Boosting targeted genome editing using the hei-tag

Abstract

Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straight-forward purification. Immediate activity is usually granted by administration of pre-assembled protein/RNA complexes. We present the 'hei-tag (high efficiency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.

De novo PAM generation to reach initially inaccessible target sites for base editing

“…To validate the efficiency of a knockout via inception, we targeted the well-described oculocutaneous albinism 2 ( oca2 ) gene responsible for the pigmentation of the retinal pigmented epithelium (RPE) in the Japanese rice fish medaka ( Oryzias latipes ) ( Cornean et al, 2022 ; Lischik et al, 2019 ). The loss of pigmentation depends on bi-allelic editing of the oca2 gene, which we use as a proxy to determine knockout efficiency via an established analysis pipeline ( Thumberger et al, 2022 ). Using base editors, we recently demonstrated that, in oca2 , non-synonymous changes of threonine 332 (T332), as well as the introduction of a PTC (glutamine>PTC, Q333*) resulted in substantial loss of pigmentation ( Cornean et al, 2022 ).…”

The inceptionist’s guide to base editing – de novo PAM generation to reach initially inaccessible target-sites

“…To address the efficiency of a knock-out via inception, we targeted the well described oculocutaneous albinism 2 ( oca2 ) gene responsible for the pigmentation of the retinal pigmented epithelium (RPE) in the Japanese rice fish medaka ( Oryzias latipes ) (Cornean et al, 2022, Lischik et al, 2019). The loss of pigmentation depends on bi-allelic editing of oca2 which we use as proxy to determine the knock-out efficiency via an established analysis pipeline (Thumberger et al, 2022). While the de novo PAM generation depends on A-to-G substitution, the subsequent PTC is introduced by C-to-T editing (Fig.…”

“…For analysis of oca2 knock-outs, the embryos were fixed 4.5 days post fertilization (dpf) (Iwamatsu, 2004) in 4 % paraformaldehyde in 1x PBS (137 mM NaCl; 2.7 mM KCl; 240 mg/l KH2PO4; 1.44 g/l Na2HPO4). Images of eyes were acquired with the ACQUIFER Imaging Machine (DITABIS AG, Pforzheim, Germany) and the mean grey value per eye was quantified as previously described (Thumberger et al, 2022). Embryos injected with guide RNAs targeting rx3 and kcnh6a were imaged 4 dpf or 9 dpf with a Nikon digital DS-Ri1 camera mounted onto a Nikon Microscope SMZ18 equipped with the Nikon Software NIS-Elements F version 4.0.…”

“…For analysis of oca2 knock-outs, the embryos were fixed 4.5 days post fertilization (dpf) (Iwamatsu, 2004) in 4 % paraformaldehyde in 1x PBS (137 mM NaCl; 2.7 mM KCl; 240 mg/l KH2PO4; 1.44 g/l Na2HPO4). Images of eyes were acquired with the ACQUIFER Imaging Machine (DITABIS AG, Pforzheim, Germany) and the mean grey value per eye was quantified as previously described (Thumberger et al, 2022). For rx3-inception two pools with four and five phenotypic editants were genotyped, for the rx3-newPAM and rx3-inception control injection one pool of five randomly picked embryos were genotyped for each injection mix.…”

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

“…Over 70% of human genes have at least one zebrafish orthologue [ 36 ], with the Xenopus genome including orthologs of ~80% of human disease genes [ 37 ]. More recently, methods to increase the genome editing efficiency of the Clustered Regularly Interspaced Short Palindromic Repeat– (CRISPR–) Cas9 system in zebrafish [ 38 , 39 , 40 , 41 , 42 , 43 ] and Xenopus [ 44 , 45 , 46 , 47 , 48 ] have led to new human disease models.…”