Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

The Identification of A ROS Responsive Motif that is regulated by snoRNP in Arabidopsis

A GGGCC motif (site II like motif) was identified from the upstream sequences of reactive oxygen species (ROS) and light induced genes in Arabidopsis. This motif is highly enriched within the −50 to −250 bp region of the induced genes, and it is also specifically distributed in the same region in mouse and human genome. EMSA experiments revealed that several nuclear factors (NFs) bind to this motif, and the binding activities altered under H2O2 treatment. Two C/D family snoRNP proteins, namely fibrillarin 2 and NOP56, were identified from the site II like motif binding NFs. Several C/D family snoRNA, including R63, U24a and Z15, were also cloned from the motif binding NFs. These data suggest new regulatory roles of snoRNP in Arabidopsis.

Real-time tracking reveals the catalytic process of Rad51-driven DNA strand exchange

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

HIV-1 Subtype C with PYxE Insertion Has Enhanced Binding of Gag-p6 to Host Cell Protein ALIX and Increased Replication Fitness

ABSTRACTHuman immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C-infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Throughin silicopredictions andin vitroexperiments, we showed that HIV-1C Gag has an increased binding to ALIX when the PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derivedgag-polsequences from HIV-1CPYxEiviruses and inserted them in a reference HIV-1 sequence. Viral growth was increased, and the sensitivity to the protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to that of wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion indicates the clinical importance of these viral variants.IMPORTANCEGenomic differences within HIV-1 subtypes is associated with various degrees of viral spread, disease progression, and clinical outcome. Viral budding is essential in the HIV-1 life cycle and mainly mediated through the interaction of Gag with host proteins. Two motifs within Gag-p6 mediate binding of host cell proteins and facilitate budding. HIV-1C has a natural deletion of one of these two motifs, resulting in an inability to bind to host cell protein ALIX. Previously, we have identified a tetrapeptide (PYxE) insertion at this deleted motif site in a subset of HIV-1C patients. Here, we report the incidence of PYxE insertions in three different HIV-1C cohorts, and the insertion restores the binding of Gag to ALIX. It also increases viral growth even in the presence of the antiretroviral drugs lopinavir and tenofovir alafenamide. Hence, PYxE insertion in HIV-1C might be biologically relevant for viruses and clinically significant among patients.

HIV-1 subtype C with PYxE insertion has enhanced binding of Gag-p6 to host cell protein ALIX and increased replication fitness

Human immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Throughin silicopredictions andin vitroexperiments, we showed that HIV-1C Gag has an increased binding to ALIX when PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derived gag-pol sequences from HIV-1CPYxEiviruses and inserted them in a reference HIV-1. Viral growth was increased, and the sensitivity to protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion could indicate the clinical importance of these viral variants.ImportanceGenomic differences within HIV-1 subtypes is associated with a varying degree of viral spread, disease progression, and clinical outcome. Viral budding is essential in the HIV-1 life cycle and mainly mediated through the interaction of Gag with host proteins. Two motifs within Gag-p6 mediate binding of host cell proteins and facilitate budding. HIV-1 subtype C (HIV-1C) has a natural deletion of one of these two motifs resulting in an inability to bind to host cell protein ALIX. Previously, we have identified a tetrapeptide (PYxE) insertion at this deleted motif site in a subset of HIV-1C patients. Here, we report the incidence of PYxE insertions in three different HIV-1C cohorts, and the insertion restores the binding of Gag to ALIX. It also increases viral growth even in the presence of antiretroviral drugs lopinavir and tenofovir alafenamide. Hence, PYxE insertion in HIV-1C might be biologically relevant for viruses and clinically significant among patients.

Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing

CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species (CRISPR from Microgenomates and Smithella, Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice (Oryza sativa). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

Systematic dissection of genomic features determining transcription factor binding and enhancer function

Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.