PMID- 35987443
OWN - NLM
STAT- MEDLINE
DCOM- 20220923
LR  - 20240104
IS  - 1095-9130 (Electronic)
IS  - 1046-2023 (Print)
IS  - 1046-2023 (Linking)
VI  - 206
DP  - 2022 Oct
TI  - Optimization of specific RNA knockdown in mammalian cells with CRISPR-Cas13.
PG  - 58-68
LID - S1046-2023(22)00177-3 [pii]
LID - 10.1016/j.ymeth.2022.08.007 [doi]
AB  - Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short 
      Palindromic Repeats (CRISPRs) and CRISPR Associated (Cas) proteins to target and 
      cleave foreign genetic elements in an RNA-guided manner [1-3]. Type VI CRISPR-Cas 
      systems contain a single effector ribonuclease, Cas13, that binds and processes a 
      CRISPR-RNA (crRNA; also known as a guide-RNA), forming an RNA-guided 
      RNA-targeting effector complex [4,5]. Previous studies have shown that Cas13 can 
      be engineered to target and modulate RNA processes in human cells, illustrating 
      the versatility and specificity of Cas13 as an RNA knockdown (KD), splicing, 
      editing, or imaging tool [6-8]. While Cas13 has been successfully used by several 
      groups, our lab has observed significant variability in Cas13 KD ability 
      depending which protocol is being followed [9-12]. To further understand this 
      variability and generate a robust Cas13 KD protocol we thoroughly tested which 
      Cas13 ortholog to use, the duration of KD experiments, the amount of plasmid DNA 
      transfected, methods for analyzing KD efficiency, and report an optimized method 
      for carrying out and analyzing Cas13 mediated RNA KD experiments. The method 
      outlined in this paper illustrates a faster and more reliable protocol to 
      iteratively test gRNA performance and target gene KD.
CI  - Copyright (c) 2022 Elsevier Inc. All rights reserved.
FAU - Burris, Brandon Joseph Davis
AU  - Burris BJD
AD  - Department of Biochemistry and Biophysics, School of Medicine and Dentistry, 
      University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, 
      University of Rochester, Rochester, NY 14642, USA.
FAU - Molina Vargas, Adrian Moises
AU  - Molina Vargas AM
AD  - Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA; 
      Department of Biomedical Genetics, School of Medicine and Dentistry, University 
      of Rochester, NY 14642, USA.
FAU - Park, Brandon J
AU  - Park BJ
AD  - Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA; 
      Department of Biomedical Genetics, School of Medicine and Dentistry, University 
      of Rochester, NY 14642, USA.
FAU - O'Connell, Mitchell R
AU  - O'Connell MR
AD  - Department of Biochemistry and Biophysics, School of Medicine and Dentistry, 
      University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, 
      University of Rochester, Rochester, NY 14642, USA. Electronic address: 
      mitchell_oconnell@urmc.rochester.edu.
LA  - eng
GR  - R35 GM133462/GM/NIGMS NIH HHS/United States
GR  - T32 GM068411/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20220817
PL  - United States
TA  - Methods
JT  - Methods (San Diego, Calif.)
JID - 9426302
RN  - 0 (RNA, Guide, CRISPR-Cas Systems)
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
RN  - EC 3.1.- (Ribonucleases)
SB  - IM
MH  - Animals
MH  - *CRISPR-Cas Systems/genetics
MH  - DNA
MH  - Humans
MH  - Mammals/genetics
MH  - RNA/genetics
MH  - *RNA, Guide, CRISPR-Cas Systems/genetics
MH  - Ribonucleases
PMC - PMC9511595
MID - NIHMS1836142
OTO - NOTNLM
OT  - CRISPR
OT  - Cas13
OT  - RNA knockdown
OT  - RNA-targeting
OT  - RfxCas13d
OT  - Type VI CRISPR-Cas systems
COIS- Declaration of Competing Interest The authors declare the following financial 
      interests/personal relationships which may be considered as potential competing 
      interests: M.R.O is an inventor on patent applications related to CRISPR-Cas 
      systems and uses thereof. M.R.O is a member of the scientific advisory boards for 
      Dahlia Biosciences and LocanaBio, and an equity holder in Dahlia Biosciences and 
      LocanaBio.
EDAT- 2022/08/21 06:00
MHDA- 2022/09/24 06:00
PMCR- 2023/01/01
CRDT- 2022/08/20 19:34
PHST- 2022/07/11 00:00 [received]
PHST- 2022/08/10 00:00 [revised]
PHST- 2022/08/15 00:00 [accepted]
PHST- 2022/08/21 06:00 [pubmed]
PHST- 2022/09/24 06:00 [medline]
PHST- 2022/08/20 19:34 [entrez]
PHST- 2023/01/01 00:00 [pmc-release]
AID - S1046-2023(22)00177-3 [pii]
AID - 10.1016/j.ymeth.2022.08.007 [doi]
PST - ppublish
SO  - Methods. 2022 Oct;206:58-68. doi: 10.1016/j.ymeth.2022.08.007. Epub 2022 Aug 17.