Biology subjects

Rozhdestvensky, T. S.

Publications and source records attributed to Rozhdestvensky, T. S..

3 recordsLinked to original sources

bioRxiv2025

CRISPR-Cas9 HDR Optimization: RAD52, Denatured and 5'-Modified DNA Templates in Knock-In Mice Generation

AbstractCRISPR/Cas9-mediated genome editing is a powerful tool for producing animal models of human diseases. However, it often encounters challenges related to low efficiency of donor DNA templates insertion through homology-directed repair (HDR) pathway or unwanted insertions and/or multiplications. Here, we present findings from multiple targeting experiments aimed at generating a Nup93 conditional knockout (cKO) mouse model. Injection of CRISPR/Cas9 components into over two thousand zygotes, resulted in 270 founder animals. Our study revealed various obstacles associated with the use of single-stranded (ssDNA) and double-stranded DNA (dsDNA) templates during cKO generation, highlighting the critical role of denaturation of long 5-monophosphorylated dsDNA templates in enhancing precise genome editing and reducing template multiplications. Application of RAD52 protein increased HDR efficiency of ssDNA integration almost 4-fold, albeit with an associated increase in template multiplication. Targeting the antisense strand of DNA using two crRNAs demonstrated better efficacy in HDR-mediated precise genome editing when compared to targeting the sense or sense-antisense strands. In addition, the application of 5-end biotin-modified donor DNA resulted in up to a 8-fold increase in HDR-mediated single-copy template integration compered to unmodified dsDNA donor. Furthermore, application of 5-end C3 spacer modified template resulted in up to a 20-fold increase in correctly HDR modified mice independent from ssDNA or dsDNA template employment. This study underscores potential pitfalls in CRISPR/Cas9-mediated genome editing and offers simple practical solutions to refine this potent tool. These findings highlight various strategies to enhance CRISPR/Cas9 HDR efficiency, providing a framework for improving precision in the generation of conditional knockout models.

molecular biology↗

bioRxiv2024

Redirecting full-length FLT1 expression towards its soluble isoform promotes postischemic angiogenesis

Vascular endothelial growth factors and their tyrosine kinase receptors are key mediators of vasculogenesis and angiogenesis with FLT1 (VEGFR1) serving as a decoy receptor. A truncated mRNA transcript encoding soluble (s) FLT1 can be generated by premature cleavage and polyadenylation (APA). Although a shortening of transcripts is described in pathological settings, including heart diseases, the functional in vivo impact of FLT1 gene isoform generation and relevance for angiogenesis remain unknown. Here, we show that specific splice site mutations within Flt1 inhibit telescripting and activate APA in vivo to efficiently modulate gene isoform expression, inducing a complete loss of full-length (fl) Flt1 and a switch towards sFlt1 in mice. FLT1 is a high-affinity decoy receptor of VEGF limiting vessel overgrowth. We show that sFLT1 was sufficient for developmental vasculogenesis, whereas flFLT1 controlled ischemia-driven angiogenesis. Our results demonstrate that telescripting is essential in vivo for controlling Flt1 isoform expression and angiogenesis and can be harnessed to improve reparative revascularization. Furthermore, given the widespread abundance of APA signals, our approach may serve as a blueprint for studying telescripting and generating other truncated gene isoforms in vivo.

cell biology↗

bioRxiv2019

Pervasive head-to-tail insertions of DNA templates mask desired CRISPR/Cas9-mediated genome editing events

CRISPR/Cas9 mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knock-out mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or non-homologous end-joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis--in most cases--failed to identify such multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential, and offer practical solutions to correctly identify precisely edited chromosomes.

genetics↗