PMID- 36493370
OWN - NLM
STAT- MEDLINE
DCOM- 20230216
LR  - 20230421
IS  - 2573-1602 (Electronic)
IS  - 2573-1599 (Linking)
VI  - 6
IP  - 1
DP  - 2023 Feb
TI  - A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity In Vivo 
      in Escherichia coli.
PG  - 43-51
LID - 10.1089/crispr.2022.0041 [doi]
AB  - Ribonuclease III (RNase III) and RNase III-like ribonucleases have a wide range 
      of important functions and are found in all organisms, yet a simple and 
      high-throughput in vivo method for measuring RNase III activity does not exist. 
      Typical methods for measuring RNase III activity rely on in vitro RNA analysis or 
      in vivo methods that are not suitable for high-throughput analysis. In this 
      study, we describe our development of a deactivated Cas9 (dCas9)-based in vivo 
      assay for RNase III activity that utilizes RNase III's cleavage of the 
      5'-untranslated region (UTR) of its own messenger RNA. The key molecule in the 
      system is a hybrid guide RNA (gRNA) between the 5'-UTR of RNase III and gGFP, a 
      gRNA that works with dCas9 to repress GFP expression. This fusion must be cleaved 
      by RNase III for full GFP repression. Our system uses GFP fluorescence to report 
      on Escherichia coli RNase III activity in culture and on an individual cell 
      basis, making it effective for selecting individual cells through 
      fluorescence-activated cell sorting. Homology between enzymes within the RNase 
      III family suggests this assay might be adapted to measure the activity of other 
      enzymes in the RNase III family such as human Dicer or Drosha.
FAU - Hauk, Pricila
AU  - Hauk P
AD  - Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 
      Baltimore, Maryland, USA.
FAU - Weeks, Ryan
AU  - Weeks R
AD  - Chemistry-Biology Interface Graduate Program, Johns Hopkins University, 
      Baltimore, Maryland, USA.
FAU - Ostermeier, Marc
AU  - Ostermeier M
AUID- ORCID: 0000-0003-0900-3665
AD  - Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 
      Baltimore, Maryland, USA.
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20221209
PL  - United States
TA  - CRISPR J
JT  - The CRISPR journal
JID - 101738191
RN  - EC 3.1.26.3 (Ribonuclease III)
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - Humans
MH  - *Escherichia coli/genetics
MH  - *Ribonuclease III/genetics/metabolism
MH  - CRISPR-Cas Systems/genetics
MH  - Gene Editing
MH  - RNA
EDAT- 2022/12/10 06:00
MHDA- 2023/02/17 06:00
CRDT- 2022/12/09 16:33
PHST- 2022/12/10 06:00 [pubmed]
PHST- 2023/02/17 06:00 [medline]
PHST- 2022/12/09 16:33 [entrez]
AID - 10.1089/crispr.2022.0041 [doi]
PST - ppublish
SO  - CRISPR J. 2023 Feb;6(1):43-51. doi: 10.1089/crispr.2022.0041. Epub 2022 Dec 9.