Targeted gene repair

This protocol describes prime editing (PE) and twinPE experiments as well as the design and optimization of pegRNAs. The authors provide guidelines for selecting the proper PE system for a given application and how to perform PE in mammalian cells.

Preliminary results from a phase 1/2 trial with 18-month follow-up show that transplantation of CRISPR–Cas9 BCL11A-edited autologous hematopoietic cells in two children with β-thalassemia was safe and achieved transfusion independence.

The identification of events occurring at target sites is critical to determine the safety of CRISPRbased DNA editing tools. Here, the authors show that LINE-1 retrotranspositions can occur frequently at canonical CRISPR/Cas9 editing sites, but are rare with prime editors and base editors.

Healthy individuals have antibodies and T cells that are reactive to the Cas13d protein from the bovine bacteria Ruminococcus flavefaciens, which may have implications for clinical testing of CRISPR–Cas13 gene editing approaches.