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Translation of the CRISPR–Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci ( HBB , IL2RG , CCR5 , HEXB , and TRAC ) in human CD34 + hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications. A bacterial screen yields a Cas9 variant that retains high on-target activity when delivered in the RNP format. As proof of principle, this Cas9 variant enables high-level correction of the sickle cell disease mutation in patient-derived HSPCs.

CRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR–Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDR

Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature inmuscle biopsies from patients with various types of muscular dystrophy. However, the consequence of disrupting DG in mature muscle is not known. Here, we investigated muscles of transgenic mice several months after genetic knockdown of DG at maturity. In our study, an increase in susceptibility to contraction-induced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contraction-induced injury was not accompanied by increased necrosis, excitation–contraction uncoupling, or fragility of the sarcolemma. Rather, disruption of the sarcomeric cytoskeleton was evident as reduced passive tension and decreased titin immunostaining. These results reveal a role for DG in maintaining the stability of the sarcomeric cytoskeleton during contraction and provide mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy after lengthening contractions.

T-cell malignancies are associated with frequent relapse and high morbidity, which is partly due to the lack of effective or targeted treatment options. To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogeneic “universal” CD2-targeting CAR-T cell (UCART2), in which the CD2 antigen is deleted to prevent fratricide, and the T-cell receptor is removed to prevent GvHD. UCART2 demonstrated efficacy against T-ALL and CTCL and prolonged the survival of tumor-engrafted NSG mice in vivo. To evaluate the impact of CD2 on CAR-T function, we generated CD19 CAR-T cells (UCART19) with or without CD2 deletion, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines (Granzyme-B and IFN-γ). We also observed that UCART19ΔCD2 had reduced anti-tumor efficacy compared to UCART19 in a CD19+NALM6 xenograft model. Of note is that the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc prolonged UCART2 persistence and increased survival in both the tumor re-challenge model and primary patient T-ALL model in vivo. Together, these data suggest that allogeneic fratricide-resistant UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines. The utility of AsCas12a can be limited to poor editing efficiency. Here the authors identify a variant, “AsCas12a Ultra”, that has high on-target specificity demonstrated through editing of clinically relevant T cell genes.

: The objective of this study was to assess the utility of the gene expression profiling technique for the preclinical evaluation of drug efficacy and safety, taking a new therapeutic approach for Duchenne muscular dystrophy (DMD) as an example. : Muscles from dystrophin-deficient (mdx) mice, a well-characterized animal model for DMD, were injected with antisense constructs that restore the open reading frame in the gene. Synthetic antisense oligonucleotides (AONs) complexed with different carriers to enhance cellular uptake and recombinant adeno-associated virus (rAAV)-expressed antisense sequences were evaluated. Muscular gene expression profiles were analyzed on oligonucleotide microarrays. Polyethylenimine (PEI)-complexed AONs restored the reading frame slightly more effectively than uncomplexed, F127- or Optison™-complexed AONs. However, PEI induced the expression of many immune genes, reflecting an aggravation of the inflammation present in untreated mice. Expression profiles in Optison and F127-injected muscles were similar to those of saline treated muscles, implying that these carriers did not evoke adverse responses. Due to moderate levels of exon skipping, a significant shift toward wild-type expression levels was not detected. Injection with rAAV vectors resulted in much higher production of dystrophin and greatly improved the histological appearance of the muscle. Depending on the efficacy of the treatment, the expression of genes previously shown to be elevated in muscular dystrophies, partly or completely returned to wild-type expression levels. Reductions in inflammation and fibrosis were among the most prominent changes observed. Expression profiling is a powerful tool for the evaluation of both desired and adverse effects of new pharmacological therapies. It is sensitive and detects changes that are not histologically visible. In addition, its ability to simultaneously monitor a large number of different biological processes not only reduces the number of different assays required in preclinical research and clinical trials, but may also assist in the early detection of potential side effects.

Summary The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post‐translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus–receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus‐bound DG. This virus‐induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process. Virus binding to cellular receptors frequently induces signaling that serves as a “knock on the door” to prime the host cell for subsequent steps of viral entry. Here, we found that binding of the highly pathogenic Lassa virus to its cellular receptor dystroglycan induces signaling, resulting in tyrosine phosphorylation of the cytoplasmic domain of the receptor. These signaling events are accompanied by the dissociation of dystroglycan from the cytoskeletal adaptor protein utrophin, likely facilitating internalization of the virus‐receptor complex.

CRISPR-Cas proteins are used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing a DNA template during repair allows for precise introduction of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR-Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein complexes with synthetic guide RNAs. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. Additionally, we employ chemically modified HDR donor templates in combination with a small molecule to boost HDR efficiency up to 10-fold. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: www.idtdna.com/HDR

Translation of the CRISPR/Cas9 system to human therapeutics holds high promise. Specificity remains a concern, however, especially when modifying stem cell populations. We show that existing rationally-engineered Cas9 high fidelity variants have reduced on-target activity using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci (HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells. We also show that the HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing Glu6Val mutation in SCD patient derived HSPCs. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome editing applications.