Nov 15, 2020
Generation of Combinatorial CRISPR Libraries
- David Adams1,
- Nicky Thompson. nt4@sanger.ac.uk. 1
- 1Wellcome Sanger Institute
External link: https://doi.org/10.1038/s41467-021-21478-9
Protocol Citation: David Adams, Nicky Thompson. nt4@sanger.ac.uk. 2020. Generation of Combinatorial CRISPR Libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.bpqhmmt6
Manuscript citation:
Thompson NA, Ranzani M, Weyden Lvd, Iyer V, Offord V, Droop A, Behan F, Gonçalves E, Speak A, Iorio F, Hewinson J, Harle V, Robertson H, Anderson E, Fu B, Yang F, Zagnoli-Vieira G, Chapman P, Velasco-Herrera MDC, Garnett MJ, Jackson SP, Adams DJ, Combinatorial CRISPR screen identifies fitness effects of gene paralogues. Nature Communications doi: 10.1038/s41467-021-21478-9
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 15, 2020
Last Modified: November 15, 2020
Protocol Integer ID: 44521
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Summary
Summary
This protocol is a step by step protocol based on the method originally described by: Vidigal, J.A. & Ventura, A. Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries. Nature Communications6, 8083 (2015).
Throughout:
All PCR clean-ups done with Monarch PCR & DNA cleanup kit
All gels run with Sybr green, not ethidium bromide, and no gels exposed to UV light to avoid DNA damage.
Vector Preparation
Vector Preparation
Backbone digestion and preparation (lenti-guide puro)
Digest 5-10ug of your vector of choice:
- lenti-guide puro 10ug (Addgene #52963)
- 10X buffer 3.1 10ul
- BsmBI 10000 U/ml 8ul
- H20 to 100ul
Incubate @ 55°C overnight
Dephosphorylation
- 49ul of digestion
- Antartic phosphatase 5ul
- Antartic phosphatase buffer 10X 6ul
37C for 45’
Then 5’ 80C
Then 4C
Gel purification: take the 8.3kb band.
PCR and oligos
PCR and oligos
PCR amplification of oligonucleotides
Supplied by TWIST bioscience
Diluted library to final concentration of 0.0045ng/ul
Do 14xPCR – each PCR 50ul
| Regent amount for 14x reactions | ||
| Oligo | 14 | |
| HF phusion buffer | 140 | |
| dNTP | 14 | |
| F primer | 17.5 | |
| R primer | 17.5 | |
| Phusion HF polymerase | 7 | |
| H2O | 490 | |
| 700 |
PCR program
a.98C 30’
i.98 10’’
ii.68 Co 35’
iii.72 C 30’’16X
b.72C 10’
c.4C 4ever
PCR cleanup and elute in 30ul
Digestion of pdonor_SU6
Digest 5ug of pDonor_sU6 (plasmid #741) with BbsI
Plasmid 5ug
Buffer 2.1 10X 5ul
Enzyme BbsI 10000 U/ml3ul
H20 up to50ul total
For 37C for three hours
Gel purification: take the 415bp band
Gibson Ligation
Donor fragment 405ng
Oligo amplicon 432ng
2xGibson MM 30 ul (NEBuilder® HiFi DNA Assembly Master Mix, E2621S)
H2O to 60 ul
Incubate @ 50°C for 2hours
Nuclease Digestion
Add to each Gibson reaction
- 10x Plasmid Safe Buffer 9 ul
- ATP (25mM) 9 ul
- Plasmid Safe nuclease 3 ul
- H2O 9 ul
àincubate @ 37°C for 1h
Clean up using PCR purification kit and elute in 50ul water
Digestion of Gibson product
Digest with BbsI
DNA 50ul
Buffer 2.1 6ul
BbsI 4ul
Incubate @ 37°C for 2.5h
Run digestion on 2.5% gel and cut ~480 bp band
Gel extract and elute the completed insert in 30ul
Ligation
Neb Quick ligation kit; M2200S; scale up as appropriate
| Amount | |||
| Digested&purified lenti-guide puro | 25ng | ||
| sgRNA insert BbsI purified | X | ||
| ligase buffer | 5ul | ||
| Ligase | 0.5ul | ||
| H2O to 10.5ul |
Incubated at 25C for 15minutes (thermocycler)
Prior to library creation controls done with vector only (lentiguide puro) to assess background.
For library used final volume of 300ul for ligation
PCR purification and elution in 30ulwater(for library)
Transformation
Transformation
Transformation
25ul bacteria/2.5ul ligation product/transformation; electroporated as per website protocol. 10 electroporations. 1hour in pre-warmed SOC shaking.
Plated at dilutions (1:1000; 1:10000) onto warmed ampicillin plates. The remainder inoculated into ampicillin containing LB broth 3 litres at 100ug/ml and shaken at 225rpm/37C overnight.
Following morning pooled spun down in Avanti centrifuge (6000g for 10 minutes)
Bacterial pellet processed using maxi/mega prep (Qiagen) ~1.5g bacteria/mega prep