MCCas is established as a promising gene editing tool with enhanced ls-KI capacity and relies on the canonical HR pathway, as inhibition of key processes such as end resection and strand invasion abolished the enhancement.
Abstract
Homologous recombination (HR) mediated large-size fragment knock-in (ls-KI) remains inefficient in mammalian cells, even with the assistance of CRISPR/Cas9. We hypothesized that adding DNA helicase activity to CRISPR/Cas9 could enhance ls-KI efficiency. To test this, we fused MCM5, a subunit of the eukaryotic MCM2-7 helicase complex, to the N-terminus of spCas9. The resulting fusion protein, termed MCCas, markedly increased knock-in rates across multiple loci in different human cell lines, significantly outperforming spCas9. Remarkably, MCCas enabled efficient ls-KI with donor templates of up to 10 kb in size. MCCas mediated ls-KI, in comparison to those mediated by spCas9, is also associated with reduced frequencies of on-target and off-target insertion and deletion (indel) events. Mechanistic investigations revealed that MCCas-mediated ls-KI relies on the canonical HR pathway, as inhibition of key processes such as end resection and strand invasion abolished the enhancement. To further validate its application, we employed MCCas to knock in the human ACE2 (hACE2) coding sequence to the rabbit genome. Consistent with our findings in human cells, MCCas led to more than two-fold increase in ls-KI rates in rabbit embryos compared to spCas9. Collectively, our results establish MCCas as a promising gene editing tool with enhanced ls-KI capacity.
Five previously uncharacterized MG102-like Cas9d orthologs are identified that share the hallmark genomic, sequence, and structural features of type II-D Cas9 and establish compact MG102-like Cas9d orthologs as robust and specific genome editors and provide promising, single-AAV– compatible scaffolds for in vivo therapeutic genome editing.
Qiaochu Wang, Ahmed Saleh, G. S. Rao et al.· bioRxiv· 0 citations
In mammalian cells, DNA double-strand breaks (DSBs) are repaired by two competing pathways-homologous recombination (HR) and non-homologous end-joining (NHEJ)-that act on the same DNA ends. Downregulation of NHEJ has been shown to enhance HR-mediated repair. Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by progressive corneal opacity and vision loss in humans. More than 180 mutations in the CHST6 gene are linked to MCD, with over 70% occurring in exon 3, making it a promising target for genome editing. In this study, we performed in vitro editing of exon 3 of CHST6 using CRISPR/Cas9 in human embryonic kidney (HEK293) cells. To promote HR, the NHEJ genes XRCC6 and XRCC5, encoding KU70 and KU80, were knocked down individually or in combination. A homologous donor template was also introduced, and HR efficiency was assessed by Western blot analysis. Results demonstrated a significant increase in HR activity following downregulation of these NHEJ components, as indicated by elevated RAD51 expression. As proof of concept, partial restoration of CHST6 protein expression was observed in edited cells compared with CHST6 knockdown controls after suppression of XRCC6 and XRCC5 along with donor template delivery. These findings suggest that targeting NHEJ to enhance HR may represent a promising therapeutic strategy for MCD.
Aparajita Baruah, Tobias Wimmer, K. Stieger et al.· Experimental Eye Research· 0 citations
This study establishes a streamlined and highly efficient framework for precise insertional mutagenesis and double-mutant generation in Ulva, thereby expanding the genetic engineering toolkit for this macroalga.
K. Ichihara, Chikako Nagasato, T. Yamazaki et al.· Phycology Journal· 0 citations
The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation and makes biomolecular condensation a general principle for enhancing CRISPR gene regulation.
Aolin Li, Congcong Cao, Chunyan Yang et al.· Theranostics· 0 citations
