A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

McGlincy, Nicholas J.; Meacham, Zuriah A.; Reynaud, Kendra; Muller, Ryan; Baum, Rachel; Ingolia, Nicholas T.

doi:10.1186/s12864-021-07518-0

Cited by 30 publications

(19 citation statements)

References 49 publications

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“…Between 0.5-10 μ g of purified genomic DNA from each library sample was sheared into ∼350 nucleotide length fragments by sonication for 10 min on ice using a Diagenode Bioruptor. Sheared gDNA was then in vitro transcribed into RNA (denoted gRNA below and in analysis code) starting from the T7 promoter region in the insert cassette, similar to previous approaches ( 85, 86 ), using a HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S). Transcribed gRNA was treated with DNase I (NEB M0303S) and cleaned using an RNA Clean and Concentrator kit (Zymo R1013).…”

Section: Materials and Methods Plasmid Constructionmentioning

confidence: 99%

A Nascent Peptide Code for Translational Control of mRNA Stability in Human Cells

Burke¹,

Park²,

Subramaniam³

2021

Preprint

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Stability of eukaryotic mRNAs is associated with their codon, amino acid, and GC content. Yet, coding sequence motifs that predictably alter mRNA stability in human cells remain poorly defined. Here, we develop a massively parallel assay to measure mRNA effects of thousands of synthetic and endogenous coding sequence motifs in human cells. We identify several families of simple dipeptide repeats whose translation triggers acute mRNA instability. Rather than individual amino acids, specific combinations of bulky and positively charged amino acids are critical for the destabilizing effects of dipeptide repeats. Remarkably, dipeptide sequences that form extended β strands in silico and in vitro drive ribosome stalling and mRNA instability in vivo. The resulting nascent peptide code underlies ribosome stalling and mRNA-destabilizing effects of hundreds of endogenous peptide sequences in the human proteome. Our work reveals an intrinsic role for the ribosome as a selectivity filter against the synthesis of bulky and aggregation-prone peptides.

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Section: Materials and Methods Plasmid Constructionmentioning

confidence: 99%

A Nascent Peptide Code for Translational Control of mRNA Stability in Human Cells

Burke¹,

Park²,

Subramaniam³

2021

Preprint

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“…McGlincy et al showed a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes, which providing a strategy for genome-wide CRISPR interference screening in budding yeast [92]. Furthermore, a short communication introduced a GDi-CRISPR system (gene drive delta site integration system by the CRISPR system) for multi-copy integration in S. cerevisiae, which holds great promising for advancing the development of S. cerevisiae multi-copy integration tools [93].…”

Section: Synthetic Genomics and The Use Of Crispr Technology In Synthetic Genomics Of S Cerevisiaementioning

confidence: 99%

Applications of CRISPR/Cas Technology to Research the Synthetic Genomics of Yeast

Lin¹,

Wang²,

Deng³

et al. 2022

Synthetic Genomics - From BioBricks to Synthetic Genomes

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The whole genome projects open the prelude to the diversity and complexity of biological genome by generating immense data. For the sake of exploring the riddle of the genome, scientists around the world have dedicated themselves in annotating for these massive data. However, searching for the exact and valuable information is like looking for a needle in a haystack. Advances in gene editing technology have allowed researchers to precisely manipulate the targeted functional genes in the genome by the state-of-the-art gene-editing tools, so as to facilitate the studies involving the fields of biology, agriculture, food industry, medicine, environment and healthcare in a more convenient way. As a sort of pioneer editing devices, the CRISPR/Cas systems having various versatile homologs and variants, now are rapidly giving impetus to the development of synthetic genomics and synthetic biology. Firstly, in the chapter, we will present the classification, structural and functional diversity of CRISPR/Cas systems. Then we will emphasize the applications in synthetic genome of yeast (Saccharomyces cerevisiae) using CRISPR/Cas technology based on year order. Finally, the summary and prospection of synthetic genomics as well as synthetic biotechnology based on CRISPR/Cas systems and their further utilizations in yeast are narrated.

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“…The arrival of CRISPR-Cas9 and related genome editing tools [394,395] is well enough known as not to need detailed review (and many are available, e.g. [369,[396][397][398][399][400][401][402][403][404][405][406][407][408][409][410]).…”

Section: Crispr-cas-based Genome Engineeringmentioning

confidence: 99%

Intelligent host engineering for metabolic flux optimisation in biotechnology

Munro

Kell

2021

Biochemical Journal

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Optimising the function of a protein of length N amino acids by directed evolution involves navigating a ‘search space’ of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is ‘making such biology predictable’. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing kcat in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering.

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A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

Cited by 30 publications

References 49 publications

A Nascent Peptide Code for Translational Control of mRNA Stability in Human Cells

A Nascent Peptide Code for Translational Control of mRNA Stability in Human Cells

Applications of CRISPR/Cas Technology to Research the Synthetic Genomics of Yeast

Intelligent host engineering for metabolic flux optimisation in biotechnology

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