Biology subjects

Snetkova, V.

Publications and source records attributed to Snetkova, V..

3 recordsLinked to original sources

bioRxiv2024

Degron-modified Cas12a enhances single-cell CRISPR screening

Single-cell CRISPR (Perturb-seq) screens have primarily relied on Cas9 whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12as guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre-crRNA sequence recovery. By developing modified pre-crRNA expression vectors and a degron-based Cas12a system, we overcome the self-processing constraint, allowing for accurate detection of pre-crRNAs at the single-cell level, thus greatly expanding possibilities for future Perturb-seq efforts.

molecular biology↗

bioRxiv2024

Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening

Cas12a is a gene-editing tool that simplifies multiplexed gene targeting through its RNase activity, enabling maturation of individual crRNAs from a pre-crRNA-encoding RNA. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene-editing in cells from enAsCas12aKI mice. To test in vivo activity, we transduced haematopoietic stem cells from E-MycT/+;enAsCas12aKI/+animals with Trp53-targeting pre-crRNAs followed by transplantation into irradiated recipient animals. Tumour development was accelerated and TRP53 protein lost. We generated compact, genome-wide Cas12a knockout libraries targeting each gene with four guide RNAs encoded on two (Menuetto) or one (Scherzo) vector. Introducing these libraries into E-MycT/+;enAsCas12aKI/+lymphoma cells followed by treatment with an MCL-1 inhibitor (S63845) or TRP53-inducer (nutlin-3a) identified known and novel drug resistance genes. Finally, we demonstrate simultaneous gene knockouts (Trp53 or combined Bax/Bak) and activation (Cd19) in primary T cells and mouse dermal fibroblasts from crosses of our enAsCas12a and CRISPR activation models (dCas9a-SAM). Our enAsCas12a mouse model and accompanying libraries enhance genome engineering capabilities and complements current CRISPR technologies.

molecular biology↗

bioRxiv2022

Widespread Increase in Enhancer-Promoter Interactions during Developmental Enhancer Activation in Mammals

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here, we investigate the 3D conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. 61% of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general significance of enhancer- promoter physical proximity for developmental gene activation in mammals.

genomics↗