Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

http://www.cell.com/article/S0092867421014847/pdf

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

Over the past decade, CRISPR has become as much a verb as it is an acronym, transforming biomedical research and providing entirely new approaches for dissecting all facets of cell biology. In cancer research, CRISPR and related tools have offered a window into previously intractable problems in our understanding of cancer genetics, the noncoding genome and tumour heterogeneity, and provided new insights into therapeutic vulnerabilities. Here, we review the progress made in the development of CRISPR systems as a tool to study cancer, and the emerging adaptation of these technologies to improve diagnosis and treatment. 

/pdf/crispr-in-cancer-biology-and-therapy-3f1wzmlu.pdf

CRISPR in cancer biology and therapy

The CRISPR-Cas toolbox and gene editing technologies

Therapeutic in vivo delivery of gene editing agents

Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3′ region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3′ terminus of pegRNAs that enhance their stability and prevent degradation of the 3′ extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3–4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3′ structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3′ motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations. Stabilizing pegRNAs with 3′ RNA structures increases prime editing efficiency.

James W. Nelson

Papers

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Engineered pegRNAs improve prime editing efficiency