Jazbec, E. | Search bioRxiv

bioRxiv2024

Rational Design of Enhanced Nme2Cas9 and Nme2SmuCas9 Nucleases and Base Editors

CRISPR-Cas genome editing tools enable precise, RNA-guided modification of genomes within living cells. The most clinically advanced genome editors are Cas9 nucleases, but many nuclease technologies provide only limited control over genome editing outcomes. Adenine base editors (ABEs) and cytosine base editors (CBEs) enable precise and efficient nucleotide conversions of A:T-to-G:C and C:G-to-T:A base pairs, respectively. Therapeutic use of base editors (BEs) provides an avenue to correct approximately 30% of human pathogenic variants. Nonetheless, factors such as protospacer adjacent motif (PAM) availability, accuracy, product purity, and delivery limit the full therapeutic potential of BEs. We previously developed Nme2Cas9 and its BE derivatives, including ABEs compatible with single adeno-associated virus (AAV) vector delivery, in part to enable editing near N4CC PAMs. Further engineering yielded domain-inlaid BEs with enhanced activity, as well as Nme2Cas9/SmuCas9 chimeras that target single-cytidine (N4C) PAMs. Here we further enhance Nme2Cas9 and Nme2SmuCas9 editing effectors for improved efficiency and vector compatibility through site-directed mutagenesis and deaminase linker optimization. Finally, we define the editing and specificity profiles of the resulting variants by using paired guide-target libraries.

molecular biology↗

bioRxiv2024

PRC1.6 localizes on chromatin with the human silencing hub (HUSH) complex for promoter-specific silencing

An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6.

microbiology↗