Overlapping genes in natural and engineered genomes

Abstract

Modern genome-scale methods that identify new genes, such as proteogenomics and ribosome profiling, have revealed, to the surprise of many, that overlap in genes, open reading frames and even coding sequences is widespread and functionally integrated into prokaryotic, eukaryotic and viral genomes. In parallel, the constraints that overlapping regions place on genome sequences and their evolution can be harnessed in bioengineering to build more robust synthetic strains and constructs. With a focus on overlapping protein-coding and RNA-coding genes, this Review examines their discovery, topology and biogenesis in the context of their genome biology. We highlight exciting new uses for sequence overlap to control translation, compress synthetic genetic constructs, and protect against mutation.

Marker-free coselection for successive rounds of prime editing in human cells

“…Although a more thorough characterisation will be required to fully establish its full potential, it is worth noting that ATP1A1 can be used as a selectable genomic safe harbor for targeted integration of transgenes via intron nesting 44 . Stable transgene expression was achieved with a near 1:1 correspondence between targeted integration and drug resistance, similar to observations made at the HBEGF locus 20 .…”

“…We then tested whether ATP1A1 could be used as a safe harbor for targeted integration of transgenes via intron nesting 44 . In our design, the outcome of HDR is the co-introduction of an ouabain resistant allele (T804N) and an expression cassette in antisense orientation within intron 17 to maintain functionality of both ATP1A1 and the transgene (Fig.…”

Spotlight on alternative frame coding: Two long overlapping genes in Pseudomonas aeruginosa are translated and under purifying selection

“…These findings support the hypothesis that OLGs are ubiquitous. Various aspects of OLGs, including potential applications in synthetic biology, have been discussed in a recent review ( Wright et al., 2021 ).…”

Creating De Novo Overlapped Genes

“…This is done through the construction of genetic circuits with natural or engineered genes controlled by regulatory elements [2]. To make the design of engineered genomes easier, most genome design approaches seek to refactor genomes to remove genetic overlaps and cryptic regulation [3][4][5][6][7][8], however this does not necessarily provide evolutionary stability to designs [9]. In fact, engineered genes and synthetic architectures often place a deleterious growth burden on the expression host [10][11][12].…”

“…A novel way to add genetic stability to engineered genomes is called Constraining Adaptive Mutations using Engineered Overlapping Sequences (CAMEOS) which seeks to emulate the condensed and overlapped coding sequence architecture found primarily in bacteriophage and bacteria [3,18,4,7,19]. This computational approach uses Hidden Markov Models (HMMs) and Random Markov Fields (MRFs) to determine protein residue diversity at a given position, and residue-residue contacts across the proteins, to generate overlapped coding sequences containing two proteins [20].…”