Multiplex base editing to convert TAG into TAA codons in the human genome

Abstract

Whole-genome recoding has been shown to enable nonstandard amino acids, biocontainment and viral resistance in bacteria. Here we take the first steps to extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and highly multiplex editing in mammalian cells.

Cryptography in the DNA of living cells enabled by multi-site base editing

“…Corresponding fasta sequences were extracted and a custom Python script was used to design gRNAs as follows: The nucleotide sequence and its reverse complement were queried for 23 nucleotide sequences that have base C at positions 4-8 and bases NGG at positions [21][22][23] where N can be any of the four bases, corresponding to the PAM site requirement for SpCas9 which of AncBE4max. Sequences were further filtered to exclude guides with homopolymer stretches of four or more nucleotides and a G/C content of lower than 30%.…”

“…for studying genetic interactions or long-range regulation of gene expression) as well as for biotechnology (e.g. for metabolic engineering), methods to introduce multiple edits remain laborious: Generating cells with multiple edits requires sequential editing cycles and isolating edited cells for subsequent editing rounds or extensive screening of edited clones 22 . To date, efforts to address this limitation have focused on using multi-gRNA arrays, frequently coupled with antibiotic selection 23,24 .…”

Strategies for precise gene edits in mammalian cells

“…showed base editing (TAG to TAA) of 33 target sites (out of the 47) via a single transfection. 85 Several other studies improved base editing by narrowing the editing window, enhancing DNA specificity, and developing different PAM compatibilities, using a different variant of APOBEC1 and small molecule dependence. 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 …”