🧬 PubMed RNA Editing Daily Digest

PIWI-interacting RNAs (piRNAs) guide PIWI endoribonucleases to destroy transposon transcripts, ensuring animal fertility. Here, we report the cryo-electron microscopy structure of the MIWI-pachytene piRNA complex isolated from mouse testes. The piRNA is held via non-specific charge-based interactions with the RNA backbone and by specific recognition of the first nucleotide uridine by residues within the MID and PIWI domains. The first six nucleotides of the guide RNA take up the A-form conformation to facilitate pairing with the target. The RNA channel is wider than that observed in insect PIWI proteins, explaining the tolerance for piRNA seed:target mismatches. The PIWI endonuclease domain is in an inactive "un-plugged" state, with the loop containing a catalytic residue (E671) requiring structural re-orientation for activity. Furthermore, the PIWI domain reveals a conserved pre-formed pocket that may serve to accommodate a conserved tryptophan from the interacting factor GTSF1 to promote small RNA-guided endoribonuclease activity.

Both RNA- and protein-based strategies have been developed to mitigate off-target cleavage by CRISPR-Cas9, yielding noncanonical guide RNAs (gRNAs) and Cas9 variants with enhanced gene-editing precision. However, the molecular mechanisms by which such PAM-distal alterations─remote from the nuclease centers─modulate Cas9 activity and specificity remain incompletely understood. Here, we performed

Muscle satellite cells are essential for skeletal muscle regeneration and represent an attractive therapeutic target for gene delivery in Duchenne muscular dystrophy (DMD). However, efficient in vivo transduction of these cells has remained challenging. Here, we demonstrate that lipid nanoparticle (LNP)-mediated delivery of Streptococcus pyogenes CRISPR-Cas9 mRNA and guide RNA (LNP-CRISPR) induces exon skipping in Pax7-positive satellite cells more efficiently than adeno-associated virus (AAV) vectors following intramuscular or intravenous administration in a DMD mouse model. Furthermore, unlike AAV-CRISPR, LNP-CRISPR-mediated genome editing showed greater resistance to repeated muscle injuries, indicating successful editing of regenerative satellite cells. These results highlight the potential of LNPs as a non-viral platform for durable genome editing in skeletal muscle and lay the foundation for developing safe and sustainable genome-editing therapies for DMD.

Genome and RNA editing modalities have revolutionized precision gene therapy, offering a safer alternative to traditional gene replacement approaches. Alpha-1 antitrypsin deficiency (AATD) is a compelling model for precision medicine because the disease mechanism is well defined-mutations in a single gene are responsible for both liver and lung pathology. In this review, we summarize the current preclinical and clinical efforts for AATD, with an emphasis on genome and RNA editing strategies.

CRISPR-Cas systems are transformative tools for gene editing that can be tuned or controlled by anti-CRISPRs (Acrs)-phage-derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that can inhibit biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful and rapid approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Here, using Leptotrichia buccalis CRISPR-Cas13a as a representative example, we demonstrate that Acrs designed using artificial intelligence (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13a nuclease activity. We present a comprehensive workflow for design validation and demonstrate AIcr functionality in controlling CRISPR-Cas13 activity in bacterial and human cells. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture and microbiology.