bims-cateng 2023-09-03 papers

Issue of 2023–09–03
two papers selected by
Chance Bowman, Dartmouth College

bioRxiv. 2023 Aug 18. pii: 2023.08.18.553620. [Epub ahead of print]

Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing.

Jacob Borrajo, Kamyab Javanmardi, James Griffin, Susan J St Martin, David Yao, Kaisle Hill, Paul C Blainey, Basem Al-Shayeb.

Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts in situ by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing.
One-sentence summary: CRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.

DOI: https://doi.org/10.1101/2023.08.18.553620

Lab Chip. 2023 Sep 01.

Ultrathin SU-8 membrane for highly efficient tunable cell patterning and massively parallel large biomolecular delivery.

Pallavi Shinde, Ashwini Shinde, Srabani Kar, Kavitha Illath, Moeto Nagai, Fan-Gang Tseng, Tuhin Subhra Santra.

Cell patterning is a powerful technique for the precise control and arrangement of cells, enabling detailed single-cell analysis with broad applications in therapeutics, diagnostics, and regenerative medicine. This study presents a novel and efficient technique that enables massively parallel high throughput cell patterning and precise delivery of small to large biomolecules into patterned cells. The innovative cell patterning device proposed in this study is a standalone, ultrathin 3D SU-8 micro-stencil membrane, with a thickness of 10 μm. It features an array of micro-holes ranging from 40 μm to 80 μm, spaced apart by 50 μm to 150 μm. By culturing cells on top of this SU-8 membrane, the technique achieves highly efficient cell patterns varying from single-cell to cell clusters on a Petri dish. Utilizing this technique, we have achieved a remarkable reproducible patterning efficiency for mouse fibroblast L929 (80.5%), human cervical SiHa (81%), and human neuroblastoma IMR32 (89.6%) with less than 1% defects in undesired areas. Single-cell patterning efficiency was observed to be highest at 75.8% for L929 cells. Additionally, we have demonstrated massively parallel high throughput uniform transfection of large biomolecules into live patterned cells by employing an array of titanium micro-rings (10 μm outer diameter, 3 μm inner diameter) activated through infrared light pulses. Successful delivery of a wide range of small to very large biomolecules, including propidium iodide (PI) dye (668.4 Da), dextran (3 kDa), siRNA (13.3 kDa), and β-galactosidase enzyme (465 kDa), was accomplished in cell patterns for various cancer cells. Notably, our platform achieved exceptional delivery efficiencies of 97% for small molecules like PI dye and 84% for the enzyme, with corresponding high cell viability of 100% and 90%, respectively. Furthermore, the compact and reusable SU-8-based membrane device facilitates highly efficient cell patterning, transfection, and cell viability, making it a promising tool for diagnostics and therapeutic applications.

DOI: https://doi.org/10.1039/d3lc00244f