Rational engineering of minimally immunogenic nucleases for gene therapy.

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases, SaCas9 and AsCas12a.

Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system.

Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp.

Transgenic Targeting of Creates a Highly Efficient Constitutively Active Microglia Cre Line with Differentiated Specificity.

Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to genetically modify them with maximum completeness in a manner that distinguishes them from closely related cell types, such as monocytes. While currently available tamoxifen-inducible CreERT2 lines can achieve the differentiation from other cells, the field needs improved and publicly available constitutively active Cre lines, especially ones with favorable efficiency and specificity profiles for studies where high recombination efficiency is imperative and where tamoxifen administration is contraindicated.

Structural basis for pegRNA-guided reverse transcription by a prime editor.

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states.

Programmable RNA writing with trans-splicing.

RNA editing offers the opportunity to introduce either stable or transient modifications to nucleic acid sequence without permanent off-target effects, but installation of arbitrary edits into the transcriptome is currently infeasible. Here, we describe Programmable RNA Editing & Cleavage for Insertion, Substitution, and Erasure (PRECISE), a versatile RNA editing method for writing RNA of arbitrary length and sequence into existing pre-mRNAs via 5' or 3' trans-splicing. In trans-splicing, an exogenous template is introduced to compete with the endogenous pre-mRNA, allowing for replacement of upstream or downstream exon sequence.

Localization of Brain Signals by Alternating Projection.

A popular approach for modeling brain activity in MEG and EEG is based on a small set of current dipoles, where each dipole represents the combined activation of a local area of the brain. Here, we address the problem of multiple dipole localization with a novel solution called Alternating Projection (AP). The AP solution is based on minimizing the least-squares (LS) criterion by transforming the multi-dimensional optimization required for direct LS solution, to a sequential and iterative solution in which one source at a time is localized, while keeping the other sources fixed.

Disentangling Fact from Grid Cell Fiction in Trained Deep Path Integrators.

Work on deep learning-based models of grid cells suggests that grid cells generically and robustly arise from optimizing networks to path integrate, i.e., track one's spatial position by integrating self-velocity signals.

Chronic metabolic stress drives developmental programs and loss of tissue functions in non-transformed liver that mirror tumor states and stratify survival.

Under chronic stress, cells must balance competing demands between cellular survival and tissue function. In metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD/NASH), hepatocytes cooperate with structural and immune cells to perform crucial metabolic, synthetic, and detoxification functions despite nutrient imbalances. While prior work has emphasized stress-induced drivers of cell death, the dynamic adaptations of surviving cells and their functional repercussions remain unclear.

Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering.

Microbial systems underpin many biotechnologies, including CRISPR, but the exponential growth of sequence databases makes it difficult to find previously unidentified systems. In this work, we develop the fast locality-sensitive hashing-based clustering (FLSHclust) algorithm, which performs deep clustering on massive datasets in linearithmic time. We incorporated FLSHclust into a CRISPR discovery pipeline and identified 188 previously unreported CRISPR-linked gene modules, revealing many additional biochemical functions coupled to adaptive immunity.

Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses.

Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence.

Characterization and discrimination of spike protein in SARS-CoV-2 virus-like particles via surface-enhanced Raman spectroscopy.

Non-infectious virus-like particles (VLPs) are excellent structures for development of many biomedical applications such as drug delivery systems, vaccine production platforms, and detection techniques for infectious diseases including SARS-CoV-2 VLPs. The characterization of biochemical and biophysical properties of purified VLPs is crucial for development of detection methods and therapeutics. The presence of spike (S) protein in their structure is especially important since S protein induces immunological response.

Drug delivery systems for CRISPR-based genome editors.

CRISPR-based drugs can theoretically manipulate any genetic target. In practice, however, these drugs must enter the desired cell without eliciting an unwanted immune response, so a delivery system is often required. Here, we review drug delivery systems for CRISPR-based genome editors, focusing on adeno-associated viruses and lipid nanoparticles.

Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells.

Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses.

TnpB proteins are RNA-guided nucleases that are broadly associated with IS200/605 family transposons in prokaryotes. TnpB homologs, named Fanzors, have been detected in genomes of some eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. We searched genomes of diverse eukaryotes and their viruses for TnpB homologs and identified numerous putative RNA-guided nucleases that are often associated with various transposases, suggesting they are encoded in mobile genetic elements.

Fanzor is a eukaryotic programmable RNA-guided endonuclease.

RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage.

The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity.

TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using purified apo TnpB, we demonstrate the ability of TnpB to generate guide omegaRNA (ωRNA) from its own mRNA through 5' processing.

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox.

CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit.

Cryo-EM structure of the transposon-associated TnpB enzyme.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide.

Programmable protein delivery with a bacterial contractile injection system.

Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. One example, the extracellular contractile injection systems (eCISs), are syringe-like macromolecular complexes that inject protein payloads into eukaryotic cells by driving a spike through the cellular membrane. Recently, eCISs have been found to target mouse cells, raising the possibility that these systems could be harnessed for therapeutic protein delivery.

A transcription factor atlas of directed differentiation.

Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts.

Monosynaptic restriction of the anterograde herpes simplex virus strain H129 for neural circuit tracing.

Identification of synaptic partners is a fundamental task for systems neuroscience. To date, few reliable techniques exist for whole brain labeling of downstream synaptic partners in a cell-type-dependent and monosynaptic manner. Herein, we describe a novel monosynaptic anterograde tracing system based on the deletion of the gene UL6 from the genome of a cre-dependent version of the anterograde Herpes Simplex Virus 1 strain H129.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes.