Mouse Genome Engineering Using Designer Nucleases

1. Identification of TALEN Target Sites

1. Visit the TAL Effector Nucleotide Targeter 2.0 website (http://tale-nt.cac.cornell.edu) and choose “TALEN Targeter”.
2. Enter the sequence of the target gene. If pCAG-T7-TALEN expression constructs is being used (Figure 1C) choose “Miller et al., 2011″ under “Use a Preset Architecture” in order to predict target sites that can be targeted with the optimal spacer length (15-20 bp) and numbers of TALE RVDs (15-20) for this TALEN architecture.
3. Choose “NN” under “G Substitute” (Guanosine-specific NH RVDs are also available but not yet extensively tested for TALEN assembly).
4. Optional: for other settings and options please follow the instructions on the website and the links provided there.
5. A textfile with potential target-sites is generated, which can be imported into a spreadsheet program for more convenient viewing. Select the TALEN pair(s) for Golden Gate assembly.
6. Optional: Sequence the genomic region of interest in the mouse strain that will be used for microinjection to detect possible single nucleotide polymorphisms (SNPs) that might not be accounted for in public databases and could potentially prevent nuclease binding.
7. Optional: Refer to Table 3 for additional designer nucleases-related online resources.
8. Most plasmids required for ZFN and TALEN construction can be obtained from Addgene:
   http://www.addgene.org/special-collections/
   Addgene IDs for the Golden Gate TALEN kit and mammalian TALEN expression constructs are provided in Table 2.
   Alternatively, particular functional modules such as zinc finger domains can be ordered from a gene synthesis service provider.

2. Golden Gate TALEN Assembly

This section describes the assembly of TALENs using a protocol published by Cermak et al.¹³ Full-length TALENs are constructed using two subsequent Golden Gate cloning steps (Figure 1). This approach allows the incorporation of any number of RVDs between 12 and 31 into the final expression constructs. The assembly protocol has been adapted to destination vectors designed for generating mRNAs (Figure 1C) that express highly active TALENs following oocyte microinjection. Please also refer to the online protocol of the Golden Gate TALEN kit for establishing and maintaining the plasmid library (http://www.addgene.org/TALeffector/goldengateV2/).

1. Day 1 – Golden Gate Reaction #1 – Assembly of RVD Arrays into pFUS Vectors
   1. Optional: For each TALEN pair designed in Protocol 1, enter the DNA sequence of the two the target sites (including the initial T, 5' to 3') into the TALEN_Voytas_Pipetting spreadsheet to generate a pipetting scheme for each Golden Gate reaction (also enter concentrations of all plasmids used for the assemblies). The spreadsheet also provides the expected DNA sequence of all RVDs that can be used for alignment of sequencing results in section 2.5.2.
   2. For every unique TALEN designed in Protocol 1:
      If TALEN length is 12-21 (standard), select repeat variable di-residues (RVDs) 1-10 and destination vector pFUS_A. Select remaining RVDs and a pFUS_B destination vector, e.g. for a TALEN with 15 RVDs (including the last repeat, which will be added to the final assembly in section 2.3.3, select RVDs 11-14 and destination vector pFUS_B4 (Figure 1A).
      If TALEN length is 22-31, use RVDs 1-10 and destination vector pFUS_A30A. Pick RVDs 11-20 and destination vector pFUS_A30B. Select remaining RVDs and a pFUS_Bdestination vector, e.g. for TALEN with 24 RVDs pick RVDs 21-23 and destination vector pFUS_B3.
   3. Set up Golden Gate reaction #1 for each pFUS + RVD combination separately, i.e. 1-10 + pFUS_A and remaining RVDs + respective pFUS_B vector or for TALEN longer than 21 RVDs, 1-10 + pFUS_A30A, 11-20 pFUS_A30B, and remaining RVDs + respective pFUS_B vector. Use 150 ng of each RVD vector, 150 ng of pFUS vector, 1 µl BsaI, 1 µl T4 DNA ligase, 2 µl 10x T4 DNA ligase buffer, and H₂O to 20 µl total reaction volume. (Using fresh aliquots of T4 ligase buffer for each round of Golden Gate assemblies is recommended since repeated thawing/freezing of T4 ligase buffer can reduce the efficiency of the reactions.)
   4. Place Golden Gate reactions in a thermo cycler.
      Program:
      37 °C 5 min
      16 °C 10 min
      10 cycles
      50 °C 5 min
      80 °C 5 min
   5. Add 1 µl 10 mM ATP and 1 µl Plasmid-Safe nuclease to each mix and incubate at 37 °C for 1 hr. This treatment will remove linear incomplete ligation products that could be cloned into the destination vectors by in vivo homologous recombination in transformed bacteria.
   6. Transform E. coli with individual ligation reactions (electrocompetent or chemically competent E. coli that facilitate α-complementation such as XL1-Blue or DH5α can be used here and in subsequent transformations).
   7. Plate bacteria on spectinomycin (50 µg/ml) plates with X-Gal and IPTG (40 µg/ml each) for blue/white colony selection.
2. Day 2 – Confirmation of Correct pFUS-RVDs Assembly
   1. By using colony PCR with primers pCR8_F1 and pCR8_R1 (See Table 1 for primer sequences) screen 1-3 white colonies from each plate. Correct pFUS-RVDs assemblies typically show a band corresponding to the combined length of all RVDs cloned (e.g. around 1.1 kb for 10 RVDs) and a “ladder” of smaller less prominent bands (Figure 1B).
      PCR program:
      95 °C 3 min
      95 °C 30 sec
      55 °C 30 sec
      72 °C 1 min 45 sec
      30-35 cycles
      72 °C 10 min
   2. Use confirmed clones to start overnight culture (2-5 ml LB with 50 µg/ml spectinomycin).
3. Day 3 – Golden Gate Reaction #2 – RVD Arrays into TALEN Expression Vectors
   1. Perform “minipreps” to isolate pFUS-RVD assemblies (depending on number of RVDs either pFUS_A and pFUS_B or pFUS_A30A, pFUS_A30B, and pFUS_B).
   2. Optional: Sequence individual pFUS vectors using primers pCR8_F1, pCR8_R1 (see Table 1 for primer sequences). Sequencing can also be performed on final TALEN constructs (section 2.5.2); however, for longer TALENs complete reads of all RVDs might not be possible using Sanger sequencing.
   3. Set up Golden Gate reaction #2 for each single TALEN. 150 ng of each pFUS vector, 150 ng of pLR-HD, pLR-NG, pLR-NI, pLR-NN (last “half-repeat”) according to the design of the RVD sequence and for the left TALEN use 75 ng of pCAG-T7-TALEN-ELD-Destination and for the right TALEN use 75 ng pCAG-T7-TALEN-KKR-Destination (or vice versa). Add 1 µl Esp3I, 1 µl T4 DNA ligase, 2 µl 10x T4 DNA ligase buffer, H₂O to 20 µl total reaction volume.
   4. Place Golden Gate reactions in a thermo cycler.
      Program:
      37 °C 5 min
      16 °C 10 min
      10 cycles
      37 °C 15 min
      80 °C 5 min
   5. Use reactions from section 2.3.4 to transform E. coli.
4. Day 4 – Confirmation of Correct TALEN Assembly
   1. Screen 1-3 white colonies from each plate by colony PCR with primers TAL_F1 and TAL_R2 (see Table 1 for primer sequences). Correctly assembled TALENs show a PCR product with a length corresponding to the total number of RVDs incorporated (Figure 1D, this band is sometimes difficult to detect while the “ladder effect” represents a robust indicator of successful assembly).
      PCR program:
      95 °C 3 min
      95 °C 30 sec
      55 °C 30 sec
      72 °C 3 min
      30-35 cycles
      72 °C 10 min
   2. Use confirmed correct clones to start overnight bacterial cultures (2-5 ml LB with 100 µg/ml ampicillin).
5. Day 5 – Confirmation of Correct TALEN Assembly
   1. Perform “minipreps” to isolate pCAG-T7-TALEN plasmids.
   2. If sequencing was not performed in section 2.3.2, use primers TAL_Seq_5-1 and TAL_R2 (see Table 1 for primer sequences) to determine correct RVD assembly in full-length TALEN.

3. Nuclease mRNA Synthesis

1. Generation of DNA Template
   1. Prepare high quality midi or maxipreps of pCAG-T7-TALEN plasmids for mRNA synthesis.
   2. Linearize 10 µg of the TALEN or ZFN mRNA synthesis plasmid using a restriction enzyme that preferentially cleaves downstream and in close proximity to the nuclease STOP codon (for pCAG-T7-TALEN vectors use SacI). mRNA synthesis plasmids typically include a T7 or SP6 phage promoter upstream of the nuclease coding sequence.
   3. Run 200-500 ng of digested plasmid on a 0.7-1% agarose gel to check for complete digestion.
   4. Remove salts from plasmid digest by precipitating the DNA with 0.1 volume sodium acetate and 3 volumes ethanol for 1 hr at room temperature. Pellet the DNA by centrifugation at 14,000 x g or more for 10 min, wash the pellet with 200 µl 70% ethanol, spin for another 5 min, remove the ethanol, air dry the pellet and resuspend in an appropriate volume of RNAse-free water. Purification of the linearized plasmid is also possible using a column-based system, e.g. QIAquick PCR Purification Kit.
   5. Determine the concentration of the linear template and use 1 µg to set up in vitro transcription.
2. mRNA Synthesis and Polyadenylation
   1. For in vitro transcription of pCAG-T7-TALEN plasmids use the mMESSAGEmMACHINE T7 Ultra Kit. Set up the transcription reaction for each TALEN on ice: to 20 µl with nuclease-free water, 10 µl T7 2x NTP/ARCA, 2 µl 10x T7 Reaction Buffer, 1 µg of DNA template, 2 µl T7 enzyme mix. Mix the reaction and incubate for 1-2 hr at 37 °C.
   2. Use the complete 20 µl transcription reaction mix to set up the polyadenylation reaction on ice: 36 µl of nuclease-free water, 20 µl 5x EPAP buffer, 10 µl 25 mM MnCl2, 10 µl 10 mM ATP. Mix and remove 2.5 µl of the reaction mix as control samples L1 and R1 (for left and right nuclease, respectively). Add 4 µl of E-PAP enzyme and incubate the reaction for 45-60 min at 37 °C. Remove another 2.5 µl of reaction mix as control samples L2 and R2.
3. mRNA Purification
   1. Use NucAway Spin Columns for mRNA purification (buffer exchange and removal of unincorporated nucleotides). Tap columns to settle dry gel in the column's bottom. Hydrate column with 650 µl of RNAse-free microinjection buffer (1 mM Tris-Cl, 0.1 EDTA, pH 7.5). Cap, vortex, tap out air bubbles, and hydrate for 5-15 min at room temperature.
   2. Place the column in a collection tube and spin at 750 x g, 4 °C for 2 min to remove excess interstitial fluid. Discard collection tube and place column in a 1.5 ml elution tube.
   3. Apply complete reaction mix from section 3.2.2 to column and spin at 750 x g, 4 °C for 2 min. Nuclease mRNA will now be dissolved in microinjection buffer. Remove 2.5 µl of purified samples L3 and R3.
   4. Store mRNA at -80 °C until microinjection aliquots are prepared.
4. mRNA Gel Electrophoresis
   1. Clean a gel chamber to remove RNAse contaminations using 10% SDS solution or RNaseZAP.
   2. Prepare a 1% agarose gel in 1xTBE running buffer.
   3. Mix each RNA sample L1/R1, L2/R2, L3/R3 with 3 volumes of NorthernMax Formaldehyde Load Dye and incubate for 15 min at 65 °C.
   4. Load samples and a RNA size ladder (e.g. RNA Millennium size marker) on the gel and run gel at 10 V/cm in 1x TBE until loading dye reaches the end of the gel.
   5. Stain the gel using SYBR green solution (Invitrogen) for 30-60 min with agitation at room temperature.
   6. Image the gel and compare sizes of L1/R1 with L2/R2 and L3/R3. Samples L2/R2 and L3/R3 should show bands of an increased size relative to L1/R1 indicating successful polyadenylation in (Figure 2).
   7. Determine mRNA concentration using a spectrophotometer.
   8. Prepare mRNA aliquots for microinjections by mixing left nuclease and right nuclease mRNA in a 1:1 ratio. We recommend preparing aliquots with a total concentration of 200 ng/µl (100 ng/µl of each nuclease) by diluting with microinjection buffer. Store mRNA aliquots at -80 °C.

4. Embryo Isolation and Microinjection

1. Embryo Isolation. This protocol has been successfully used with C57BL/6J and BDF1 strains and can most likely be adapted to other strains commonly used in microinjection experiments such as FVB or CBF1. The mice are housed under a 12 hr-12 hr light-dark cycle in a temperature- and humidity-controlled facility with food and water ad libitum.
   1. Superovulate donor females to increase the yield of embryos. 16 females (4 weeks old) are superovulated by intraperitoneal (i.p.) injection of 5 IU pregnant mare's serum gonadotropin (PMSG), followed by i.p. injection of 5 IU human chorionic gonadotropin (hCG) 48 hr later.
   2. Mate the 16 superovulated females to 16 breeding age (2-8 months) males immediately after hCG injection.
   3. Sacrifice the females using an approved euthanasia protocol such as CO₂ inhalation.
   4. Recover the fertilized oocytes. Oocytes are collected from oviducts the next morning and then freed from any remaining cumulus cells by a 3-5 min treatment in 0.1% bovine hyaluronidase dissolved in M2 medium. Depending on the mating performance and the strain used the embryo yield may vary. 16 C57BL/6J females usually produce 150-250, whereas BDF1 yield 300-400 fertilized oocytes, respectively.
2. Embryo Microinjection
   1. Inject the nuclease mRNA. Pronuclear stage oocytes are typically injected in the early afternoon. Cytoplasmic mRNA microinjection is carried out in M2 medium under mineral oil using an inverted microscope equipped with Nomarski DIC using 20X objective and with embryo micromanipulators as well as an injection unit. We recommend commencing the injections of TALEN mRNA at a concentration of 10 ng/µl (20 ng/µl in total).
   2. Aspirate the oocyte with a holding capillary and inject the nuclease mRNA in the cytoplasm of the embryo avoiding contact with the pronuclei. The injection should be shallow, close to the plasma membrane. The injection volume should be kept low and the microinjection needle should be withdrawn at the first sign of cytoplasmic distension. A microinjection series will normally consist of 100-300 oocytes. Typically, at least 80-90% of the embryos should survive the injection without immediate lysis.
   3. Following microinjection, place the embryos for 1 hr in M16 (Sigma) medium at 37 °C and 5% CO₂ and transfer the surviving embryos into pseudopregnant foster mothers.

5. Surgical Embryo Transfer

1. Prepare pseudopregnant embryo recipients (foster mothers) one day before mRNA microinjection. Mature females (3-6 months old) of a robust outbred strain, such CD-1, are well suited for the role. Induce pseudopregnancy by mating the females on a previous day with either surgically or genetically vasectomized males²⁸. Only 0.5 dpc females with a clear copulatory plug are used as embryos recipients. In unused females the pseudopregnancy disappears after around 3 weeks allowing their repeated use.
2. Anaesthetize the 0.5 dpc foster mother females by i.p. injection of ketamine, xylazine (120 mg/kg and 16 mg/kg, respectively). This formulation guarantees ~30 min surgical tolerance time is more than sufficient for the projected operation time of 5-10 min.
3. Position the anaesthetized animal on its abdomen on a warm surface and disinfect the area of incision with an appropriate disinfectant such as 70% ethanol or chlorhexidine and alcohol mixture.
4. Incise the skin on the back of the animal and open the peritoneal cavity with sterile scissors.
5. Visualize and externalize the uterine horn by pulling on the fat pad attached to the ovary. Keep the organs moist using warm 0.9% NaCl solution. Keep in mind that the surgical site may have to be draped with sterile dressing or shaved in order to conform to local veterinary guidelines.
6. Immobilize the reproductive organs by clipping the ovary fat pad with a bulldog clamp.
7. Gently grasp the oviduct just upstream of the ampulla with watchmaker forceps and use a 30 G hypodermic needle to create a small hole in the oviduct wall.
8. Withdraw the needle and reinsert a thin glass capillary containing the embryos into the hole allowing the placement of the embryos in the ampulla by gently blowing into the mouthpiece of the capillary holder. Withdraw the capillary once the embryos are deposited in the ampulla and replace the reproductive organs back in the body cavity.
9. Close the peritoneal cavity with a series of sterile sutures (Prolene 6-0).
10. Close the skin using 1-2 wound clips (Autoclip 9 mm) depending on the size of the opening.
11. Following the operation return the animals to their home cage and supervise them until the effect of anesthesia wear off.
12. In order to minimize stress, house the mice in stable social groups (2-4 animals/type III cage) whenever possible.
13. Apply post-operative analgesics in the form of Dafalgan added to the drinking water (Paracetamol 200 mg/kg BW) for 3 days after the surgery.

6. Analysis of Founders by PCR and T7 Endonuclease or Restriction Enzyme Digestion

1. Design primers to amplify a region between 200-700 bp around the nuclease binding site. The distances forward primer-nuclease spacer region and nuclease spacer region-reverse primer should be sufficiently different to allow detection of two separate bands for digested products on an agarose gel (see Figures 3 and 4 for examples).
2. Run PCR with optimized conditions. Check PCR amplicon size on an agarose gel.
3. Optional: Purify PCR products, e.g. with QIAquick PCR Purification Kit to remove salts and nucleotides from the PCR mix. Many restriction enzymes are active in PCR samples complemented with the appropriate restriction enzyme buffer and purification is not needed. We have also successfully used T7 endonuclease in QiagenTaq PCR buffer complemented with NEBuffer 2.
4. 1. T7 endonuclease assay. Mix 17 µl of PCR product with 2 µl of NEBuffer 2 and run heteroduplex formation (Figure 3b) program in a thermocycler:
      95 °C 2 min
      95 °C to 85 °C (-2 °C/sec)
      85 °C to 25 °C (-0.1 °C/sec)
      4 °C hold.
      Add 1 µl of T7 endonuclease to each sample and incubate at 37 °C for 20 min. (The surveyor assay (Transgenomic), which relies on CEL 1 nuclease, can also be used here. Please refer to the manufacturer's instructions.)
   2. Restriction digest of PCR product. Mix 17 µl of PCR product with 2 µl of 10x restriction enzyme buffer and 1 µl of restriction enzyme. Digest at appropriate temperature for 1 hr or more.
5. Add DNA loading dye to samples and run on a 2% agarose gel to detect distinct digestion patterns for wild-type animals and founders carrying mutated alleles. See Figures 3 and 4 for expected results.
6. Clone PCR products of founders positive for mutated alleles for Sanger sequencing (e.g. by TA-cloning into pGEM-T Easyor directly sequence mixtures of PCR products using next-generation sequencing.