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A study of T7 RNA polymerase‐Template interaction utilizing CRISPR/dCas9 protein
Author(s) -
Enkhbaatar Khulan,
Rohlman Christopher E.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.07163
Subject(s) - polymerase , transcription (linguistics) , rna , crispr , t7 rna polymerase , dna , biology , trans activating crrna , nucleic acid , rna polymerase , rna dependent rna polymerase , computational biology , microbiology and biotechnology , cas9 , chemistry , genetics , gene , bacteriophage , linguistics , philosophy , escherichia coli
The CRISPR system is an adaptive immune mechanism present in many bacteria and the majority of characterized Archaea . This system binds to nucleic acids, which can be RNA or DNA and modifies specific sequences at desired locations. Although the CRISPR system can form the basis of a flexible genomic engineering toolkit, its molecular basis of interaction with DNA, RNA and other proteins during transcription is not yet clear. This research is investigating the structural and chemical interaction between dCas9, DNA and RNA polymerase at the point of the transcription blockade. This modified CRISPR system consists of two components. dCas9 protein has the ability to bind DNA at a specific location when guided by a complementary RNA molecule. This complementary RNA is called guide RNA (gRNA). T7 RNA polymerase in vitro transcription of purified U5 DNA template was optimized with respect to time, template and rNTP concentration. This established conditions under which to pause transcription, utilizing dCas9 protein, and analyze protein, polymerase, and nucleic acid interactions.