CRISPR-mediated engineering of mouse knockins was performed using the workflow described by the following figure:

Figure 1. Workflow for efficient genome engineering via ex vivo microinjection of mouse embryos at the one-cell stage. First, the CRISPR editing components (Cas9/sgRNA [crRNA:tracrRNA duplex]), RNP complex, and ssDNA HDR templates are prepared. The CRISPR components are then microinjected into one-day-old mouse zygotes and transferred to pseudopregnant foster mothers. Seven weeks after microinjection, biopsy samples are collected from the pups derived from the microinjected zygotes. The genotyping step includes two rounds of PCR with primers designed to confirm whether the knockin insertions are complete and there are no truncations or duplications. Finally, the DNA from positive founders are sequenced by Sanger sequencing.

• Microinjection buffer (T₁₀ E_0.1 buffer) (10 mM Tris-HCl and 0.1 mM EDTA prepared with sterile filtered, embryo-tested water; e.g., Millipore Sigma, Cat. # W1503)
• Target-specific crRNA and tracrRNA (each stock 100 µM, suspended in microinjection buffer [T₁₀ E_0.1 buffer]; alternatively, in vitro-transcribed or synthesized single-guide RNA [sgRNA] can be used)
• SpCas9 protein (1 µg/µl working stock diluted in SpCas9 dilution buffer and stored at –20°C)
• SpCas9 dilution buffer (300 mM NaCl, 10 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 500 µg/ml BSA, and 50% glycerol prepared in embryo-tested water, pH 7.4 at 25°C) (NEB, Cat. # B8002S)
• Single-stranded DNA (ssDNA) HDR template generated using the Guide-it Long ssDNA Production System (Cat. # 632644)
• Urea-containing loading dye (e.g., Novex TBE-Urea Sample Buffer (2X), Thermo Fisher, Cat. # LC6876)
• Centrifugal filters (low-protein-binding PVDF membrane; e.g., Ultrafree-MC Centrifugal Filter, Millipore Sigma, Cat. # UFC30VV25)
• Certified sterile, nuclease-free 0.5-ml microcentrifuge tubes (e.g., Eppendorf Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes, Thermo Fisher, Cat. # 022600001)
• Materials needed for microinjection and embryo transfer (please see Wefers et al. 2017 for details)

This protocol is for the generation of transgenic knockin mice containing a fluorescent protein in frame with an endogenous gene separated by a P2A signal. Gene editing was performed on one-day-old mouse zygotes via microinjection of the RNP complex together with a 1,200-nt long ssDNA HDR template encoding the insert sequence (~800 nt) flanked on either side by homology arms of ~200 nt.

Preparation of editing reagents

1. Preparation of ssDNA HDR template
   
   
   1. Preparation of dsDNA substrate by PCR

      The dsDNA substrate was generated by PCR using a forward and a reverse primer complementary to the donor template. The reverse primer was designed with a 5' phosphorylation allowing for the generation of the ssDNA encoding the sense strand in the next step.

      PCR reaction:		Cycling conditions:
      50 µl	PrimeSTAR Max Premix (2X)	98°C 10 sec	40 cycles
      40 ng	Template DNA (encoding for HDR template)	55°C 5 sec
      2 µl	Forward primer	72°C 15 sec
      2 µl	Reverse primer (5' phosphorylated)
      X µl	RNase-Free Water
      100 µl	Total volume

      The specificity and yield of the PCR product were confirmed by agarose gel electrophoresis prior to further processing (Figure 2, Panel A). The PCR product was column purified, yielding approximately 10.7 µg of dsDNA.

   2. Generation of ssDNA

      To generate ssDNA, the PCR product obtained in the previous step was subjected to two short, consecutive strandase reactions (Strandase A and Strandase B) to yield ssDNA as specified by the kit protocol, using the conditions indicated below.

      Strandase A reaction:		Incubate as follows:
      10 µg	dsDNA substrate	37°C   6 min (5 min/kb) 80°C   5 min 4°C     until next step
      5 µl	Strandase A Buffer (10X)
      5 µl	Strandase Mix A
      X µl	RNAase-Free Water
      50 µl	Total volume

      Strandase B reaction (processing Strandase A reaction mixture):		Incubate as follows:
      50 µl	Strandase A reaction mixture	37°C   6 min (5 min/kb) 80°C   5 min 4°C     until next step
      50 µl	Strandase B Buffer (2X)
      1 µl	Strandase Mix B
      101 µl	Total volume

      The ssDNA generated from the Strandase B reaction above was purified using the NucleoSpin Gel and PCR Clean-Up kit (supplied with the ssDNA kit) and eluted in 30 µl of the microinjection buffer (in substitution of the standard NE buffer provided by the kit). The ssDNA concentration was measured by NanoDrop spectrophotometry (1 OD₂₆₀ = 33 ng/µl) and was approximately 66 ng/µl (total yield of ~2 µg).

      NOTE: The ssDNA is eluted directly from the DNA purification column using microinjection buffer, allowing the direct use of the ssDNA for microinjection after purification.

   3. Quality control of ssDNA template

      Following purification of the strandase reactions, the successful production of ssDNA HDR templates was confirmed via two different methods:

      Agarose gel electrophoresis:

      Strandase products were treated with urea-containing loading dye at 95°C for 5 min to eliminate secondary structures prior to electrophoresis and were run side-by-side with corresponding undigested dsDNA PCR products for size comparison purposes on a 1.5% TAE agarose gel using 0.005% (v/v) SERVA HiSens Stain G (Figure 2, Panel B, below).

      

      Figure 2. Production of ssDNA HDR template. Panel A. Analysis of the dsDNA PCR template prior to strandase digestion. Left lane: 1-kb ladder; Lane 1: dsDNA PCR amplification product. Panel B. Analysis of ssDNA product following strandase treatment and purification. Left lanes: 100-bp ladder; 1-kb ladder. Lane 1: dsDNA (1,206 bp, 100 ng); Lane 2: sense ssDNA (200 ng); Lane 3: RiboRuler ladder.

      Sanger sequencing:

      To confirm that the strandase treatment did not result in truncation of the generated ssDNA and that there was no trace of dsDNA, three independent sequencing reactions were performed with different annealing primers:

      • The reverse primer used in the PCR (Section 1.A) to sequence the end of the right homology arm (the sequencing would fail if the right arm is truncated)
      • Internal reverse primer running upstream to the PCR forward primer to sequence the end of the left homology arm
      • Internal forward primer running downstream and complementary to the antisense strand which should fail if there is no trace of dsDNA

      The sequencing results indicated that there was no obvious size reduction or presence of dsDNA (data not shown).

2. Preparation of microinjection aliquots containing the editing reagents
   
   

   Aliquots of the microinjection reagents (containing the editing components) can be prepared beforehand and stored at –80°C until use. One injection capillary (filled with 3 µl of the microinjection mix) can be used to inject >100 embryos at once; however, sometimes capillaries break or get clogged. Therefore, it is advisable to prepare single-use, 10-µl aliquots of the reagents to have enough to fill two new capillaries for each session of microinjections.

   NOTE: Pronuclear injection efficiency is variable from day to day (due to the yield of embryo collection, implantation of embryos in fosters, number of pups born, etc.). Therefore, it is advisable to inject on several days to ensure a sufficient number of pups (and consequently pups carrying the desired edit). Since identical microinjection aliquots are used on 2–3 different injection days, the aliquot mix might be scaled up by 2–3 fold and frozen in 10-µl single-use aliquots. This will reduce variation between the aliquots and their degradation due to repeated freeze/thaw cycles.

   1. Preparation of crRNA:tracrRNA mix
      1. Combine the following in a sterile, nuclease-free microcentrifuge tube:
         • 1 µl   crRNA (10 µM)
         • 1 µl   tracrRNA (10 µM)
         • 8 µl   microinjection buffer
      2. Incubate the crRNA:tracrRNA mix in a thermal cycler running the following program:
         • 95°C 5 min
         • Cool to 25°C (decrease temperature at a rate of –5°C/min)
      3. Store mix on ice until use.
   2. Preparation of pronuclear injection (PNI) aliquot mix
      1. Prepare a 10-µl aliquot of microinjection mix for each PNI day/session in a sterile, nuclease-free microcentrifuge tube as shown below, and incubate the mixture at RT for 15 min.
         
         

         NOTE: Scale up according to the total number of PNI day/sessions as explained in the previous section.

         Reagent	Volume per aliquot	Concentration (stock)	Concentration (final)
         crRNA:tracrRNA mix (previous section)	0.6 µl	10 µM	0.6 µM
         SpCas9 protein	0.3 µl	1 µg/µl (6.1 µM)	30 ng/µl (0.2 µM)
         Microinjection buffer (T10E0.1)	9.1 – X µl*	–	–

         
         

         *The amount of microinjection buffer (T₁₀E_0.1) included per aliquot varies depending on the volume of ssDNA template (X) added following the 15-min incubation (Step 2, below).

      2. After incubation, add X µl of ssDNA (or single-stranded donor oligonucleotides, ssODN) template per 10 µl aliquot to obtain a final concentration of 10 ng/µl. For example, in the case of an ssDNA with a stock concentration of 66 ng/µl, add 1.5 µl of ssDNA and 7.6 µl of microinjection buffer.
         
         

         NOTE: If an ssODN (<200 nt) is injected, its final concentration must be 25 ng/µl.

      3. Filter the PNI aliquot mix using a centrifugal filter for 2 min at 12,000g.
      4. Transfer 10-µl aliquots of PNI mix into separate 0.5-ml microcentrifuge tubes.
      5. Store aliquots at –80°C until the day of injection.
3. Microinjection and implantation of mouse embryos
   
   

   On the day of injection:

   1. Thaw PNI aliquots on ice.
   2. Spin the aliquots for 5 min at 20,000g.
   3. Transfer 9.5 µl of supernatant from each aliquot to a fresh, nuclease-free 0.5-ml microcentrifuge tube.
   4. Keep isolated supernatant from PNI aliquot on ice until injection.

For the exact procedure of the microinjection and implantation of mouse embryos, please follow the protocol explained in Wefers et al. 2017.

Seven weeks after microinjection and implantation of the embryos, genotyping was performed on the F₀ founders via both PCR analysis and Sanger sequencing of the genomic target region.

The first round of genotyping consisted of three independent PCR reactions (Figure 3, Panel A) designed to amplify the junctions between the targeted sequence and insert (Out-In/Fw 1-Rev 1 and In-Out/Fw 2-Rev 2) as well as the whole insert sequence (In-In/Fw 2-Rev 1). The correct band amplification in all three PCR reactions confirms the presence of the insert and its correct orientation.

Figure 3, Panel B shows a representative agarose gel of samples analyzed using the primer pair Out-In/Fw 1-Rev 1. The analysis identified founders carrying full-length insertions at the correct genomic locus and in the correct orientation (Founders 2, 4, and 6) as well as other founders carrying truncated inserts (Founders 1 and 3).

From the positive samples (F₀ mice) identified from the first PCR, a second round of PCR genotyping was performed using the primer set Fw 1-Rev 2 (Out-Out). These primers anneal outside the homology arms and help confirm whether the entire insert sequence was incorporated successfully without any truncations or duplications. A representative agarose gel of samples analyzed using this primer pair Out-Out/Fw 1-Rev 2 is presented below (Figure 3, Panel C).

Samples that tested positive for successful integration of the insert were then confirmed by Sanger sequencing of both the 5' and 3' junctions. The final genotyping results are shown in Table I. Overall, 48% of the pups were born with a knockin and from this subpopulation, 70% had a correct insertion of the sequence encoding for the fluorescent protein.

Figure 3. PCR genotyping experiment and results. Different sets of primers were used to identify the founders (F₀) carrying the correct insertion of the fluorescent protein at the target site. Panel A. The locations of the primers and the expected amplicon sizes for the four different PCR reactions. Panel B. Representative results for Out-In/Fw 1-Rev 1 PCR. Lanes are marked with founder numbers and correspond to amplicon sizes of ~510 bp (Founder 1), 1,054 bp (Founders 2, 4, and 6), ~880 bp (Founder 3); PC: positive control; NTC: no template control. Panel C. Example of results for Out-Out/Fw 1-Rev 2 PCR. Lane 1: 1-kb ladder; Lane 2: positive founder; Lane 3: WT control; Lane 4: no template control; Lane 5: 100-bp ladder.

Wefers, B., Bashir, S., Rossius, J., Wurst, W., & Kühn, R. Gene editing in mouse zygotes using the CRISPR/Cas9 system. Methods 121, 55–67 (2017).