Transposons and CRISPR: Rewiring Gene Editing

Abstract

CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposonassociated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.

Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles

“…11,13 Newer gene-editing strategies may require even more complex mixtures of additional proteins or large donor-DNA sequences, which require additional optimization. 14 There is, therefore, a significant need for modular, high-capacity, cargo-agnostic delivery systems for gene editing.…”

“…RNPs thus present challenges due to a more diverse charge distribution and Cas9 enzyme sensitivity to denaturation. It has been recently shown that LNP formulations should therefore be independently optimized for RNP delivery rather than directly using systems previously optimized for RNA delivery. , Newer gene-editing strategies may require even more complex mixtures of additional proteins or large donor-DNA sequences, which require additional optimization . There is, therefore, a significant need for modular, high-capacity, cargo-agnostic delivery systems for gene editing.…”

RNA-guided genome engineering: paradigm shift towards transposons

“…In 2017, the connection between Tn7-like transposons (See Glossary) and CRISPR is discovered [16], with subsequent observations revealing associations with various CRISPR types, including I-B, I-D, I-F, and V-K [17][18][19][20][21][22]. In 2019, the type V-K [17] and type I-F3 [18] CRISPRassociated transposons (termed CASTs) are first repurposed for DSB-free, RNA-guided DNA insertion into E. coli, which implicates the potentials of CASTs to obviate the requirement of DSB induction in CRISPR-Cas systems.…”

“…Several articles have comprehensively reviewed CASTs, either offering a general introduction [25][26][27][28] or delving into specific aspects like molecular mechanisms [20,21,29] and applications related to DSB-free DNA integration [28,30,31]. Yet OMEGA system is reviewed in only two papers [21,32] and several research spotlight articles [33][34][35].…”

Editorial: Overcoming genome editing challenges in plants: new tools and nanotechnologies

“…As a result, we have witnessed many breakthroughs, from the development of designer nucleases to their use in microbes, animals, humans, and agricultural plants ( Adli, 2018 ; Zhang et al, 2019 ; Anzalone et al, 2020 ; Li et al, 2023 ; Wang and Doudna, 2023 ). More recently, to improve the editing accuracy and precision and reduce dependence on the cell’s developmental state, new approaches, such as the OMEGA (obligate mobile element-guided activity), CAST (CRISPR-Cas-associated transposon), and INTEGRATE (insertion of transposable elements by guide RNA-assisted targeting), were developed and tested in different organisms ( Tenjo-Castaño et al, 2022 ). Further, CRISPR technology was deployed in imaging, diagnostics ( Wang and Doudna, 2023 ), and treatment of major human disorders ( Wang and Doudna, 2023 ).…”