Best Practices for Precision in Genome Editing

A method called 𝐏𝐫𝐢𝐦𝐞 𝐀𝐬𝐬𝐞𝐦𝐛𝐥𝐲 (𝐏𝐀) - combined prime editing and Gibson assembly - enables scarless, efficient, and programmable insertion of large DNA fragments - all without double-strand breaks, recombinases, or plasmids. In this study by Liu et al. (2025) - PREPRINT - , PA leverages the programmable flaps of twin prime editing and matches them with linear DNA donors to support in vivo Gibson-like assembly. Using this approach, the team achieved precise insertions up to 11.3 kb, including full-length DMD, CD19 CAR, and reporter genes like GFP.   𝐏𝐫𝐢𝐦𝐞 𝐀𝐬𝐬𝐞𝐦𝐛𝐥𝐲 (𝐏𝐀) - Step-by-Step Mechanism: 1. Twin Prime Editing Setup - Two specially designed pegRNAs (called twinPE) guide a prime editor (a Cas9 nickase fused to a reverse transcriptase) to two nearby locations in the genome. - Each pegRNA creates a short single-stranded DNA “flap” (~30–50 nt) that becomes incorporated into the genome. 2. Designing the DNA Donor - A linear DNA fragment (dsDNA, 3'-overhang dsDNA, or ssDNA) is generated by PCR. - Its ends are designed to match the sequence of the flaps generated by the pegRNAs. 3. Flap Annealing and Insertion - The flaps from the genome anneal to the complementary ends of the donor DNA. - This brings the donor into position for in vivo assembly—similar to how Gibson Assembly works in vitro. 4. Cellular Polymerases Extend and Seal - Endogenous DNA polymerases likely fill in any gaps. - The donor becomes seamlessly incorporated into the genome, with precise junctions and no unwanted sequences ("scars"). 5. Optional Enhancement - Using a DNA-PK inhibitor (e.g., AZD-7648) further improves the precision and efficiency by reducing interference from unwanted DNA repair pathways like NHEJ.   Key Advantages: - No double-strand breaks = safer for therapeutic use. - No plasmid or integrase delivery needed. - Works with multi-part donors for complex insertions. - Can target clinically relevant genes like DMD and CD19 CAR.   Limitations & Future Work - In vivo delivery remains a challenge. - The exact DNA repair pathways involved remain unclear. - Further optimization could improve efficiency and reduce off-target effects. - Chemical modification of donors and donor tethering strategies may enhance outcomes. Summary Prime Assembly represents a powerful, modular, and efficient genome engineering platform that enables large and precise genomic insertions without the drawbacks of traditional DSB-based or integrase-based systems. Read here: https://lnkd.in/e_25YxfN   #GenomeEditing #PrimeEditing #SyntheticBiology #CRISPR #GeneTherapy #DNAAssembly #Biotech

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here the researchers improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. https://lnkd.in/g7_kh79V

I’m trying something new—highlighting research that I find particularly enjoyable or useful, especially on CRISPR, molecular/protein engineering, and cardiometabolic disease space. To kick things off, here are two new papers: one unveiling a clever single-cell off-target detection method and another exploring how CRISPR fusions that write DNA may globally affect DNA repair—an important and underexplored off-target topic. 1. A New Off-Target (OT) Identification Method by Lorenzini et al. (Preprint) I always appreciate some good ole method development, and this one is satisfying. They’ve taken a “GUIDE-seq” style off-target identification (oligo drop-in) and upgraded it by adding a barcoded T7 promoter as the oligo. Essentially, it's an updated version that can amplify OT signals for single-cell analysis. Feels useful for capturing rare off-target events in cell types that can incorporate an oligo at dsDNA breaks. Why I like it: Print your Off-Targets as RNA transcripts, both simple and—in hindsight—obvious! Check it out: https://lnkd.in/gQTu-4Bp 2. A Deeper Look at Prime Editors’ Reverse Transcriptase OTs (Zheng et al., Nature Biotechnology) This paper examines prime editors, Cas9 nickases fused to a reverse transcriptase (RT), and how they can override normal DNA repair processes —even beating endogenous repair proteins to the punch.  Prime editors are often billed as more precise than standard Cas9, yet many of us have wondered about the impact of an always-active RT on genome-wide changes—especially after Gurnewald et al. (2023) and Liu et al. (2022) showed prime editors function well even when RT isn’t fused to Cas9. Zheng et al. reveal that these non-endogenous RTs can cluster at DNA breaks (Cas9-induced or not) and alter repair outcomes—often writing in random nucleotides. Why I like it: This work demonstrates that prime editing may create yet-to-be-characterized global off-target effects, similar to recent base editing insights where a Deaminase-Cas9 fusion can modify R-loops or ssRNA without any guide homology. This is important! There may be a need to think more deeply about identifying and tracking the tricky, random, and hard-to-capture off-target/genotoxic impacts of Cas9 base and prime editors and, as they do here, engineer the CRISPR systems to limit these deleterious effects! Read it here: https://lnkd.in/ghjQXxQh