Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers.

COVID-19 infection and vaccination rarely impact HLA antibody profile in waitlisted renal transplant candidates- a multicenter cohort.

Although rare, infection and vaccination can result in antibodies to human leukocyte antigens (HLA). We analyzed the effect of SARS-CoV-2 infection or vaccination on HLA antibodies in waitlisted renal transplant candidates. Specificities were collected and adjudicated if the calculated panel reactive antibodies (cPRA) changed after exposure.

Tebentafusp in the Treatment of Metastatic Uveal Melanoma: Patient Selection and Special Considerations.

Uveal melanoma (UM) is a rare type of melanoma with distinct features from cutaneous melanoma, low response rates to immune checkpoint inhibition, and poor survival rates. Tebentafusp, a bispecific antibody engaging T cells with gp 100 on HLA-A*02:01, was recently approved by the FDA as the first drug of its class and the first treatment approved by the FDA to treat UM. In this review, we summarize the preclinical and clinical data on tebentafusp for UM.

CRISPR-Cas9 treatment partially restores amyloid-β 42/40 in human fibroblasts with the Alzheimer's disease M146L mutation.

Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-β (Aβ) peptide that deposits as plaques in the Alzheimer's disease (AD) brain. The M146L mutation in the PS1 gene () leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aβ42. This change is evident not only in the brain but also in peripheral cells of mutation carriers.

Non-Invasive Monitoring for Rejection in Kidney Transplant Recipients After SARS-CoV-2 mRNA Vaccination.

Introduction: Studies have shown reduced antiviral responses in kidney transplant recipients (KTRs) following SARS-CoV-2 mRNA vaccination, but data on post-vaccination alloimmune responses and antiviral responses against the Delta (B.1.617.

Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito.

CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair ("homing"). Understanding whether CRISPR-induced off-target mutations are generated in mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome.

A Code of Ethics for Gene Drive Research.

Gene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity.

Association between Human Leukocyte Antigen Type and Keratinocyte Carcinoma Risk in Renal Transplant Recipients.

Keratinocyte carcinoma (KC), defined as squamous cell carcinoma and basal cell carcinoma, is the most common malignancy among white, non-Hispanic renal transplant recipients. Although recent genome-wide association studies reported that class II HLA is associated with KC risk, epidemiologic data on HLA type and KC risk in renal transplant recipients is limited. Using an institutional cohort of white, non-Hispanic renal transplant recipients transplanted between 1993 and 2017, we examined the association between pretransplant molecular HLA types and KC risk.

Use of Routine Complete Blood Count Results to Rule Out Anaplasmosis Without the Need for Specific Diagnostic Testing.

Background: Anaplasmosis presents with fever, headache, and laboratory abnormalities including leukopenia and thrombocytopenia. Polymerase chain reaction (PCR) is the preferred diagnostic but is overutilized. We determined if routine laboratory tests could exclude anaplasmosis, improving PCR utilization.

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts.

Small molecule-triggered Cas9 protein with improved genome-editing specificity.

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

Determining the specificities of TALENs, Cas9, and other genome-editing enzymes.

The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases, transcription activator-like effectornucleases, and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction, and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety.

Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity.

Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells.

In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites.

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications.

High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences.

Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection.

Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 10(11) DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively.

Observed versus predicted structure of fluorescent self-quenching reporter molecules (SQRM): caveats with respect to the use of "stem-loop" oligonucleotides as probes for mRNA folding.

We developed self-quenching reporter molecules (SQRMs), oligodeoxynucleotides with fluorophore and quencher moieties at the 5' and 3' ends respectively, to probe mRNAs for single-stranded, hybridization accessible sequences. SQRMs and their homologous antecedents, Molecular Beacons (MB), are designed with the assumption that they adopt a stem-loop structure thought critical for regulating their reporter function. Recently, we observed that stem-loop structures are not required for SQRM function, and on this basis proposed that these reporter molecules be classified according to whether they were stemmed (Type I) or not (Type II).

Directed evolution and substrate specificity profile of homing endonuclease I-SceI.

The laboratory evolution of enzymes with tailor-made DNA cleavage specificities would represent new tools for manipulating genomes and may enhance our understanding of sequence-specific DNA recognition by nucleases. Below we describe the development and successful application of an efficient in vivo positive and negative selection system that applies evolutionary pressure either to favor the cleavage of a desired target sequence or to disfavor the cleavage of nontarget sequences. We also applied a previously described in vitro selection method to reveal the comprehensive substrate specificity profile of the wild-type I-SceI homing endonuclease.

Stemless self-quenching reporter molecules identify target sequences in mRNA.

The design of oligonucleotides for gene silencing requires a rational method for identifying hybridization-accessible sequences within the target RNA. To this end, we have developed stem-loop self-quenching reporter molecules (SQRMs) as probes for such sequence. SQRMs have a 5' fluorophore, a quenching moiety on the 3' end, an intervening sequence that forms an approximately 5-basepaired stem, and a loop sequence of approximately 20-30 bases.