Biology subjects

Sanchez-Mejias, A.

Publications and source records attributed to Sanchez-Mejias, A..

4 recordsLinked to original sources

bioRxiv2022

Synergic homology directed recombination by PRDM9 meiotic factor

Genome editing requires precision to broadly move on to industrial and clinical applications. For this reason, homologous directed repair (HDR) is one of the preferred methods for small edits, other than knock-outs. However, HDR has low efficiency. Current investigations to enhance HDR have mainly gone in the direction of finding non-homologous end joining (NHEJ) inhibitors. NHEJ is crucial for cellular integrity, then the inhibition of this pathway is detrimental for the correct survival of living entities. In other studies, a second opportunity is given to HDR by targeting the byproducts of NHEJ, using an extra gRNA. In this study, we propose the use of a meiotic factor, PRDM9, to directly enhance homology recombination. Through the exploration of combinatorial factors and donor design, we have established an optimized protocol for HDR. PRDM9-Cas9 fusion combined with CtIP improves HDR/NHEJ ratio. In addition, we have validated this combinatorial approach for small edits through a traffic light reporter system, as well as for longer edits with a split-GFP reporter system.

synthetic biology↗

bioRxiv2022

CRISPR-Analytics (CRISPR-A): a platform for precise analytics and simulations for gene editing

Gene editing characterization with currently available tools does not always give precise relative proportions among the different types of gene edits present in an edited bulk of cells. We have developed CRISPR-Analytics, CRISPR-A, which is a comprehensive and versatile genome editing web application tool and a nextflow pipeline to give support to gene editing experimental design and analysis. CRISPR-A provides a robust gene editing analysis pipeline composed of data analysis tools and simulation. It achieves higher accuracy than current tools and expands the functionality. The analysis includes mock-based noise correction, spike-in calibrated amplification bias reduction, and advanced interactive graphics. This expanded robustness makes this tool ideal for analyzing highly sensitive cases such as clinical samples or experiments with low editing efficiencies. It also provides an assessment of experimental design through the simulation of gene editing results. Therefore, CRISPR-A is ideal to support multiple kinds of experiments such as double-stranded DNA break-based engineering, base editing (BE), primer editing (PE), and homology-directed repair (HDR), without the need of specifying the used experimental approach.

bioinformatics↗

bioRxiv2022

INSERT-seq enables high resolution mapping of genomically integrated DNA using nanopore sequencing

Comprehensive characterization of genome engineering with viral vectors, transposons, CRISPR/Cas mediated DNA integration and other DNA editors remains relevant for their development and safe use in human gene therapy. Currently, described methods for measuring DNA integration in edited cells rely on short read based technologies. Due to the repetitive nature of the human genome, short read based methods can potentially overlook insertion events in repetitive regions. We modelled the impact of read length in resolving insertion sites, which suggested a significant drop in insertion site detection with shorter read length. Based on that, we developed a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration in a genome. This method, called INSERT-seq, is capable of detecting events occurring at a frequency of up to 0.1%. INSERT-seq presents a complete handling of all insertions independently of repeat size. The experimental pipeline improves the number mappable insertions at repetitive regions by 7.3% and repeats larger than the long read sequencing size are processed computationally to perform a peak calling in a repeat database. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex-vivo and in-vivo samples.

genomics↗

bioRxiv2022

Evolution of CRISPR-associated Endonucleases as Inferred from Resurrected Proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 protein is an effector that plays a major role in a prokaryotic adaptive immune system, by which invading DNA can be targeted and cut for inactivation. The Cas9 endonuclease is directed to target sites by a guide RNA (gRNA) where Cas9 can recognize specific sequences (PAMs) in foreign DNA, which then serve as an anchoring point for cleavage of the adjacent RNA-matching DNA region. Although the CRISPR-Cas9 system has been widely studied and repurposed for diverse applications (notably, genome editing), its origin and evolution remain to be elucidated. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species as old as 2600 myr to the current day. Surprisingly, we demonstrate that these ancient forms were much more flexible in their PAM and gRNA scaffold requirements compared to modern day Cas9 enzymes. In addition, anCas portrays a gradual paleoenzymatic adaptation from nickase to double-strand break activity, suggesting a mechanism by which ancient CRISPR systems could propagate when harboring Cas enzymes with minimal PAMs. The oldest anCas also exhibit high levels of activity with ssDNA and ssRNA targets, resembling Cas nucleases in related system types. Finally, we illustrate editing activity of the anCas enzymes in human cells. The prediction and characterization of anCas proteins uncovers an unexpected evolutionary trajectory leading to ancient enzymes with extraordinary properties.

evolutionary biology↗