Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases

Abstract

Genome editing technologies that install diverse edits can widely enable genetic studies and new therapeutics. Here we develop click editing, a genome writing platform that couples the advantageous properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of edits including substitutions, insertions, and deletions. Click editors (CEs) leverage the 'click'-like bioconjugation ability of HUH endonucleases (HUHes) with single stranded DNA substrates to covalently tether 'click DNA' (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs (DNA polymerase and HUHe orthologs, architectural modifications, etc.) and their clkDNAs (template configurations, repair evading substitutions, etc.), we demonstrate the ability to install precise genome edits with minimal indels and no unwanted byproduct insertions. Since clkDNAs can be ordered as simple DNA oligonucleotides for cents per base, it is possible to screen many different clkDNA parameters rapidly and inexpensively to maximize edit efficiency. Together, click editing is a precise and highly versatile platform for modifying genomes with a simple workflow and broad utility across diverse biological applications.

Harnessing RNA-based DNA repair pathways for targeted gene editing

“…The high efficiency of rep-editing derives from the co-localization of the RNA repair template and DSB by genetically encoding the repair template and gRNA as a single unit. This strategy offers advantages in terms of simplicity and delivery over other methodologies that co-localize a DNA repair template and Cas9 8, 48, 49 . Rep-editing is also differentiated from size-constrained or multi-component Cas9-editing strategies in a number of ways.…”

“…However, a major challenge in the gene-editing field is the heterogeneous nature of repair events that impact the efficiency, fidelity, and spectrum of editing outcomes 5–7 . Recently developed strategies to enhance editing include Cas9-fusion proteins with added biochemical activities 8–10 , co-delivery of exogenous DNA polymerases or single-strand DNA repair templates 8, 11 , and knockdown of competing DNA repair pathways or over expression of repair factors 12–15 . These strategies come at the cost of increasing the number, size, complexity and potential toxicity of components that need to be delivered to cells.…”

DNA on the move: mechanisms, functions and applications of transposable elements

“…In general, introduction of genomic DSBs can lead to detrimental rearrangements as a consequence of both on‐target and off‐target chromosomal DNA breakage events [ 46 , 47 , 48 ]. Fusion strategies of nickase Cas9 to deaminases (base editing [ 49 , 50 ]), reverse transcriptases (PRIME editing [ 42 ]), non‐LTR retrotransposon enzymes [ 51 ] or DNA polymerases (click editing [ 52 , 53 ]) do not rely on the introduction of double‐stranded breaks any longer but are still limited in the size of the DNA sequences to be inserted. Hence, major limitations are still in place to fully revolutionize the field of genome engineering using TEs and CRISPR‐Cas systems.…”

HUHgle: An Interactive Substrate Design Tool for Covalent Protein-ssDNA Labeling Using HUH-tags

“…They have been used to tether DNA templates to Cas9 in order to improve homology directed repair efficiency and gene knock in [12][13][14]. Additionally, HUH-tags play a critical role in 'click editing' [15], an emerging alternative to prime editing [16], in which an HUH-endonuclease covalently tethers a ssDNA template encoding user-specified mutations to a Cas9-Klenow fragment fusion for site-specific rewriting of genetic information. Beyond genome engineering applications, HUH-tags have been utilized in a user-friendly molecular tension sensing tool called RAD-TGTs ("Rupture And Deliver Tension Gauge Tethers") [17], which provide a simple platform for characterizing cellular tension profiles.…”