Construction of a Tl-CRISPRi Genetic Circuit in Bacteria for Translation-Level Gene Knockdown.

Giho Kim, Ho Joon Kim, Sang Woo Seo

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Article in Methods in molecular biology (Clifton, N.J.), 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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5 · Who and what money

Authors and funding

3 authors.

Giho KimInstitute of Chemical Processes, Seoul National University, Seoul, South Korea.

Ho Joon KimSchool of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea.

Sang Woo SeoInstitute of Chemical Processes, Seoul National University, Seoul, South Korea. swseo@snu.ac.kr.

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

The Tl-CRISPRi system, which harnesses the specific RNA-binding activity of CRISPR-dCas13, has been recently developed for translation-level gene knockdown in bacteria. By introducing spacers complementary to the translation initiation region of the mRNA, dCas13 can be directed to block the ribosome and inhibit the translation of that mRNA. Here, we discuss how to construct the Tl-CRISPRi genetic circuit and implement this system for gene knockdown. This chapter describes how to design spacer sequences and install them into the guide RNA expression plasmid. Also, we describe how to mutate the handle of gRNA to achieve tunable knockdown of a target gene. By following the method described in this chapter, we anticipate that a precise and controllable knockdown of a target gene in bacterial cells can be performed in a programmable manner.

Indexed as

BacteriaClustered Regularly Interspaced Short Palindromic RepeatsCRISPR-Cas SystemsGene Knockdown TechniquesGene Regulatory NetworksProtein BiosynthesisGenetic EngineeringPlasmidsRNA, Guide, CRISPR-Cas SystemsRNA, Guide, CRISPR-Cas SystemsCRISPR-dCas13Guide RNA engineeringSynthetic biologyTranslation-level CRISPRiTunable gene regulation

Identifiers

PMID42420723

DOI10.1038/nmeth.2515

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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.