Dual sgRNA cloning

Purpose:

    To construct dual sgRNA expressing vector together with spCas9.

 

Materials:

    2x CloneAmp HiFi PCR Premix (Clonetech Cat.#: 639298)

    Primers (dissolved in DNase-free water or TE buffer at 100 mM, dilute to 10 mM before use!)

    pLD1991: pLenti-2x promoters, provides H1 + U6 promoters

    pLD2008 (Lenti), pLD2126 (GFP) or pLD2349 (Lenti & GFP), provides scaffolds + SpCas9

    Esp3I restriction enzyme (NEB, enzyme box @ -20°C)

    T7 ligase (NEB, enzyme box @ -20°C)

    10 mM ATP (NEB, enzyme box @ -20°C)

    Gel purification kit (Thermo, @ RT)

 

Procedure:

  •     Design DNA primers for gene of interest with Esp3I cutting sites (Reverse Complement the sgRNA!!!)

        GOI-U6-F: AGCGcgtctctTCTAAAAC[20 bp reverse complement sgRNA#1]Cggtgtttcgtcctttccac

​        GOI-H1-R: GGCAcgtctccAAAC[20 bp reverse complement sgRNA#2]Tgggaaagagtggtctcatacaga

  •     Order the designed primers, dissolve in ddH2O to a concentration of 100 mM;

  •     For each primer, dilute 20 uL primers with 180 uL ddH2O in a 1.5 mL tube  (f.c.=10 mM) for PCR;

  •     PCR sgRNA1-U6-H1 sgRNA2 from pLD1991 using designed primers in 20 uL reaction (product size ~770 bp)

  •     Run the whole sample onto 0.7-1.0% agarose gel to verify the PCR product, 180 V, 20-30 min;

  •     Purify the PCR product by gel purification, measure the concentration by nanodrop;

  •     Set up digestion and ligation in a 200 uL PCR tube with the following order:

  1.           ddH2O                                                5.0 uL

  2.           10XSmartCut Buffer (NEB)                1.0 uL

  3.           10 mM ATP (NEB)                              1.0 uL

  4.           pLDxxxx scaffold (100 ng/mL)            1.0 uL

  5.           sgRNA PCR product (100 ng/mL)      1.0 uL

  6.           Esp3I (NEB)                                        0.5 uL

  7.           T7 DNA ligase (NEB)                          0.5 uL

  •     Incubate @ 37°C for at least 60 min, better do 3 h reaction;

  •     Transform 5 uL reaction into DH5alpha or Stbl3 competent cells, select on LB+AmpR plate;

  •     Pick 3 colonies for validation and mini-prep.

Transfect cells to produce virus

If 6-well plate is used for viral packaging, just use 1/5 of the components below!!!

10-cm dish = 10 mL media; 6 well plate = 6 x 2 mL/well

  • Prepare a large beaker with 10% bleach solution and place in viral hood

  • Get a 10-cm plate ~70-80% confluent with 293FT cells

  • Check whether viral expression plasmid is for retrovirus or lentivirus and obtain suitable packaging plasmids 

  • Make two 1.5 ml eppendorf tubes (for Tube #2, choose either retrovirus or lentivirus):

Tube #1: Lipofectamine mix:

              500 ul Opti-MEM                               For 6-well plate: 100 ul

              60 ul Lipofectamine 2000                                            12 ul

 

Tube #2: DNA mix (retrovirus):

              500 ul Opti-MEM                               For 6-well plate: 100 ul

              6 ug retroviral expression plasmid                                1.2 ug

              4.5 ug gag-pol plasmid (pLD424)                            0.9 ug

              1 ug VSV-g plasmid      (pLD421)                             0.2 ug

 

Tube #2: DNA mix (lentivirus):                                  

              500 ul Opti-MEM                              For 6-well plate: 100 ul

              12 ug lentiviral expression plasmid                              2.4 ug

              7.5 ug psPAX2 (pLD422)                                        1.5 ug

              6 ug pMD2.G   (pLD423)                                        1.2 ug

 

  • Gently mix each individually and then combine (= transfection mix), incubate for 5 min

  • Aspirate medium from 293FT plate, add 9 ml (2 mL for 6-well) fresh DMEM +FBS +P/S

  • Add the transfection mix dropwise around the plate, and mix VERY gently

  • Place in the viral incubator for 6 hours up to overnight

  • Rinse with 10 mL (2 mL for 6-well)  fresh medium, aspirate, then apply 10 ml (2 mL for 6-well) fresh medium

  • Return the plate to incubator

  • After 24 hours, collect the medium into a sterile 50-ml conical tube (14 mL tube for 6-well)  (store at 4C) and add fresh medium

  • Collect medium again after another 24 hours, combine together into the previous tube

  • Filter through a syringe-driven 0.45-um filter unit

  • Use filtered virus that day and/or store in 1- or 1.5-ml aliquots at -80C