Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

. Author manuscript; available in PMC: 2023 Jan 3.

Published in final edited form as: Cell Rep. 2022 Aug 16;40(7):111226. doi: 10.1016/j.celrep.2022.111226

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Mouse monoclonal α-MBNL1 (MB1a(4A8))	MilliporeSigma	Cat#MABE70; RRID:AB_2618248
Chicken polyclonal α-GFP	Abcam	Cat#ab13970; RRID:AB_300798
Alexa Fluor 647 conjugated goat α-mouse secondary	Life Technologies	Cat#A21235; RRID:AB_2535804
Alexa Fluor 488 conjugated goat α-chicken secondary	Life Technologies	Cat#A11039; RRID:AB_2534096
Rabbit monoclonal α-HA (C29F4)	Cell Signaling Technology	Cat# 3724S; RRID:AB_1549585
Mouse monoclonal α-HSP70 (5A5)	Invitrogen	Cat#MA3007; RRID:AB_325455
IRDye 800CW donkey α-rabbit secondary	LI-COR	Cat#926-32213; RRID:AB_621848
IRDye 680RD donkey α-mouse secondary	LI-COR	Cat#926-68072; RRID:AB_10953628
Alexa Fluor 568 conjugated donkey α-rabbit secondary	Invitrogen	Cat#A10042; RRID:AB_2534017

Bacterial and virus strains

NEB Stable chemically competent E. coli	New England Biolabs	Cat#C3040
AAV-CMV-unreg-NT	This paper	N/A
AAV-CMV-GENO-CUG1	This paper	N/A
AAV-CMV-GENO-NT	This paper	N/A

Chemicals peptides and recombinant proteins

Fluoroshield antifade mounting medium	Sigma-Aldrich	Cat#F6182
ProLong Diamond antifade mounting medium	ThermoFisher Scientific	Cat#P36965
4′,6-diamidino-2-phenylindole (DAPI)	Invitrogen	Cat#D1306
HCS CellMask Green cell stain	ThermoFisher Scientific	Cat#H32714
T4 DNA ligase	New England Biolabs	Cat#M0202
TransIT-X2 transfection reagent	Mirus Bio	Cat#MIR6005
Paraformaldehyde (PFA)	Electron Microscopy Sciences	Cat#15710
Ribonucleoside vanadyl complex	New England Biolabs	Cat#S1402S
Puromycin	AG Scientific	Cat#P-1033-SOL
Doxycycline	Sigma	Cat#D3447-500MG
TRI Reagent	Zymo Research	Cat#R2050-1-200
dsGreen DNA detection dye	Lumiprobe	Cat#11010
SIGMAFAST Protease Inhibitor Cocktail	Sigma-Aldrich	Cat#S8830
Phenylmethylsulfonyl fluoride (PMSF)	Sigma-Aldrich	Cat#10837091001
4X NuPage LDS Sample Buffer	Invitrogen	Cat#NP0008
Dithiothreitol (DTT)	New England Biolabs	Cat#B1034A
SEA BLOCK Blocking Buffer	Thermo Scientific	Cat#37527

Critical commercial assays

PrestoBlue resazurin cell viability assay	Invitrogen	Cat#A13261
Taq 2X master mix	New England Biolabs	Cat#M0270L
Phusion High-Fidelity DNA polymerase	New England Biolabs	Cat#M0530L
Gibson assembly master mix	New England Biolabs	Cat#E2611
Zyppy Plasmid Miniprep kit	Zymo Research	Cat#D4036
Direct-Zol RNA Miniprep kit	Zymo Research	Cat#R2051
iScript Reverse Transcription Supermix	Bio-Rad	Cat#1708841
NEBNext rRNA Depletion Kit	New England Biolabs	Cat#E6310L
NEBNext Ultra II Directional RNA Library Prep Kit for Illumina	New England Biolabs	Cat#E7760L
Pierce BCA Protein Assay	Thermo Scientific	Cat#23225
Dynabeads Oligo(dT)₂₅ magnetic beads	Invitrogen	Cat#61005

Deposited data

GEO (RNA-seq of repeat-targeting Cas13d, dCas9 and shRNA in HeLa)	This paper	GSE191329

Experimental models: Cell lines

Human: HeLa	ATCC	CCL-2
Human: HEK293	ATCC	CRL-1573
Human: DM-05 adult DM1 patient-derived myoblasts	(Xia et al., 2013)	N/A
Mouse: Neuro2a	ATCC	CCL-131
Human: HeLa-tet:Cas13d-mCherry	This paper	N/A

Oligonucleotides

gRNA spacer sequences, see Table S1	This paper	N/A
PCR primers, see Table S1	This paper	N/A
Alexa Fluor 594 conjugated CAG10 FISH probe	Biosearch Technologies	Cat#SS151541-01
Cas13d mRNA HCR FISH probe set, B1 amplifier	Molecular Instruments	N/A
PPIB mRNA HCR FISH probe set, B2 amplifier	Molecular Instruments	N/A
POLR2A mRNA HCR FISH probe set, B5 amplifier	Molecular Instruments	N/A

Recombinant DNA

pXR001 (NLS-RfxCas13d-NLS-HA-T2A-EGFP)	(Konermann et al., 2018)	Addgene #109049
pXR002 (NLS-dRfxCas13d-NLS-HA-T2A-EGFP)	(Konermann et al., 2018)	Addgene #109050
pXR003 (RfxCas13d gRNA cloning plasmid)	(Konermann et al., 2018)	Addgene #109053
pXR-dSpCas9	This paper	N/A
pCR-Blunt II-TOPO- U6-(CAG)₆ gRNA	(Pinto et al., 2017)	N/A
pLKO.1 (shRNA cloning plasmid)		Addgene #10878
DMPKS (CUG₀ target plasmid)	Gift from Thomas Cooper	N/A
DT480 (CUG₄₈₀ target plasmid)	(Ho et al., 2004)	Addgene #80413
dCas13d-EGFP (NLS-dRfxCas13d-(GGGGS)₂-EGFP)	This paper	N/A
pmCherry	This paper	N/A
pB-Tet-Cas13d	This paper	N/A
pB-mCherry	This paper	N/A
pXR001-GENO-CUG1	This paper	N/A
pXR001-GENO-NT	This paper	N/A
RG6-MBNL1e5	(Orengo et al., 2011)	N/A
mPB (PiggyBac transposase)	(Cadiñanos and Bradley, 2007)	N/A
pUC19	New England Biolabs	Cat#N3041S

Software and algorithms

Code generated in this study	This paper	https://github.com/cpkelley94/geno
Python 3.6	Python Software Foundation	https://www.python.org/
ZEN v2.1 SP3 FP3 black 14.0.20.201	ZEISS	https://www.micro-shop.zeiss.com/en/us/softwarefinder/software-categories/zen-black/
Cellpose 0.0.2.0	(Stringer et al., 2021)	https://www.cellpose.org/
kallisto 0.43.0	(Bray et al., 2016)	https://pachterlab.github.io/kallisto/
HISAT2 2.0.0-beta	(Kim et al., 2019)	https://daehwankimlab.github.io/hisat2/
DESeq2 1.30.1	(Love et al., 2014)	https://bioconductor.org/packages/release/bioc/html/DESeq2.html
htseq-count 0.11.2	(Anders et al., 2015)	https://htseq.readthedocs.io/en/master/
PANTHER gene ontology analysis, annotation v.2021-05-01	(Mi et al., 2019)	http://www.pantherdb.org/
Fiji v2.0.0-rc-69/1.52p	(Schindelin et al., 2012)	https://imagej.net/software/fiji/
Bio-Rad CFX Manager 3.1	Bio-Rad	https://www.bio-rad.com/en-us/sku/1845000-cfx-manager-software?ID=1845000

Other

Cytation 3 plate reader	Bio-Tek	N/A
NextSeq 500	Illumina	N/A
C1000 Touch thermocycler	Bio-Rad	N/A
Odyssey CLx scanner	LI-COR	N/A
EVOS FL digital inverted fluorescence microscope	Life Technologies	Cat#AMF4300PM
LSM 880 confocal microscope with Airyscan	ZEISS	N/A
Fragment Analyzer	Advanced Analytical	1.1.0.11
iBlot 2 dry transfer system	ThermoFisher Scientific	Cat#IB21001