for the CRISPR/Cas9 system to become feasible for single-step embryo editing in a commercial system.

Intracorneal delivery of HSV-targeting CRISPR/Cas9 mRNA prevents herpetic stromal keratitis

ABSTRACTHerpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus and are incapable of modulating the virus reservoir. Here, we target HSV-1 genome directly using mRNA-carrying lentiviral particle (mLP) that simultaneously delivers spCas9 mRNA and two viral genes-targeting gRNAs (designated HSV-1-erasing lentiviral particles, HELP). We showed HELP efficiently blocked HSV-1 replication in both acute and recurrent infection models, and prevented occurrence of herpetic stromal keratitis (HSK). We further showed retrograde transportation of HELP from corneas to trigeminal ganglia (TG) where HSV-1 established latency and found evidence of HELP modulating herpes reservoir. Additionally, the potent antiviral activity of HELP was also replicable in human-derived corneas. These results strongly support clinical development of HELP as a new antiviral therapy and may accelerate mRNA-based CRISPR therapeutics.

Gene Editing and mRNA-Based Therapy: Two Complementary Therapeutic Approaches for the Treatment of Patients with Xmen Disease

Introduction 'X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia' (XMEN) disease is a primary immunodeficiency disease caused by loss-of-function mutations in the MAGT1 gene encoding for the magnesium transporter 1. This leads to the absence of expression of the "Natural-Killer Group 2, member D" (NKG2D) receptor in natural killer (NK) and CD8+ T cells, which is essential for their antiviral and antitumoral cytotoxic activity. In consequence, XMEN patients develop chronic EBV infections and EBV-related lymphoproliferative disorders. Allogeneic bone marrow transplant has been associated with significant mortality, and there are no other effective treatments. In that context, we aimed at developing two complementary approaches to treat XMEN patients: 1) Adoptive transfer of XMEN T/NK cells corrected by transient mRNA therapy or longer-lasting gene editing therapy in order to control infections, and 2) Transplantation of gene-edited CD34+ cells in order to permanently restore production of functional immune cells. Material and methods CD34+ cells and PBMCs were collected from XMEN patients and healthy donors (HD) (NIH Protocol 94-I-073). For mRNA therapy, we expanded T cells with anti-CD3/anti-CD28 beads in RPMI + 10% serum supplemented with 200 IU/mL IL2 for 5-7 days and NK cells with 100 IU/mL IL2 and 10 ng/mL IL15 in culture with K562-mb15-41BBL for 10-15 days. Both XMEN T and NK cells were electroporated (EP) with MAGT1 mRNA and cultured for up to 28 days. For gene editing, XMEN CD34+ or stimulated T cells were electroporated with Cas9 mRNA and sgRNA; a rAAV6 donor encoding for the codon-optimized MAGT1 cDNA was added after EP. Two days post-EP, CD34+ cells were differentiated into NK cells for 35 days in vitro. Results MAGT1 mRNA-based therapy. We first showed a restored MAGT1 expression by western blot at 6h and 24h post-EP of the MAGT1 mRNA. In consequence, NKG2D expression analyzed by flow cytometry was restored in expanded CD8+ T and NK cells starting within the 6h post-EP (20-40%), with a peak at 48h (>85%) and a progressive decrease of the expression over time (still 40% and 75%, respectively, of CD8+ T and NK cells of cells at day 14 post-EP respectively). The cytotoxic activity of mRNA-corrected XMEN NK cells was analyzed by culture with K562 target cells at several effector:target (E:T) ratios and shown to be restored at a level similar to HD NK cells (mRNA-treated: 66.7% ±5.8%; HD: 67.8% ±5.9% at E:T 2:1 ratio) compared to untreated cells (49.0% ±7.2%) (Fig 1a). Anticipating the potential use of these cells for repeated infusions as a treatment modality to control infections, we demonstrated that MAGT1 mRNA-corrected CD8+ T and NK cells that have been cryopreserved and thawed exhibit the same NKG2D expression kinetics following thaw and culture. Gene editing therapy. XMEN CD34+ cells electroporated with Cas9 mRNA and a sgRNA targeting exon 1 of MAGT1 gene showed an in vitro average integration rate of the MAGT1 cDNA AAV donor of 35.6% (range: 33.8-41.9%). The NKG2D expression in AAV-treated CD34+-derived NK cells was approximatively of 23% (range: 14.2-27.9%). Interestingly, their cytotoxic activity was similar to the level of NKG2D expression (23.1% ±4.3%), significantly higher than in untreated cells (9.7% ±2.8%) (Fig 1b). Similar rates of targeted integration and NKG2D expression were also obtained in AAV-treated CD8+ T cells. Conclusion For the first time, we demonstrate the efficiency of two approaches for development of potential cell therapy treatments of XMEN patients. MAGT1 mRNA electroporation can restore efficient transient expression of NKG2D in CD8+ T and NK cells, thus fully restoring the cytotoxic activity of NK cells. In addition, cells electroporated with MAGT1 mRNA can be cryopreserved, thus allowing repeated infusions. In parallel, we showed that efficient targeted insertion can be achieved in CD8+ T cells and CD34+ cells by using an AAV donor although the level of NKG2D expression is lower. Optimizations are currently ongoing in order to reach higher levels of correction. Both approaches could be combined in order to propose a new therapeutic strategy for the treatment of XMEN patients: repetitive adoptive transfer of mRNA-corrected autologous T/NK cells for the prevention or control of intractable infections, and transplantation of gene-edited CD34+ cells for the definitive treatment of these patients. Disclosures Meis: CELLSCRIPT, LLC: Employment. Li:MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Clark:CELLSCRIPT, LLC: Employment. Dahl:CELLSCRIPT, LLC: Other: Owner and officer.