Accelerated genome engineering through multiplexing

Abstract

Throughout the biological sciences, the past fifteen years have seen a push towards the analysis and engineering of biological systems at the organism level. Given the complexity of even the simplest organisms, though, to elicit a phenotype of interest often requires genotypic manipulation of several loci. By traditional means, sequential editing of genomic targets requires a significant investment of time and labor, as the desired editing event typically occurs at a very low frequency against an overwhelming unedited background. In recent years, the development of a suite of new techniques has greatly increased editing efficiency, opening up the possibility for multiple editing events to occur in parallel. Termed as multiplexed genome engineering, this approach to genome editing has greatly expanded the scope of possible genome manipulations in diverse hosts, ranging from bacteria to human cells. The enabling technologies for multiplexed genome engineering include oligonucleotide-based and nuclease-based methodologies, and their application has led to the great breadth of successful examples described in this review. While many technical challenges remain, there also exists a multiplicity of opportunities in this rapidly expanding field.

Orthogonal Genetic Regulation in Human Cells Using Chemically Induced CRISPR/Cas9 Activators

“…A significant advantage of using dCas9 as DNA binding domain is the great ease of multiplexing. 21 By simply coexpressing multiple gRNAs targeting multiple genes, dCas9 activators can be recruited to multiple loci across the genome and activate expression of multiple genes simultaneously. This is particularly important in applications where multiple genes have to be activated to carry out a certain biological process.…”

Potential Biotechnological Strategies for the Cleanup of Heavy Metals and Metalloids

“…A more recent study reported improved heavy metal biosorption capacities for E. coli cells engineered with mice MT1, demonstrating the potential of the genetic engineering approach in developing organisms with tailored and improved biosorption capacities ( Almaguer-Cantú et al, 2011 ). The introduction of modern research technologies in genomics such as next generation sequencing ( El-Metwally et al, 2014 ) and high throughput genome editing techniques ( Bao et al, 2016 ) allowed the study of organisms with potential biosorption capacities that are expected to have several bioremediation applications in future.…”

Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system

“…The clustered regularly interspaced short palindromic repeats (CRISPR) system has been recently developed for multiplex genome engineering in nearly all kingdoms of life 7 – 10 . In this modular and highly efficient system, a guide RNA (gRNA) recruits a CRISPR nuclease to a specific region of the genome to create in a double strand break.…”