Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing

Abstract

The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.

Subunits of an E3 Ligase Complex as Degrons for Efficient Degradation of Cytosolic, Nuclear, and Membrane Proteins

“…To ensure that the substrate is connected to degrons, we used designed coiled-coil (CC) heterodimers. CC heterodimers have been previously used to build complex modular 3D structures , and logic circuits , and to regulate cellular processes. − They can be designed to be highly orthogonal to other CC pairs and natural proteins and have a tunable affinity between partner peptides . We prepared genetic fusions of all degrons with a P4 CC-forming peptide and luciferase substrate with a complementary P3 peptide (Figure a).…”

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

“…m, Schematic of different CE1 architectures tested. CC, coiled-coil domains N5/N6 64,65 ; EcKlenow, Klenow fragment from E.coli DNA polymerase I (D355A, D357A); Phi29, DNA polymerase from bacteriophage <29; Phi29 (D169A), 3’-5’ exonuclease-deficient Phi29 DNA polymerase; ePhi29, engineered thermostable Phi29 DNA polymerase (M8R, V51A, M97T, G197D, E221K, Q497P, K512E, F526L); ePhi29 (D169), 3’-5’ exonuclease-deficient ePhi29 (D169A, M8R, V51A, M97T, G197D, E221K, Q497P, K512E, F526L); eB103, engineered thermostable B103 DNA polymerase (a Phi29 ortholog) (H73R, A147K, R221Y, A318G, M339L, E359D, K372E, F383L, D384N, A503M, I511V, R544K, T550K). n-p, Percentage of sequencing reads with edits in experiments targeting ACTB , PRNP and DNMT1 ( panels n-p , respectively) when using CE1.n2 constructs encoding different DNA-dependent polymerases and different construct architectures.…”

“…4c,d ), we recognized that our previous N-terminal fusion could be detrimental to polymerization activity 66–68 , and that Phi29 is optimally active at 30 °C rather than 37 °C 66,67 . To explore whether Phi29 or similar polymerases could support click editing, we tested wild-type Phi29, an engineered thermostable Phi29 (ePhi29) 69 , and an engineered thermostable Phi29 ortholog (eB103) 70 in fused, unfused, or polymerase recruited CE architectures (the latter via N5/N6 coiled-coil domains 64,65 ) ( Fig. 4m ).…”

Coiled Coils as Versatile Modules for Mammalian Cell Regulation

“…Further expanding the versatility of coiled coil peptides, Lainšček et al [38] designed an enhanced gene editing method by recruiting exonuclease to Cas9/gRNA via coiled coil interaction, termed CCExo (Figure 2g). This method robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, decreasing the re-ligation of the non-edited DNA.…”