Incorporation of a Synthetic Amino Acid into dCas9 Improves Control of Gene Silencing

Koopal, Balwina; Kruis, Aleksander J.; Claassens, Nico J.; Nóbrega, Franklin L.; Oost, John van der

doi:10.1021/acssynbio.8b00347

Cited by 13 publications

(11 citation statements)

References 54 publications

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“…Therefore, a recent study attempted to introduce a TAG stop codon in the dcas9 gene and a tRNA CUA that recognizes the UAG codon as well as an aminoacyl-synthetase which attaches the tRNA with a synthetic amino acid, l-biphenylalanine (BipA) into E. coli. [191] Typically, the full translation of dCas9 is completely abolished in the absence of BipA. When BipA was added, the translation of dCas9 continued to form a functional full-length dCas9.…”

Section: Constructing Gene Knockdown Librarymentioning

confidence: 99%

“…Thus, any leakage level expression of dCas9 and sgRNA would lead to a potent transcriptional inhibition on the target gene, even when the inducer of the promoter was not added. Therefore, a recent study attempted to introduce a TAG stop codon in the dcas9 gene and a tRNA CUA that recognizes the UAG codon as well as an aminoacyl‐synthetase which attaches the tRNA with a synthetic amino acid, l ‐biphenylalanine (BipA) into E. coli . Typically, the full translation of dCas9 is completely abolished in the absence of BipA.…”

Section: Crispr‐dcas Tools For Targeted Gene Regulationmentioning

confidence: 99%

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Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

Wang

Zhang

et al. 2019

Small Methods

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Gene editing is a fundamental technique for the identification of the linkages from genetic determinants to significant biological phenotypes, and the engineering of industrial strains to produce fine chemicals. Recently, the primitive bacterial immunity systems, clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas systems, have been engineered as programmable nucleases to deliver double‐strand breaks (DSBs) at user‐defined loci. The DSB is repaired via either homology‐direct repair or the non‐homologous end joining pathway, generating a mutant in various organisms, and leading a revolution in the field of gene editing. This paper reviews the structural and molecular details of CRISPR‐Cas systems, describing their applications and challenges of genome targeting in bacterial cells. Moreover, the DSB repair pathways in bacteria are summarized and a guideline for selecting repair machines and maximizing the recombination efficiency is presented. In addition, the plasmid curing approaches in bacteria are outlined for iterative gene editing or downstream biological applications. Furthermore, CRISPR‐based transcriptional regulation, base editing, and transposition are also introduced. Finally, this paper prospects the future directions for improving CRISPR‐based gene editing methods in bacteria.

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Section: Constructing Gene Knockdown Librarymentioning

confidence: 99%

Section: Crispr‐dcas Tools For Targeted Gene Regulationmentioning

confidence: 99%

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

Wang

Zhang

et al. 2019

Small Methods

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“…One straightforward approach to reduce the background level of repression by dCas9 in E. coli is to introduce a stop codon in the dcas9 gene in order to insert a synthetic amino acid, named l ‐biphenylalanine (BipA), via a suppressor tRNA for that codon and a cognate aminoacyl‐tRNA synthetase [150]. In the absence of BipA, a nonfunctional, truncated dCas9 is synthesized, and only when BipA is present, the UAG codon is translated resulting in a functional, full‐length dCas9 protein.…”

Section: Limitations Of Crispri and Future Directionsmentioning

confidence: 99%

“…In the absence of BipA, a nonfunctional, truncated dCas9 is synthesized, and only when BipA is present, the UAG codon is translated resulting in a functional, full‐length dCas9 protein. Using dCas9‐BipA repression approach, mrfp gene transcription was fully repressed with 14‐fold reduced background [150]. Certainly, sequence‐specific off‐target effects are more prominent in organisms with genomes that are larger than those of bacteria [142,143,151].…”

Section: Limitations Of Crispri and Future Directionsmentioning

confidence: 99%

Impact of CRISPR interference on strain development in biotechnology

Schultenkämper

Brito

Wendisch

2020

Biotech and App Biochem

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Genetic perturbation systems are of great interest to redirect metabolic fluxes for value‐added production, as well as genetic screening for the development of new drugs, or to identify new targets for biotechnological applications. Here, we review CRISPR interference (CRISPRi), a method for gene expression using a catalytically inactive version of the CRISPR‐associated protein 9 (dCas9) of the widely applied CRISPR‐Cas9 genome editing system. In combination with the appropriate sgRNA, dCas9 binds to specific DNA sequences without causing double‐stranded DNA breakage but interfering with transcription initiation or elongation. Besides manifold uses to interrogate the physiology of a bacterial cell, CRISPRi is used in applications for metabolic engineering and strain development in industrial biotechnology. Albeit in its infancy, CRISPRi has already delivered the first success stories; however, we also analyze limitations of the CRISPRi system and give future perspectives.

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“…The ACRs that were found to inhibit Cas nuclease activity, have been reported to be useful for controlling CRISPR-Cas-based genome editing [21][22][23][24][25][26] . Aside from these naturally occurring ACRs, several other strategies have been devised to control Cas enzyme activity at the level of transcription [27][28][29] , translation [30][31][32] , protein state [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] , and guide RNA 14,48 .…”

Section: Introductionmentioning

confidence: 99%

Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

Swartjes

Shang

Berg

et al. 2022

Preprint

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CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

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Incorporation of a Synthetic Amino Acid into dCas9 Improves Control of Gene Silencing

Cited by 13 publications

References 54 publications

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

Impact of CRISPR interference on strain development in biotechnology

Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

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