Evidence map›Paper›PMID 36404524›Full record

ArticleMetabolic engineering2023

Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria.

Jian Wang, Yuxi Teng, Xinyu Gong, Jianli Zhang, Yifei Wu, Lei Lou, Michelle Li, Zhong-Ru Xie, Yajun Yan

Open access · greenAbstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Metabolic engineering, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.

0numbers the graph read from it
0cells of the map it votes in
6citing papers in PubMed
1.3field-weighted citation impact, top 20% of its field
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

6 citing papers in PubMed, 16 citations in OpenAlex.

  1. Review
  2. Article
  3. Article
  4. Review
  5. Review
  6. Engineering aACS synthetic biology · 2023
    Article
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

9 authors at 1 institution in 1 country.

Jian WangSchool of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
Yuxi TengSchool of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
Xinyu GongSchool of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
Jianli ZhangSchool of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
Yifei WuSchool of Electrical and Computer Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
Lei LouSchool of Electrical and Computer Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
Michelle LiNorth Oconee High School, Bogart, GA, 30622, USA.
Zhong-Ru XieSchool of Electrical and Computer Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.
Yajun YanSchool of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA. Electronic address: yajunyan@uga.edu.
University of Georgia · US

Funding

Engineering Dynamic Control of Natural Product Biosynthesis in BacteriaR35GM128620 · NIGMS · UNIVERSITY OF GEORGIA · PI Yajun Yan · 2018 to 2026
$3.0M
NIGMS NIH HHS R35 GM128620
6 · The paper itself

Abstract

The RNA-guided Cas9s serve as powerful tools for programmable gene editing and regulation; their targeting scopes and efficacies, however, are always constrained by the PAM sequence stringency. Most Streptococci Cas9s, including the prototype SpCas9 from S. pyogenes, specifically recognize a canonical NGG PAM via a conserved RxR PAM-binding motif within the PAM-interaction (PI) domain. Here, SpCas9-based mining unveils three distinct and rarely presented PAM-binding motifs (QxxxR, QxQ and RxQ) among Streptococci Cas9 orthologs. With the catalytically-dead QxxxR-containing SedCas9 from S. equinus, we dissect its NAG PAM specificity and elucidate its underlying recognition mechanism via computational prediction and mutagenesis analysis. Replacing the SedCas9 PI domain with alternate PAM-binding motifs rewires its PAM specificity to NGG or NAA. Moreover, a semi-rational design with minimal mutation creates a SedCas9-NQ variant showing robust activity towards expanded NNG and NAA PAMs, based upon which we engineered a compact ω-SedCas9-NQ transcriptional regulator for PAM-directed bifunctional and titratable gene control. The ω-SedCas9-NQ mediated metabolic reprogramming of endogenous genes in Escherichia coli affords a 2.6-fold increase of 4-hydroxycoumarin production. This work reveals new Cas9 scaffolds with distinct PAM-binding motifs for PAM relaxation and creates a new PAM-diverse Cas9 variant for versatile gene control in bacteria.

Indexed as

CRISPR-Associated Protein 9CRISPR-Cas SystemsGene EditingMutagenesisStreptococcus pyogenesCRISPR-Associated Protein 94-hydroxycoumarinCas9 engineeringCRISPR interference and activationGene controlMetabolic engineering

Identifiers

PMID36404524
PMCPMC10947553
OpenAlexW4307812286

What OpenQuestion holds

Textmetadata
LicenceTDM
Read underepoch 390

Registered trials

None linked

Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.