TITLE: Generation of Combinatorial CRISPR Libraries
AUTHORS: David Adams, Nicky Thompson. nt4@sanger.ac.uk. 

[STEPS]
?. [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 kithttps://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ugAll gels run with Sybr green, not ethidium bromide, and no gels exposed to UV light to avoid DNA damage.
?. [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 10ulBsmBI 10000 U/ml 8ulH20 to 100ulIncubate @ 55°C overnightDephosphorylationhttps://www.neb.com/protocols/1/01/01/vector-dephosphorylation-protocol49ul of digestionAntartic phosphatase 5ulAntartic phosphatase buffer 10X 6ul37C for 45’Then 5’ 80C Then 4CGel purification: take the 8.3kb band.
?. [PCR and oligos]
PCR amplification of oligonucleotidesSupplied by TWIST bioscienceDiluted library to final concentration of 0.0045ng/ul Do 14xPCR – each PCR 50ul https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530  AB1     Regent amount for 14x reactions2  Oligo    14  3  HF phusion buffer    140  4  dNTP    14  5  F primer    17.5  6  R primer    17.5  7  Phusion HF
  polymerase    7  8  H2O    490  9       700  PCR program a.98C 30’i.98 10’’ii.68 Co 35’iii.72 C 30’’16Xb.72C 10’c.4C 4everPCR cleanup and elute in 30ulDigestion of pdonor_SU6Digest 5ug of pDonor_sU6 (plasmid #741) with BbsIPlasmid 5ugBuffer 2.1 10X 5ulEnzyme BbsI 10000 U/ml3ulH20 up to50ul totalFor 37C for three hoursGel purification: take the 415bp bandGibson Ligationhttps://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ugDonor fragment 405ng Oligo amplicon 432ng 2xGibson MM 30 ul (NEBuilder® HiFi DNA Assembly Master Mix, E2621S)H2O to 60 ulIncubate @ 50°C for 2hoursNuclease Digestion http://www.epibio.com/enzymes/nucleases-glycosylases-dna-binding-proteins/dna-exonucleases/plasmid-safe-atp-dependent-dnase?detailsAdd to each Gibson reaction 10x Plasmid Safe Buffer 9 ulATP (25mM) 9 ulPlasmid Safe nuclease 3 ulH2O 9 ulàincubate @ 37°C for 1hClean up using PCR purification kit and elute in 50ul waterDigestion of Gibson productDigest with BbsIDNA 50ulBuffer 2.1 6ulBbsI 4ulIncubate @ 37°C for 2.5hRun digestion on 2.5% gel and cut ~480 bp bandGel extract and elute the completed insert in 30ulLigationhttps://www.neb.com/products/m2200-quick-ligation-kitNeb Quick ligation kit; M2200S; scale up as appropriate  ABC1Amount2  Digested&amp;purified
  lenti-guide puro     25ng  3  sgRNA insert BbsI
  purified    X  4  ligase buffer    5ul  5  Ligase    0.5ul  6  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 ligationPCR purification and elution in 30ulwater(for library)
AB1     Regent amount for 14x reactions2  Oligo    14  3  HF phusion buffer    140  4  dNTP    14  5  F primer    17.5  6  R primer    17.5  7  Phusion HF
  polymerase    7  8  H2O    490  9       700
ABC1Amount2  Digested&amp;purified
  lenti-guide puro     25ng  3  sgRNA insert BbsI
  purified    X  4  ligase buffer    5ul  5  Ligase    0.5ul  6  H2O to 10.5ul
?. [Transformation]
Transformationhttps://www.neb.com/products/c3020-neb-10-beta-electrocompetent-e-coli25ul 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