| Bacterial and Virus Strains |
| BL21-CodonPlus (DE3)-RIL Competent cell |
Agilent Technologies |
Cat# 230245 |
| Rosetta (DE3)pLysS Competent cell |
Novagen |
Cat# 70956 |
| One Shot PIR1 Chemically Competent cell |
Thermo fisher scientific |
Cat# C101010 |
| Stellar Competent Cell |
Takara Bio |
Cat# 636763 |
| Chemicals, Peptides, and Recombinant Proteins |
| Terrific Broth (TB) |
Thermo fisher scientific |
Cat# BP97285 |
| isopropyl β-D-thiogalactopyranoside (IPTG) |
Thermo fisher scientific |
Cat# R0393 |
| Tris-Base |
Thermo fisher scientific |
Cat# BP152–1 |
| HEPES |
Thermo fisher scientific |
Cat# BP310–1 |
| Sodium chloride |
Thermo fisher scientific |
Cat# S271–10 |
| Glycerol |
Thermo fisher scientific |
Cat# G33–500 |
| 2-Mercaptoethanol |
Thermo fisher scientific |
Cat# O3446I-100 |
| Magnesium chloride |
Thermo fisher scientific |
Cat# M33–500 |
| PMSF |
Sigma-Aldrich |
Cat# P7626 |
| Imidazole |
Sigma-Aldrich |
Cat# I202–2KG |
| DTT |
Thermo fisher scientific |
Cat# AAJ1539722 |
| EDTA |
Thermo fisher scientific |
Cat# AM9261 |
| Potassium Chloride |
Thermo fisher scientific |
Cat# P217–500 |
| Magnesium acetate tetrahydrate |
Millipore Sigma |
Cat# M5661–250G |
| Agarose |
Thermo fisher scientific |
Cat# BP1356–500 |
| SYBR safe DNA Gel Stain |
Thermo fisher scientific |
Cat# S33102
|
| Tobacco Etch Virus (TEV) protease |
Homemade |
N/A |
| FspI |
New England Biolabs |
Cat# R0135L |
| HindIII-HF |
New England Biolabs |
Cat# R3104S |
| BseRI |
New England Biolabs |
Cat# R0581L |
| BsaI |
New England Biolabs |
Cat# R3733S |
| Proteinase K |
Thermo fisher scientific |
Cat# EO0491 |
| Benzonase Nuclease |
Millipore Sigma |
Cat# E1014–25KU |
| ATP |
Thermo fisher scientific |
Cat# BP413–25 |
| Phusion Hot Start II High-Fidelity PCR Master Mix |
Thermo fisher scientific |
Cat# F565L |
| Phusion High-Fidelity PCR Master Mix with HF Buffer |
Thermo fisher scientific |
Cat# F531L |
| PrimeSTAR Max Premix |
Takara Bio |
Cat# R045A |
| In-Fusion HD Cloning Kit |
Takara Bio |
Cat# 639650 |
| Q5® Site-Directed Mutagenesis Kit |
New England Biolabs |
Cat# E0554 |
| HiScribe T7 Quick High Yield RNA Synthesis Kit |
New England Biolabs |
Cat# E2050S |
| QIAprep Spin Miniprep Kit |
QIAGEN |
Cat# 27106 |
| MinElute PCR Purification Kit |
QIAGEN |
Cat# 28006 |
| ZymoPURE II Plasmid Gigaprep Kit |
Zymo Research |
Cat# D4204-B |
| Mini-PROTEAN TGX Precast Gels |
Bio-rad |
Cat# 4561086 |
| Amicon Ultra-15 Centrifugal Filter Units 3kDa NMWL |
Millipore sigma |
Cat# UFC900324 |
| Amicon Ultra-15 Centrifugal Filter Units 10kDa NMWL |
Millipore sigma |
Cat# UFC901024 |
| Amicon Ultra-15 Centrifugal Filter Units 30kDa NMWL |
Millipore sigma |
Cat# UFC903024 |
| Amicon Ultra-15 Centrifugal Filter Units 50kDa NMWL |
Millipore sigma |
Cat# UFC905024 |
| NuPAGE LDS Sample Buffer (4X) |
Thermo fisher scientific |
Cat# NP0007 |
| Gel Loading Dye, Purple (6X) |
New England Biolabs |
Cat# B7024S |
| Bovine Serum Albumin |
Sigma-Aldrich |
Cat# A2153–10G |
| Ampicillin, sodium salt |
Thermo fisher scientific |
Cat# BP1760–25 |
| Chloramphenicol, 98%, ACROS Organics |
Thermo fisher scientific |
Cat# AC227920250 |
| Kanamycin Monosulfate, USP Grade |
Gold Biotechnology |
Cat# K-120–5 |
| SnakeSkin Dialysis Tubing, 7K MWCO, 22 mm |
Thermo fisher scientific |
Cat# 68700 |
| Diethyl pyrocarbonate |
Millipore sigma |
Cat# D5758–25ML |
| Critical Commercial Assays |
| Ni-NTA agarose resin |
QIAGEN |
Cat# 30430 |
| HiTrap Heparin HP affinity column |
Cytiva |
Cat# 17040701 |
| RESOURCE Q anion exchange column |
Cytiva |
Cat# 17117701 |
| Superdex 200 10/300 column |
Cytiva |
Cat# 28990944 |
| InstantBlue Coomassie Protein Stain |
Abacam |
Cat# ISB1L |
| Deposited Data |
| Cas12k–sgRNA–target DNA coordinate |
This study |
PDB: 7N3P |
| Cas12k–sgRNA–target DNA map |
This study |
EMDB: EMD-24143 |
| Cas12k–sgRNA coordinate |
This study |
PDB: 7N3O |
| Cas12k–sgRNA map |
This study |
EMDB: EMD-24142 |
| Oligonucleotides |
| DNA primers |
IDT |
Table S1
|
| DNA oligos (for structure determination) |
IDT |
Table S1
|
| Cas12k sgRNA |
IDT |
Table S1
|
| Cas12k sgRNA variants |
IDT |
Table S1
|
| Recombinant DNA |
| pDonor_ShCAST_kanR |
Addgene |
#127924 |
| pTarget_CAST |
Addgene |
#127926 |
| pET28-MKH8SUMO-shTnsB |
This paper |
N/A |
| pET28-MKH8SUMO-shTnsC |
This paper |
N/A |
| pET28-MKH8SUMO-shTniQ |
This paper |
N/A |
| pET-His6-StrepII-TEV LIC-Cas12k |
This paper |
N/A |
| pET-His6-StrepII-TEV LIC-Cas12k variant mutants |
This paper |
N/A |
| Software and Algorithms |
| PyMOL |
Schrodinger LLC |
https://pymol.org/2/
|
| UCSF Chimera |
(Pettersen et al., 2004) |
https://www.cgl.ucsf.edu/chimera/
|
| Phenix |
(Afonine et al., 2018) |
https://phenix-online.org/
|
| Coot |
(Emsley et al., 2010) |
https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
|
| CryoSPARC |
(Punjani et al., 2017) |
https://cryosparc.com/
|
| Leginon |
(Suloway et al., 2005) |
https://emg.nysbc.org/redmine/projects/leginon/wiki/Leginon_Homepage
|
| CTFFIND4 |
(Rohou and Grigorieff, 2015) |
https://grigoriefflab.umassmed.edu/ctffind4
|
| MotionCor2 |
(Zheng et al., 2017) |
https://msg.ucsf.edu/em/software/motioncor2.html
|
| PROMALS3D |
(Pei et al., 2008) |
http://prodata.swmed.edu/promals3d/promals3d.php
|
| ESPript |
(Robert and Gouet, 2014) |
https://espript.ibcp.fr/ESPript/ESPript/
|
| Sequence Manipulation Suite |
(Stothard, 2000) |
https://www.bioinformatics.org/sms2/ident_sim.html
|
| GraphPad Prism 8 |
GraphPad Software |
https://www.graphpad.com/scientific-software/prism/
|
| National Center for Biotechnology Information (NCBI) |
(Sayers et al., 2021) |
https://www.ncbi.nlm.nih.gov
|
