A single CRISPR base editor to induce simultaneous C-to-T and A-to-G mutations

Abstract

While several Cas9-derived base editors have been developed to induce either C-to-T or A-to-G 1 mutation at target genomic sites, the possible genome editing space when using the current base editors 2 remains limited. Here, we present a novel base editor, Target-ACE, which integrates the abilities of both 3 of the previously developed C-to-T and A-to-G base editors by fusing an activation-induced cytidine 4 deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired 5Streptococcus pyogenes Cas9. In mammalian cells, Target-ACE enabled heterologous editing of multiple 6 bases in a small sequence window of target sites with increased efficiency compared with a mixture of 7 two relevant base editor enzymes, each of which may block the same target DNA molecule from the 8 other. Furthermore, by modeling editing patterns using deep sequencing data, the editing spectra of 9Target-ACE and other base editors were simulated across the human genome, demonstrating the 10

Multiple gene substitution by Target-AID base-editing technology in tomato

“…Another BE technique has been developed based adenosine deaminase (ADA) 5 but has only been tested in bacteria. Recently, the CDA and ADA technologies were fused in a single plasmid to form a protein complex that is able to specifically modify nucleotides 6 . Such technologies have been studied in organisms such as bacteria 3,6 , animals [7][8][9] and plants 5,[10][11][12] , with a variable range of efficiency.…”

CRISPR Start-Loss: A Novel and Practical Alternative for Gene Silencing through Base-Editing-Induced Start Codon Mutations

“…We supposed that non-ATG-initiated translation is after all a rare occurrence and it does not hinder CRISPR-SL from inactivating genes. To eliminate the possibility of initiating translation using a non-start codon, recently developed Target-ACE, a novel BE that tethers activation-induced cytidine deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired SpCas9, 64 can be used. With Target-ACE, CRISPR-SL could disrupt T and G simultaneously, converting ATG into ACA, a non-start codon that has not been proven to enable translation, with only one sgRNA.…”

CRISPR-Mediated Engineering across the Central Dogma in Plant Biology for Basic Research and Crop Improvement

“…Integration of emerging CRISPR toolkits with non-tissue culture approaches, such as nanoparticles (Doyle et al, 2019), speed breeding (Watson et al, 2018;Hickey et al, 2019), or de novo meristem induction (Maher et al, 2020), promises rapid gene identification in crops with complex genomes. The CRISPR tools designed for directed evolution include EvolvR (Halperin et al, 2018), Target-G (Nishida and Kondo, 2017), and dual BEs (Sakata et al, 2019;Gr€ unewald et al, 2020;Zhang et al, 2020). Prime editor (PE), one of the latest additions to the CRISPR toolset, performs RNA template-based DNA modification using an engineered reverse transcriptase (Anzalone et al, 2019).…”