In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells.

: The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, transcribe single guide RNAs, and transfect RNPDs into various mammalian cells. Abstract The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, in vitro transcribe single guide RNAs, and transfect RNPDs into various mammalian cells. show that repair oligos can also be purified from excess BG using Gel-Pak cartridge purification in combination with EtOH purification. FACS analysis of the fluorescent reporter cells transfected with different RNPD systems: Non-treated, no RFP HPLC Glen RFP RFP ethanol precipitation followed by Gel-Pak cartridge


Background
The CRISPR-Cas9 system efficiently induces site-directed DSBs, which are repaired by cell-autonomous mechanisms. Since mammalian cells predominantly repair DSBs by NHEJ, * For correspondence: schwankg@ethz.ch.

Competing interests
The authors declare no competing interests. the CRISPR-Cas9 system mainly causes indel mutations at targeted loci. For many applications, precise repair via HDR from template DNA is however desired. Hence, several attempts have been made to increase the efficiency of accurate DSB repair, including biochemical alteration the repair pathways (Chu et Savic et al., 2018). Importantly, these approaches do not involve any potentially harmful chemical treatment to alter endogenous cellular processes, and due to the shorter half-life of ribonucleoprotein complexes compared to DNA, they exhibit reduced risks of generating off-target mutations (Kim et al., 2014). In addition, procedures for clinical-grade recombinant protein production are well established, and progress has been made to deliver Cas9 RNP complexes in vivo in animal models (Zuris et al., 2015;Wang et al., 2016;Lee et al., 2017;Staahl et al., 2017). In summary, using Cas9 complexes with linked DNA repair templates is a promising approach for gene editing therapies, where high rates of precise repair is desired.
Here, we provide a detailed protocol for linking the DNA repair template to Cas9 as demonstrated in Savic et al., 2018. The described approach is based on the SNAP-tag technology, which enables the generation of a covalently linked Cas9-DNA complexes in vitro. We explain how to label repair-oligonucleotides with Benzylguanine (BG), how to produce Cas9-SNAP fusion proteins, how to produce single guide (sg)RNAs, how to generate DNA-ribonucleoprotein complexes in vitro, how to transfect them into mammalian cells, and how to analyze correction efficiencies ( Figure 1).

22.
Liquid chromatography-mass spectrometry system and software for peak analysis (e.

7.
GraphPad's Prism7 software for statistical analyses To purchase software, please visit the respective company webpage.

A.
Generation of Benzylguanine (BG)-coupled repair template oligos

c.
Spin the Eppendorf tube(s) for 15 min at 13,500 x g at 4 °C.

e.
Spin for 10 min at 13,500 x g at 4 °C.

g.
Spin for 10 min at 13,500 x g at 4 °C.

h.
Remove the supernatant and air dry the pellet for 10-15 min.

i.
Dissolve the pellet in 200 μl RNase-free sterile water when proceeding with purification of the BG-coupled DNA on reversed phase HPLC. When using the Glen Gel-Pak cartridge purification method, dissolve the pellet in 1,500 μl RNase-free sterile water.

3.
Purification of BG-coupled DNA on reversed phase HPLC Inject residual 190 μl of the reaction mixture and collect the product containing fraction using the threshold estimated above.

e.
Remove solvent under vacuum from product containing fractions using a centrifugal concentrator, such as Genevac  Figure 2.
Note: If the lab has no access to a suitable HPLC system, the ethanol precipitated BG-coupled oligos can also be further separated from unreacted BG by using a size-exclusion based desalting column, such as Glen Gel-Pak 1.0. For Glen Gel-Pak based purification follow the manufacturer's protocol. In our hands, the combination of ethanol precipitation and Glen Gel-Pak based purification resulted in only a slightly lower coupling efficiency compared to the purification on reversed phase HPLC ( Figure 3). In addition, when we functionally tested RNPD complexes purified with different methods in fluorescent HEK reporter cells, we found that the correction efficiencies were comparable ( Figure 4).

4.
Quality control of purified BG-coupled DNA by LC/MS i.

ii.
Tap gently to mix and incubate tube on ice for 30 min.

iii.
Incubate cells at 42 °C for 45 s in a heat block.

iv.
Immediately place tube back on ice for 3 min.

v.
Add 350 μl of room temperature sterile LB medium.

vi.
Incubate at 37 °C while shaking for 60 min.
vii. Plate 100 μl of the mixture on an LB agar plate containing 50 μg/ml of kanamycin and 33 μg/ml of chloramphenicol. Use sterile glass beads or a Drigalski spatula for spreading the cells.

b.
Preparation of media and stock solutions (Day 1)

i.
Prepare 8 x 750 ml LB media in 2 L Erlenmeyer flasks.

ii.
Prepare 1 x 100 ml LB media in a 250 ml Erlenmeyer flask for pre-culture.

iii.
Prepare antibiotic stock solutions.

i.
Add 100 μl of 50 mg/ml kanamycin stock solution and 33 μl of 50 mg/ml chloramphenicol stock solution to 100 ml of sterile LB medium prepared on Day 1.

ii.
Pick a single colony from the LB agar plate of E. coli Rosetta 2 (DE3) cells transformed with P120 on Day 1. For picking, you can use a sterile pipette tip or an autoclaved tooth pick.

iii.
Incubate pre-culture at 37 °C overnight while shaking at 220 rpm. Start pre-culture in the late afternoon.

iv.
Prepare 10 ml of IPTG stock solution. i.
Add 750 μl of 50 mg/ml kanamycin stock solution and 750 μl of 33 mg/ml chloramphenicol stock solution to each of the 750 ml LB media from Day 1.

ii.
Inoculate each flask with 10 ml of the overnight preculture prepared on Day 2.

iii.
Incubate the cultures at 37 °C while shaking at 110 rpm.

iv.
Check the optical density at a wavelength of 600 nm (OD 600 ) of the culture every hour until it reaches 0.6-0.8.

v.
Reduce the temperature in the incubator to 18 °C.

vi.
Remove a 1 ml sample of the culture and centrifuge for 10 min at 17,000 x g. Europe PMC Funders Author Manuscripts iv.

v.
Cell pellets can be stored at -20 °C (pellets can be also frozen and stored after resuspension in Lysis buffer).

vi.
Prepare buffers for Ion exchange and Size exclusion chromatography (500 ml of Heparin buffer A, 500 ml of Heparin buffer B, 1 L of SEC buffer).
If cell pellets were frozen after harvesting, thaw the cell pellets on ice.

b.
Resuspend pellets in a total volume of 40 ml of Lysis buffer (~2.5-5 ml Lysis buffer per gram of cell pellet).

c.
To lyse the cells use a sonicator with the following settings: 6 min total time, 1 s on, 2 s off, 20% amplitude. The cell suspension noticeably changes color and viscosity during lysis.

d.
Transfer the lysed cells into Oak Ridge centrifuge tubes and centrifuge for 45 min at 30,000 x g at 4 °C.

f.
Use a peristaltic pump to wash a 10-ml self-packed Ni-NTA Superflow column (or two 5 ml QIAGEN Ni-NTA Superflow prepacked cartridges connected in series) with 5 column volumes of water at a flow rate of 3 ml/min.

g.
Wash the column with another 5 column volumes of Wash buffer.

h.
Load the cleared lysate (~50 ml) onto the equilibrated column with a flow rate of 3 ml/min.
Note: There might be still unbound SpCas9-SNAP protein left in the flow-through. It can be loaded again on the Ni-NTA column for a second round of purification. Equilibrate the Ni-NTA column in Wash buffer before reloading the flowthrough.

i.
Remove a 5 μl sample of the flow-through for SDS-PAGE analysis ("flow-through" sample, Figure 8) and mix it with 20 μl 1x Laemmli buffer. Store at -20 °C.

j.
Wash the pumps of the Äkta FPLC system with Wash buffer (pump A) and Elution buffer (pump B) and flush the flow-path of the system with Wash buffer until the absorbance signal (280 nm wavelength, A 280 ) in the UV detector reaches a stable baseline.

k.
Connect the loaded Ni-NTA column to the Äkta FPLC and wash the column with Wash buffer with a flow rate of 3 ml/min until A 280 reaches baseline level ( Figure 5).

l.
Elute the protein with 50% Elution buffer and collect in 1.8 ml fractions in a 96-well 2 ml deep-well block, or in tubes with appropriate capacity ( Figure 5).

m.
Pool the peak fractions in a clean 50 ml tube and determine the concentration by measuring the absorbance at 280 nm using a NanoDrop UV-VIS spectrophotometer. The extinction coefficient of His 6 -MBP-TEV-SpCas9-SNAP is 1416140 M -1 cm -1 .

n.
Remove a 5 μl sample of the pooled fractions for SDS-PAGE analysis ("Ni-NTA elution" sample, Figure 8) and mix it with 20 μl 1x Laemmli buffer. Store at -20 °C.

o.
Add 1 mg of recombinant, His-tagged TEV per 50 mg of Cas9 SNAP (as determined from absorbance measurement).

Purification of Cas9 SNAP: Part II (Day 5)
a.
Remove a 5 μl sample of the overnight TEV cleavage reaction for SDS-PAGE analysis ("TEV cleavage" sample, Figure 8) and mix it with 20 μl 1x Laemmli buffer. Store at -20 °C.

b.
Heat the collected samples for SDS-PAGE analysis for 5 min at 95 °C.

c.
Load 3 μl on a 12% SDS PAGE gel with 15 wells to check if cleavage is complete.

d.
Connect a 5 ml Heparin FF column to a peristaltic pump and wash with at least 5 column volumes of water at a flow rate of 3 ml/min.

e.
Equilibrate the column with at least 5 column volumes of Heparin buffer A.

f.
Load the overnight cleavage reaction onto the column and collect the flow-through in a clean 50 ml tube.

g.
Remove a 5 μl sample of the flow-through for SDS-PAGE analysis ("Heparin flow-through" sample, Figure 8) and mix it with 20 μl 1x Laemmli buffer. Store at -20 °C.

h.
Wash the pumps of the Äkta FPLC with Heparin buffer A and Heparin buffer B and flush the flow path of the system with Heparin buffer A until it reaches a stable baseline at the absorbance of 280 nm.

i.
Connect the loaded Heparin FF column to the Äkta FPLC system and wash the column with Heparin buffer A at a flow rate of 3 ml/min until the A 280 signal reaches a steady baseline ( Figure 6).

j.
Elute with a linear gradient to 50% Heparin buffer B over 20 column volumes (100 ml).

k.
Collect eluted protein in 1.8 ml fractions in a 96-well 2 ml deep-well block, or in tubes with appropriate capacity ( Figure  6).

m.
Remove a 20 μl sample of the pooled fractions for SDS-PAGE analysis ("Heparin peak" sample, Figure 8) and mix it with 5 μl 5x Laemmli buffer. Store at -20 °C.

o.
Transfer concentrated sample into a 15 ml tube and centrifuge for 5 min at 4,500 x g at 4 °C to remove any precipitated material.

p.
In the meantime, equilibrate a HiLoad 16/600 Superdex 200 pg with 1 column volume of SEC buffer on the Äkta FPLC at a flow rate of 1 ml/min.

q.
Prepare a 5 ml injection loop by washing with 15 ml of SEC buffer on the Äkta FPLC. Flush the injection port with 10 ml of SEC buffer using a 10 ml syringe.

r.
Load the concentrated sample on the HiLoad 16/600 Superdex 200 pg via the 5 ml injection loop at a flow rate of 1 ml/min. Elute with SEC buffer at a flow rate of 1 ml/min (Figure 7).

s.
Collect elution in 1.8 ml fractions in a 96-well 2 ml deep-well block, or in tubes with appropriate capacity (Figure 7).

t.
Pool the peak fractions and determine the concentration by measuring the absorbance at 280 nm using the NanoDrop UV-VIS spectrophotometer. Remove a 20 μl sample of the pooled fractions for SDS-PAGE analysis ("SEC peak" sample, Figure  8) and mix it with 5 μl 5x Laemmli Buffer. Store at -20 °C.

u.
Concentrate the SpCas9-SNAP protein to the required concentration (~1 mg/ml) by using an Amicon Ultra-15 Centrifugal Filter Unit (MWCO of 100 kDa). SpCas9-SNAP can be concentrated up to 10-20 mg/ml as long as the SEC buffer contains at least 500 mM KCl.

v.
Prepare 50 μl aliquots of purified SpCas9-SNAP protein, freeze in liquid nitrogen and store at -80 °C.

C.
Production of sgRNA Guide RNAs can be produced via in vitro transcription from a template DNA (see protocol below), or purchased from various sources (e.g., Integrated DNA Technologies).

a.
Purify the sgRNA by DNase I treatment followed by ethanol precipitation: i. Add 1 μl DNase I and incubate for 15 min at 37 °C in the Thermal Cycler.

ii.
Add DEPC-treated water up to 100 μl.

v.
Wash the pellet with 500 μl 75% ethanol and centrifuge at 17,000 x g for 15 min at 4 °C using Microlitre Centrifuge.

b.
Purify the sgRNAs further using RNA Clean and Concentrators, according to the manufacturer's protocol.
Measure the eluted RNA concentration on NanoDrop spectrophotometer. Store the RNA at -80 °C.

a.
Control the sgRNA on a denaturing 2% MOPS gel: i. Prepare the gel Heat 2 g agarose in 80 ml double distilled and deionized water using a microwave oven until dissolved, and then cool to 60 °C. Add 10 ml 10x MOPS running buffer, and 10 ml 37% formaldehyde (12.3 M), pre-warmed to 60 °C using a water bath. Mix and pour the gel using a comb that will form wells large enough to accommodate at least 25 μl. Let the gel settle for 30 min. Assemble the gel in the tank, and add enough 1x MOPS running buffer to cover the gel by a few millimeters. Remove the comb.
Note: Prewash the chamber, tray, and comb with soapy water/tween/SDS, wipe with RNase AWAY, and remove the leftover RNase AWAY with a paper tissue. Use autoclaved or UV-treated Erlenmeyer flask.
Warning: Formaldehyde is toxic through skin contact and inhalation of vapors. Manipulations involving

ii.
Prepare RNA samples and ladder

2)
Heat denature samples at 65 °C for 15 min using a Thermoshaker.

3)
Briefly spin down samples in a microcentrifuge and place on ice. iii.

RNA electrophoresis
Load the gel and run at 5-6 V/cm (approx. 80 V) until the bromophenol blue (the faster-migrating dye) has migrated at least 2-3 cm into the gel, then raise to 120 V and let run as far as 2/3 the length of the gel.

iv.
Stain the RNA gel using Sybr Gold

2)
Incubate for 45 min, shaking at room temperature. Wash 1 x 5 min in TE buffer followed by 1 x 5-min wash in double distilled and deionized water.
Note: Protect the gel from the light during incubation.

v.
Image on ChemiDoc imaging System to confirm the concentration and the RNA quality.
Note: An example of denaturing RNA gel with in vitro transcribed sgRNAs can be found in Figure

D.
Cell culturing

Culture HEK293T line
Notes:

i.
Change cell culture medium (for HEK293T medium, see Recipes) every other day.

ii.
Passage cells before they become fully confluent, approximately every 4 days. iii.
Cells grow at 37 °C in a humidified 5% CO 2 environment.

a.
Aspirate the medium from dish.

b.
Add 1 ml of TrypLE Express per 10 cm (for a 10 cm dish).

c.
Place the plate in the 37 °C incubator for 5 min.

d.
Once the cells detach, add 9 ml of DMEM medium and transfer the cells to a 15 ml conical tube.

e.
Centrifuge at 250 x g for 5 min at 4 °C.

f.
Aspirate the medium from the conical tube.

g.
Add 10 ml of fresh DMEM medium and split cells at a 1:10-1:15 dilution factor on to a new 10 cm dish and add enough additional medium to each plate for 10 ml total.

i.
Change cell culture medium (for K562 medium, see Recipes) every other day.

ii.
Passage cells before they become fully confluent, approximately every 4 days.

iii.
Cells grow at 37 °C in a humidified 5% CO 2 environment.

a.
Collect the K562 cells from the dish in a 15 ml conical tube.

b.
Centrifuge at 250 x g for 5 min at 4 °C.

c.
Aspirate the medium from the tube.

d.
Add 10 ml of fresh RPMI medium and split cells at a 1:5-1:10 dilution factor on to a new 10 cm dish and add enough additional medium to each plate for 10 ml total.

3.
Culture mouse embryonic stem cell line Notes:

i.
Change cell culture medium (for mESC proliferative growth medium, see Recipes) daily.

ii.
Passage cells every 2-3 days depending upon the growth rate of cells. The optimal condition is to maintain cells at approximately 80% confluency on Day 2 or 3.

iii.
Cells grow at 37 °C in a humidified 8% CO 2 environment.
iv. mECS grow on 0.2% gelatin-coated flasks/plates in absence of feeder cells.

a.
Aspirate the medium from the T75 flask.

d.
Place the flask in the 37 °C incubator for 5 min.

e.
Once cells begin to dissociate from the flask, transfer the detached cell aggregates to a 15 ml conical tube containing 5 ml mESC DMEM growth medium.

f.
Rinse the flask with an additional 3 ml of DMEM growth medium to collect any remaining aggregates. Add the rinse to the conical tube that contains the cells.

g.
Centrifuge the conical tube at 250 x g for 2 min at room temperature.

h.
Aspirate the medium from the conical tube and add the desired amount of growth medium. Split the cells at a 1:4 to 1:10 dilution factor into a new flask or dish and add enough additional growth medium to each flask or plate.

Covalent binding of Cas9-SNAP protein and BG-coupled oligonucleotide
Complex the BG-coupled repair oligo templates with Cas9-SNAP proteins directly before performing the transfection.

1.
Thaw the SpCas9-SNAP protein on wet ice.
Note: Very small samples such as 10 μl aliquots can be thawed by holding the tube in your hand.

2.
Once thawed, gently mix the sample using a micropipettor with a polypropylene tip to make sure the solution is homogeneous. Be careful not to introduce bubbles into the solution. When necessary, dilute the protein in Protein buffer.

4.
Mix by flicking the microcentrifuge tubes.

5.
Briefly spin down samples in a microcentrifuge.

7.
Briefly spin down samples in a microcentrifuge.

8.
Place the sample on ice until used for cell transfection.
Note: An example of silver-stained SDS-PAGE gels showing band shifts that confirm covalent linkage of Cas9-SNAP proteins to BG-coupled oligos can be found in Figures 3e and 3f

Seeding HEK293T cells
One day prior to transfection (24 h): Seed HEK293T cells at a density of 120,000-140,000 cells per well in 24-well plates.

a.
Aspirate the medium from dish.

b.
Add 1 ml of TrypLE Express (for a 10 cm dish).

c.
Place in a 37 °C incubator with 5% CO 2 environment for 5 min.

d.
Once the cells detach, add 9 ml of DMEM medium and transfer the cells to a 15 ml conical tube.

e.
Pipette up and down several times with 5 ml Serological pipette to ensure single cell suspension.
f. Perform a cell count using a hemocytometer or automated cell counter.

g.
Dilute the cells with DMEM medium at the desired final concentration of 120,000-140,000 cells/well.

Seeding mES cells
One day prior to transfection (24 h): Seed mES cells at a density of 180,000-190,000 cells per well in 6-well plates.

a.
Aspirate the medium from a flask or dish.

c.
Add 1 ml 0.05% Trypsin containing EDTA to the flask or dish.

d.
Place the flask in the 37 °C incubator with 5% CO 2 environment for 5 min.

e.
Once cells begin to dissociate from the flask, transfer the detached cell aggregates to a 15 ml conical tube containing 5 ml mESC growth medium.

f.
Rinse the flask with an additional 3 ml of growth medium to collect any remaining aggregates. Add the rinse to the conical tube containing the cells.

g.
Perform cell count using a hemocytometer or automated cell counter.

h.
Dilute the cells with DMEM growth medium without PenStrep at the desired final concentration of 180,000-190,000 cells/ well.

3.
Seeding K562 cells On the day of transfection: K562 cells (suspension culture) can be distributed 6 h prior to transfection in 24-well plates at a density of 220,000-240,000 cells per well.

a.
Collect the K562 cells from the dish in a 15 ml conical tube.

b.
Centrifuge at 250 x g for 5 min at 4 °C.

c.
Aspirate the medium from the tube.

d.
Add fresh RPMI medium and pipette up and down several times with 5 ml Serological pipette to ensure a homogeneous cell suspension.

e.
Perform cell count using a hemocytometer or automated cell counter. Dilute the cells with RPMI medium at the desired final concentration of 120,000-140,000 cells/well.

Preparation of transfection reactions
Prepare transfection reactions by mixing following components in a laminar flow hood (see Table 3 for amounts, this example is for 24-well plate format): a.

c.
Briefly vortex Tube A and B, and spin down samples in a microcentrifuge.

d.
Incubate for 5 min at room temperature.

e.
Add the content of Eppendorf Tube B to Eppendorf Tube A, briefly vortex and spin the content down in a microcentrifuge.

f.
Incubate the complexes at room temperature for 15 min, to allow lipid particle formation.

g.
Add the transfection mixture to the desired wells, by slowly dropping the mixture on the medium.

h.
Gently mix the media via back and forth motion.
Note: The medium of the mES transfected cells should be replaced to mES culture medium with Pen-Strep 8 h posttransfection.

i.
Incubate at 37 °C with 5% CO 2 environment for 24 h.

j.
Next day (post-transfection), HEK293T and K562 cells can be transferred to 10 cm dish for further expansion of the transfected cells.

k.
The mES cells can be collected after 48 h post transfection for extracting genomic DNA.

l.
At Day 5 post-transfection, HEK293T and K562 cells can be analyzed by different methods. When a reporter-line is used, the correction and indel formation events can be visualized by fluorescence imaging and quantified by FACS. In case no reporter line used, cells can be collected for DNA extraction and analysis via next-generation sequencing (NGS).

G.
Correction efficiency analysis The protocols described here allow to assess the correction rates of the RNPD system in the cell line of interest. The protocols are designed for HEK293T cells, and would have to be adapted for the cell line of interest.

Microscopy imaging
As mentioned above, when a fluorescent reporter cell line is used, correction and indel formation events can be visualized by either fluorescence imaging or flow cytometry. One day post-transfection, the HEK reporter cell line can be transferred to Poly-L-lysine coated glass chamber slides and can be analyzed 5-7 days post-transfection.
Conformation and visualization by microscope imaging can be achieved as followed:

a.
A few hours before transferring the transfected cells, coat the glass chamber slides with Poly-L-lysine. Pour a few hundred microliters of 0.2% Poly-L-lysine solution on the bottom of the chamber slides, distribute homogeneously (the entire surface must be covered, e.g., 150 μl for 8-well chamber slide), incubate for minimum 1 h at 37 °C and finally remove the leftover Poly-L-lysine solution that is on the chamber slides.
Note: Store the chamber slides until use in a 37 °C incubator when using them on the same day or else store the chamber slides at 4 °C.

b.
Aspirate the medium of the HEK293T cells that got transfected the day before.

c.
Add 100 μl of TrypLE Express to each well (in case of 24-well plate).

d.
Pipette up and down several times with a P200 pipet to ensure single cell suspension.

e.
Add 240 μl pre-warmed DMEM medium (HEK293T cells) to the coated chamber slides.
f. Take 10 μl of the trypsinized HEK293T suspension and add this to the coated chamber slide. Let the HEK293T cells grow until they reach a confluency of 80%-90%. This will be probably around Days 5-7 post-transfection.

g.
Prior to the image session, add Hoechst 33342 to the cell culture medium to a final concentration of 0.1 μg/ml.

h.
Incubate the cells for 10 min at 37 °C with 5% CO 2 environment.
i. Start imaging session.

Flow cytometry analysis
Depending on the type of experiment, the best time point for flow cytometry analysis should be determined by the experimenter. In this protocol, we analyze HEK293T reporter cells at 5 days posttransfection. Important note when performing flow cytometry: Make use of proper controls for setting up your gating.
Quantification of correction and indel formation events by flow cytometry analysis can be achieved as followed:

a.
Aspirate the medium of dishes.

b.
Add 1 ml of TrypLE Express per 10 cm dish.

c.
Incubate for 3 min at 37 °C.

d.
Once the cells detach, add 4 ml of DMEM medium and transfer the cells to a 15 ml conical tube.

e.
Centrifuge at 250 x g for 5 min at 4 °C.

f.
Aspirate the supernatant.

g.
Add 1 ml FACS buffer with or without Sytox Red to the cell pellet and pipette up and down several times with P1000 pipet to ensure single cell suspension. Sytox Red can be added for excluding dead cells from the populations.

h.
Transfer the cell suspension to 5 ml test tubes with cell strainer cap.

i.
Place samples on ice until starting with the flow cytometry analysis on the BD LSR Fortessa cell analyzer.
Gently mix the reaction and briefly spin down content in a microcentrifuge.
iii. PCR cycling conditions used in this protocol: Step Note: Annealing temperatures should be optimized for each primer set to ensure that a single amplicon will be produced.

iv.
Run 10 μl of the PCR products on agarose gel (2%) to confirm correct amplicon size.

v.
Purify PCR amplicons by solid phase reversible immobilization (SPRI) bead cleanup using Agencourt AMPure XP reagent, according to the manufacturer's protocol.

vi.
Run 5 μl of the purified samples on agarose gel (2%) to confirm the purification step succeeded.

b.
Generation of pooled sequencing libraries ii.
Gently mix the reaction and briefly spin down content in a microcentrifuge.

iii.
PCR cycling conditions used were as follows in this protocol: Step Note: Before sequencing the libraries, it is recommended to perform a quality check on, e.g., Agilent 2200 TapeStation system.

Data analysis
Correction efficiencies in HEK293T fluorescent reporter cells are available in Savic et al., 2018. For obtaining correction rates in other cell types the here described protocols would have to be adapted.

A.
Flow cytometry
Forward versus side scatter (FSC-A vs. SSC-A) gating should be used to identify cells of interest.

b.
Doublets should be excluded using the forward scatter height versus forward scatter area density plot (FSC-H vs. FSC-A).

c.
Live cells should be gated based on Sytox-Red-negative staining.

d.
Live-gated cells should be further used to quantify the percentage of eGFP negative and turboRFP positive populations, in this example.