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Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double‐Stranded DNA Donors

Abstract Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genomes of mammalian cells has required the construction of plasmids containing selection markers and/or extended sequences homologous to the site of insertion (homology arms). Here we describe a streamlined protocol that eliminates all cloning steps by taking advantage of the high propensity of linear DNAs to engage in homology‐directed repair of DNA breaks induced by the Cas9 RNA‐guided endonuclease. The protocol uses PCR amplicons, or synthetic gene fragments, with short homology arms (30‐40 bp) to insert fluorescent reporters at specific genomic locations. The linear DNAs are introduced into cells with preassembled Cas9‐crRNA‐tracrRNA complexes using one of two transfection procedures, nucleofection or lipofection. The protocol can be completed under a week, with efficiencies ranging from 0.5% to 20% of transfected cells depending on the locus targeted. © 2019 The Authors.


INTRODUCTION A Fast and Cloning-Free Genome Engineering Method
The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technique has emerged as a powerful tool for genome engineering (Doudna & Charpentier, 2014). The Cas9 protein is an RNA-guided endonuclease that can be programmed to target a unique DNA sequence in the genome for cleavage. Such programming requires two small Step 1 (left): Design, ordering, and synthesis of required RNA components (the constant tracrRNA component and the variable crRNA component programmed to cut at the target locus), the Cas9 protein and the donor DNA repair template. It takes approximately 1 week between crRNA order and delivery.
Step 2 (middle): Generation of the active Cas9 ribonucleoprotein (RNP) complex containing Cas9 protein, crRNA, and tracRNA, followed by the addition of DNA repair template, and cell transformation.
Step 3 (right): Identification of targeted genome engineering events by fluorescent reporter expression using cytometry and microscopy.
RNAs: a CRISPR RNA (crRNA), comprising a nucleotide sequence complementary to the target DNA, and a trans-activating crRNA (tracrRNA) that serves as a binding scaffold for the Cas9 nuclease and the crRNA. The crRNA and tracrRNA are sometimes combined in a single RNA, called a guide RNA (gRNA; Jinek et al., 2012). The Cas9-crRNA-tracrRNA complex creates a double-strand break that is repaired by the cell's endogenous homology-directed repair (HDR) system using a synthetic donor DNA containing the desired edit. This method allows deletion, tagging, or mutation of any locus in the genome. One powerful application of genome engineering is the tagging of proteins with fluorescent reporters such as green fluorescent protein (GFP). For this application, the donor DNA consists of a fluorescent reporter-encoding sequence flanked by homology arms matching the sequence around the Cas9-induced cleavage site. Previously published GFP tagging protocols require cloning to create GFP donors with long homology arms or selection schemes to identify homozygous edits (Bollen, Post, Koo, & Snippert, 2018;Danner et al., 2017;Koch et al., 2018;Kwart, Paquet, Teo, & Tessier-Lavigne, 2017), or else require engineered cell lines (Leonetti, Sekine, Kamiyama, Weissman, & Huang, 2016).
The method described here bypasses the cloning, selection, and engineered cell line requirements by taking advantage of the high propensity of linear DNA fragments with short homology arms to act as templates for the homology-directed repair of Cas9induced DNA breaks (Figs. 1 and 2; Arbab, Srinivasan, Hashimoto, Geijsen, & Sherwood, 2015;Liang, Potter, Kumar, Ravinder, & Chesnut, 2017;Paix, Folkmann, Goldman, et al., 2017;Paix, Folkmann, Rasoloson, & Seydoux, 2015;Paix, Schmidt, & Seydoux, 2016;Paix et al., 2014). The high editing frequency allows genomic modifications to be identified directly without selection by screening for cells with fluorescent reporter expression. Procedures requiring cloning are also eliminated by using recombinant Cas9 protein and chemically synthesized crRNAs and tracrRNAs that are assembled with Cas9 Paix et al.

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Current Protocols in Molecular Biology (D) Synthesis: The 3 overhangs are used as primers to begin DNA synthesis (dotted lines) using the repair donor as a template. (E) Template switching: The newly synthesized strands dissociate from the template and pair with each other at the locus. Note that the repair donor is not inserted at the locus. This diagram depicts an idealized situation where both overhangs pair with the repair donor. Pairing could also initiate on only one side of the Cas9-induced break to generate only one newly synthesized strand. That strand would then be replicated after template switching back to the locus. See Paix, Folkmann, Goldman, et al. (2017) for more details.

Figure 3
Reagents for Cas9 RNP genome engineering. (A) Cas9 RNP: simplified schematic of CRISPR/Cas9 components (Cas9 protein, tracrRNA, and crRNA) required to target a specific genomic locus. The crRNA contains a nonvariable sequence (blue line) and a sequence complementary to the locus (bold) and immediately upstream of a protospacer-adjacent motif (PAM) in the locus (CGG with overbar in the example shown). The tracrRNA (purple) binds Cas9 and the nonvariable sequence of the crRNA to form a functional Cas9 ribonucleoprotein complex (RNP) that will cleave the targeted locus. See Basic Protocol 1 for assembly of the Cas9 RNP. (B) Repair donor: in this example, the donor is designed to insert the GFP coding sequence just upstream of the STOP codon ("taa"). The sequence targeted by the crRNA (spacer) is in bold and the PAM is underlined. In this example, the Cas9 cleavage site is 26 nt away from the desired GFP insertion site, which is at the upper limit for successful editing. Mutations introduced to prevent donor DNA cleavage by Cas9 or to reduce internal homology are indicated. Note that these mutations are not required in donors where the site for GFP insertion resides in the PAM or the sequence targeted by the crRNA. (C) Primers to generate the repair donor using PCR: the GFP coding sequence (green) in a plasmid template (black, Figure 7 and Table S2) is amplified by PCR using forward and reverse primers. The primers are complementary at their 3 ends with the GFP sequence (green) and contain flanking sequences homologous to the targeted locus as shown in (B). See Basic Protocol 2 for details. Note that the repair donor can also be obtained commercially as a gBlock (Alternate Protocol 1).
multiple fusions in parallel (Tables 1 and 2). The protocol can also be used to estimate the targeting efficiency of a particular crRNA, because the percentage of cells expressing the fluorescent reporter is a function of targeting efficiency at the locus. Several guide RNAs can be easily tested in parallel to identify the most efficient crRNA for subsequent targeting and integration of other tags. Prior knowledge of guide efficiency is particularly useful when choosing guides to create edits that cannot be identified by fluorescence, including insertion of short immunogenic tags and point mutations. Such edits need to be screened by PCR or sequencing using clonal populations derived from the edited population (Koch et al., 2018;Kwart et al., 2017;Paix, Folkmann, Goldman, et al., 2017;Yang, Yang, Byrne, Pan, & Church, 2014).
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Limitations of the Method
The protocol relies on an efficient type of homology-dependent repair, synthesisdependent strand annealing (SDSA), in which donor DNAs are used as templates for repair DNA synthesis ( Fig. 2; Jasin & Haber, 2016;Mehta, Beach, & Haber, 2017;Paques & Haber, 1999). This type of repair makes for an efficient and simple protocol, but some limitations need to be kept in mind: r SDSA repair is most efficient when the Cas9 cleavage site is within 10 bases of the desired integration site (Paix, Folkmann, Goldman, et al., 2017;Paquet et al., 2016).
r Editing efficiency varies with different cell types, guide RNAs, and transformation methods (details are provided in Figs. 4, 5, 6, and 7, Tables 1 and 2, and Supporting Information,Tables S1-S3). Nucleofection (Basic Protocol 3) is more efficient than lipofection (Alternate Protocol 2) for most cell lines. Some users, however, may prefer lipofection for its ease of use and workable efficiency, especially in HEK293T cells.
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Current Protocols in Molecular Biology r Occasionally, repair using linear templates leads to small insertions or deletions, especially at junctions between the fluorescent reporter and the genomic region around the Cas9 cleavage site (Paix et al., 2017). This is less of a concern when working with polyclonal cell populations, which contain multiple independent edits. When working with clonal populations, however, we recommend sequencing the edited locus (both alleles) to ensure that there are no undesired mutations.
r The method presented here typically results in the tagging of only one allele.
Heterozygous tagging is sufficient to analyze protein localization and dynamics, but not to quantify protein levels or to verify the functionality of the tagged protein. For experiments that require homozygous edits, other approaches can be used (Koch et al., 2018;Paquet et al., 2016).

crRNA DESIGN
Cas9 is an RNA-guided endonuclease that requires a crRNA for targeting to a specific genomic site (Figs. 1-3 and Supporting Information, Table S3; Jinek et al., 2012). The crRNA consists of a sequence of 20 nt homologous to the targeted genomic region (spacer sequence; Fig. 3) and a nonvariable sequence (repeat sequence). The spacer sequence is designed in function of the targeted genomic region and must be upstream of a so-called protospacer-adjacent motif (PAM) in the targeted genomic region. The PAM for the Cas9 protein from Streptococcus pyogenes (spyCas9) is the trinucleotide NGG Paix et al.

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Current Protocols in Molecular Biology ( Fig. 3). The nonvariable repeat sequence of the crRNA is necessary to bind to a second RNA called the tracrRNA. The tracrRNA is a nonvariable RNA molecule that contains a sequence complementary to the crRNA repeat sequence and a sequence required to bind Cas9 protein. The same tracrRNA is used for all experiments; only the crRNA needs to be custom designed. Because single gRNAs (combined crRNA and tracrRNA in one molecule; Jinek et al., 2012) are longer than crRNAs and thus more expensive to synthesize, they are not recommended for this protocol.

Materials
Milli-Q-filtered H 2 O, sterile (stored at room temperature) Ultrapure 1 M Tris·Cl buffer, pH 7.5 (e.g., Thermo Fisher Scientific, cat. no. 15567027) and 5 mM working solution (diluted in sterile Milli-Q-filtered H 2 O and stored at room temperature) Custom-synthesized crRNA (e.g., Integrated DNA Technologies [IDT] Alt-R CRISPR-Cas9 crRNA) tracrRNA (e.g., IDT CRISPR-Cas9 tracrRNA) 1. Identify the best site for GFP insertion for your particular locus. A C-terminal fusion is often considered least likely to interfere with possible localization sequences that Paix et al.

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Current Protocols in Molecular Biology This protocol  List of plasmids to create donor DNAs by PCR. Repair donors can be amplified using PCR primers annealing with the beginning and the end of the fluorescent reporter (green) and containing short flanking sequences (ß35 nt) homologous to the gene to edit (homology arms [HA], blue). Plasmids containing various fluorescent reporters and epitope tags are used as PCR template for amplification (Supplementary Information File S1).

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Current Protocols in Molecular Biology are often located at the N terminus. N-terminal and internal insertion sites can also be considered as long as functional domains are not disturbed.
We recommend identifying protein domains using online resources such as SMART (http://smart.embl-heidelberg.de/) in order to choose an insertion site that will not disturb an essential domain (Letunic & Bork, 2018). 2. Design the crRNA following these guidelines: r Choose a crRNA that will cause Cas9 to cleave the genomic DNA as close as possible to the desired GFP insertion site. Cas9 will generate a blunt cut 3 bp upstream of the PAM sequence. For best results, we recommend staying within a window of ±10 bp from the edit (Paix, Folkmann, Goldman, et al., 2017). r When possible, choose a crRNA containing a guanine nucleotide immediately before the PAM. Avoid having a cytosine at that position (Doench et al., 2014).
r A GC content of 50%-70% GC in the crRNA spacer sequence also improves cutting efficiency (Gagnon et al., 2014). r Make sure that the crRNA targets a unique sequence (20 bp) and has few, if any, predicted off-target potential editing sites. Generally, two or more mismatches within the 10 bp upstream of the PAM sequence are sufficient to avoid targeting by Cas9. We recommend using online resources to identify potential off-target sites for crRNAs (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrnadesign; Doench et al., 2016).
3. Order the crRNA: Several commercial sources are available, including IDT Alt-R CRISPR-Cas9 crRNA. Two nanomoles is sufficient for 60 experiments using lipofection or six experiments using nucleofection. If more experiments are planned, order 10 nmol. Use the provider's website to enter the desired sequence (with IDT, only the 20-nt spacer sequence needs to be entered; do not include the PAM or repeat sequence). Briefly spin the crRNA in a bench microcentrifuge at room temperature and add 30.8 µl 5 mM Tris·Cl, pH 7.5, to the 2 nmol of crRNA provided (final concentration: 65 µM). Mix well by pipetting and store at −80°C.
4. Order the tracrRNA: Use the same provider as for your crRNA to make sure that the crRNA and tracrRNA will be able to pair. Twenty nanomoles is sufficient for 600 experiments using lipofection or 60 experiments using nucleofection. Briefly spin the tracrRNA in a bench microcentrifuge at room temperature and add 308 µl 5 mM Tris·Cl, pH 7.5, to the 20 nmol of tracrRNA provided (final concentration: 65 µM). Mix well by pipetting and store at −80°C.

PCR DONOR DESIGN AND SYNTHESIS
Insertion of a fluorescent reporter is achieved using a linear donor DNA that will function as a template to repair the Cas9-induced break through homology-directed repair (Paix, Folkmann, Goldman, et al., 2017). Linear donors are synthesized in the lab using the polymerase chain reaction (PCR) or can be purchased as gene fragments (e.g., IDT gBlocks; Alternate Protocol 1).
The donor template consists of the insert (the coding sequence of GFP or other fluorescent protein) and sequences homologous to genomic sequences on the left and right sides of the desired site of insertion ( Fig. 3; Paix, Folkmann, Goldman, et al., 2017;Paix, Folkmann, & Seydoux, 2017). Use the genomic sequence (not the cDNA sequence) when designing the donor template, as the cutting site can be close to an exon/intron junction. In addition, we recommend the following.
r The insert should be <1 kb in length, as editing frequency decreases significantly with increasing insert size (Paix, Folkmann, Goldman, et al., 2017). Most Paix et al.

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Current Protocols in Molecular Biology fluorescent proteins are small enough to fit this size restriction (e.g., GFP without start and stop codons is 714 bp; Supporting Information, File S1).
r The insertion site should be as close as possible to the site of Cas9-induced cleavage (Paix, Folkmann, Goldman, et al., 2017;Paquet et al., 2016). The sequence between the Cas9 cleavage site and the insertion site is homologous to the genomic region targeted (internal homology) and should be recoded with silent mutations to prevent premature recombination that would result in low insertion efficiency of the fluorescent reporter gene (incomplete editing; Fig. 3; Paix, Folkmann, Goldman, et al., 2017). r Make sure that the donor template cannot be cut by Cas9. Mutations in the PAM sequence or in the sequence immediately upstream of the PAM in the crRNA spacer sequence are generally sufficient to prevent cleavage by Cas9 ( Fig. 3; Hsu et al., 2013). Note that spyCas9 also weakly cleaves sequences with NAG or NGA PAMs, so avoid recoding the PAM to NAG or NGA (Kleinstiver et al., 2015).
r We recommend testing the donor template in silico by translating the predicted cDNA fusion in order to rule out any unwanted codon changes or frameshifts due to errors in the design of the donor template (use cloning software or online resources such as https://web.expasy.org/translate/; Gasteiger, 2003). Eight-PCR-tube strips with thin walls (e.g., VWR, cat. no. 93001-118) Bench microcentrifuge (e.g., Fisher Scientific Accuspin Micro17) PCR thermocycler with temperature-gradient functionality (e.g., Bio-Rad C1000 thermal cycler) Agarose minigel electrophoresis system (e.g., Fisher Scientific 14-955-170) NanoDrop spectrophotometer (e.g., Thermo Fisher Scientific ND-1000) 1. Design the PCR primers: Design forward and reverse primers containing ß19 nt homologous to the 5 and 3 ends of the desired fluorescent protein coding sequence. Add the homology arms (35-40 nt of sequence flanking the edit and cleavage site) to the 5 end of each primers and, if necessary, for one primer, add the sequence including silent mutations between the edit and the cleavage site (Fig. 3). Purchase or synthesize the primers (e.g., 25 nmol from IDT, desalted), reconstitute each primer at 100 µM with Milli-Q-purified water, and store at −20°C.

Materials
2. Prepare PCR master mix: In a 1.5-ml microcentrifuge tube, mix 0.8 µl template plasmid, 2 µl each of 100 µM stocks of the forward and the reverse primers, We do not recommend using agarose gel purification, as this procedure results in impure and low-yield DNA preparations.

ALTERNATE PROTOCOL 1 gBLOCK DONOR DESIGN
As an alternative to PCR fragments (Basic Protocol 2), commercial synthesized gene fragments can be used as donors (e.g., gBlocks from IDT). gBlocks can be purchased commercially, but typically are provided in a limited quantity (1 µg for 751-1000 bp gBlocks, sufficient for one editing experiment).

Additional Materials (see Basic Protocol 2)
Gene fragment (e.g., IDT gBlock) 1. Design the gBlock using the same design considerations as for a PCR donor (Basic Protocol 2; Fig. 3). The sequence complexity of the homology arms may prevent efficient gBlock synthesis. If so, extend the length of the homology arms while maintaining the total size of the gBlock at <1 kb (to obtain a yield of 1 µg).
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Cas9 PROTEIN PURIFICATION
Relatively large amounts of spyCas9 protein are required for each editing experiment (80 µg/experiment for nucleofection and 8 µg/experiment for lipofection). Cas9 can be purchased from commercial suppliers, but it is also straightforward to purify in the laboratory. The procedure requires only few days and can yield enough Cas9 to perform >50 genome editing experiments using nucleofection (Basic Protocol 3).

Materials
Rosetta 2  13. Elute protein with buffer C. Determine which fractions contain Cas9 protein using a NanoDrop or by running a small amount on SDS gel, and pool these fractions together.
14. To remove contaminating DNA from the prep: Prepare and equilibrate a 5-ml Q Sepharose column with 25 ml 1 M KCl to charge the column. Equilibrate column with buffer C (25 ml). Flow eluent (from step 13) over prepared Q Sepharose column. Collect flowthrough and dialyze into 1 L buffer D at 4°C overnight.

Optional:
To remove Cas9 aggregates from the sample, the pooled fractions (from step 14) can be further fractionated via size-exclusion chromatography: a. Equilibrate a 26/60 Sephacryl S-200 column with 1.5 column volumes buffer D. b. Concentrate the pooled fractions from step 14 to 5-ml volume using a 100K centrifugal filter, run the 5-ml concentrated sample over the S200 column, and collect the monomeric 280 nm absorbance peak (from the FPLC spectrometer) using the fraction collector (Fig. 8).

GENE TAGGING USING NUCLEOFECTION
Nucleofection is an efficient method for delivery of programmed Cas9 and donor templates into HEK293T, DLD1, or U2OS cells (Table 1). This method relies on a solution that permeabilizes the cells along with an electroporator device (Amaxa Nucleofector) used to introduce programmed Cas9 and donor template in the permeabilized cells. Lipofection is an alternative delivery technique that does not require specialized equipment, but is less efficient (Alternate Protocol 2).

Materials
Culture of the cells of interest, grown to 70%-80% confluence (Support Protocol 2) crRNA and tracrRNA (Basic Protocol 1) Cas9 protein (from Support Protocol 1 for recombinant Cas9 preparation, or purchased from commercial sources: e.g., IDT, cat. no. 1081058) Donor DNA: PCR donor DNA (Basic Protocol 2) or gBlock donor DNA (Alternate Protocol 1) Filtered sterile glycerol/KCl buffer (see recipe) Filtered sterile HEPES/MgCl 2 /KCl buffer (see recipe) Milli-Q-filtered sterile H 2 O (store at 4°C) Amaxa Cell Line Nucleofector Kit V (Lonza, cat. no. VCA-1003; add the 500 µl of the provided nucleofection supplement solution to the 2.25 ml of nucleofection reagent and store at 4°C; can be used for up to 3 months after adding the supplement solution) Penicillin/streptomycin (Pen/Strep; e.g., Thermo-Fisher Scientific, cat. no. 15140-122; store at −20°C; prepare working aliquot in 15-ml Falcon tubes and store at −20°C, then transfer 0.5 ml in 50 ml culture medium into a 50-ml Falcon tube and store at 4°C) Paix et al.
2. From a culture grown to 70%-80% confluence, harvest the cells with trypsin (Support Protocol 2). Seed the cells into the two T75 flasks (500,000 HEK293T cells, 650,000 DLD1 cells, or 800,000 U2OS cells per flask). Place the cells back in the cell incubator.

Prepare the dsDNA donor mix for nucleofection (on the day of nucleofection)
3a. If using a PCR donor: Dilute 6-8 µg of the purified PCR product in 10 µl H 2 O final. Keep on ice or at room temperature. For a PCR donor containing GFP and ß35-bp homology arms, this concentration will be sufficient to obtain a final concentration of 0.2-0.3 µM in the final 50 µl Cas9 RNP/donor mix. To adjust for larger or small donor templates, use the following formula: 1 µg of X bp dsDNA = 1624/X pmol.
A final donor DNA concentration of 0.3 µM is recommended for highest yield (Fig. 4).
3b. If using a gBlock donor (1 µg): Spin briefly in a bench microcentrifuge at room temperature. Keep at room temperature.

Prepare the Cas9 RNP mix for nucleofection (on the day of nucleofection)
4. Prepare the Cas9 RNP mix immediately before nucleofection. Place the crRNA, tracrRNA, and Cas9 protein on ice. The homemade Cas9 aliquots (Support Protocol 1) are intended to be single use but can be flash frozen again at least one more time without loss of activity. Take the number of Cas9 aliquots needed, briefly spin them in a bench microcentrifuge at room temperature, pool Cas9 into one tube, and store on ice.

Harvest cells for nucleofection (the day of nucleofection)
8. Resuspend the cells at 50%-70% confluence from two T75 flasks: use trypsin as described in Support Protocol 2 (except using 3× more PBS, trypsin, and culture medium for T75 flasks than T25 flasks).
9. Resuspend the pellet in 1 ml PBS and transfer into a 1.5-ml microcentrifuge tube.
11. Count the cells (the cell concentration is 10 times that from the count because of the dilution in step 10). Spin down the cells for 3 min at 350 × g (1815 rpm with FA-45-30-11 rotor), room temperature (1 ml in PBS in a 1.5-ml microcentrifuge tube).
13. Aliquot 80 µl of resuspended cells (from step 12) each into 1.5-ml microcentrifuge tubes (one tube per editing experiment + one additional tube for a notransformation negative control), and spin down the cells for 3 min at 350 × g (1815 rpm with FA-45-30-11 rotor), room temperature. Keep the tubes at room temperature without removing the supernatant.
14. Prepare 6-well plates to receive the transformed cells: one plate per experiment + one for no-transformation negative control. Fill each of four wells per plate with 2 ml culture medium. The wells will be used for cell counting by cytometer (well no. 1) and for microscopy analysis (well no. 2; add coverslip to the well). The two extra wells can be used to passage the transformed cells and/or to derive single-cell clones. Also prepare 1.5-ml microcentrifuge tubes containing 600 µl culture medium (to recover the cells after nucleofection; one tube per experiment).
Keep the plates and tubes in the cell incubator.

Nucleofection procedure
15. Equilibrate the nucleofector solution at room temperature.
16. Move the 6-well plates and the 1.5-ml microcentrifuge tubes with culture medium (prepared to receive the transformed cells) from the cell incubator into the cell culture hood.
17. Remove the supernatant of the cell pellet from step 13 (80 µl), and add 80 µl of nucleofection solution (solution V with supplement solution added). Mix by pipetting.
18a. If using a PCR donor: Take 40 µl Cas9 RNP mix (from step 7) and add it to 10 µl of the PCR donor mix, and mix by pipetting.
18b. If using a gBlock donor: Take 40 µl Cas9 RNP mix (from step 7) and add it to the tube containing the dried gBlock donor, spin briefly in a bench microcentrifuge at room temperature, wait 1 min, and mix by pipetting.
19. Take 40 µl of the Cas9 RNP/donor mix and add it to the 80 µl of cells in the nucleofection solution.
20. Using a narrow-end pipet, gently mix the nucleofection mix containing the cells + nucleofection solution and Cas9 RNP/donor mix, and transfer it in the bottom of an Amaxa 2b electroporation cuvette.
Do not make bubbles and avoid transferring the mix outside the space between the two electrodes.
21. Put the lid on the cuvette and tap it on a solid surface in order to remove any bubbles, and place the cuvette in the Amaxa 2b machine and run the appropriate electroporation program (A023 for HEK293T and DLD1 cells, X001 for U2OS cells).
22. Remove the cuvette from the Amaxa 2b machine, remove the lid, and immediately add the 600 µl of culture medium from the 1.5-ml microcentrifuge tube (using a micropipet with 1000-µl tip) and mix by pipetting. Transfer the culture medium with cells back into the 1.5-ml microcentrifuge tube (using a fresh narrow-end pipet). Next, transfer 150 µl cells in culture medium into each of four wells of the 6-well plate (using a micropipet with 200-µl tip) and replace the plates in the cell incubator.
23. Repeat with other Cas9 RNP/donor mixes as needed (from step 16). For the negative control, resuspend the 80 µl of cells and place 10 µl in the wells of a 6-well plate.
24. The next day, replace the culture medium of the transformed cells with fresh medium containing Pen/Strep. If necessary, the cells can also be split into several wells using trypsin treatment.

Note for two-genes or two-colors tagging using nucleofection 25a.
To tag two genes with two fluorescent proteins (Fig. 9): For the Cas9 RNP, mix the two different crRNAs targeting the two desired genes (2.5 µl of each crRNA at 65 µM). For the donor mix, use two repair donors encoding the two different fluorescent proteins at a 1:1 molar ratio. Be sure that the two donors do not contain any similar sequences and encode fluorescent proteins with distinct excitation and emission spectra (such as eGFP and mCherry).
25b. To tag one locus with two fluorescent proteins ( Fig. 9): Prepare the Cas9 RNP with a single crRNA. For the dsDNA donor mix, use two repair donors coding for the two different fluorescent proteins at a 1:1 molarity ratio.

GENE TAGGING USING LIPOFECTION
Lipofection relies on liposome particles that passively fuse with cells to deliver the Cas9 RNP/Donor mix. This method does not require specialized equipment but is less efficient than nucleofection (Basic Protocol 3). We recommend lipofection for cell types that transform efficiently, such as HEK293T (Table 2).
IMPORTANT NOTE: To tag two genes with two fluorescent proteins using lipofection, or one gene with two fluorescent proteins (Fig. 9), see Basic Protocol 3. When tagging two genes, use 0.25 µl of each crRNA at 65 µM.

Materials
Culture of the cells of interest, grown to 70%-80% confluence (Support Protocol 2) Cas9 protein (from Support Protocol 1 for recombinant Cas9 preparation, or purchased from commercial sources, e.g., IDT, cat. no. 1081058) Paix et al.

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Current Protocols in Molecular Biology Tagged cells exhibit GFP signal in endosomes (green) and/or nuclear membrane RFP signal (red). Misinsertion indicates rare events where the wrong tag was inserted at either locus. Because each editing event is independent, co-edits are rare but do occur. In the example shown, a few co-targeted cells are visible (red and green in same cell). (B) Experiment targeting the same locus (Lamin A/C, HEK293T cells) with GFP and RFP. This approach can be used to generate cells where the targeted locus is tagged with GFP or RFP in one experiment. Occasionally, cells where both alleles are targeted by the different tags can be recovered, but this is a rare event (see graph). No such cells are visible in the example shown.
2. Add 2 ml culture medium per well into a 6-well plate. Each well corresponds to one editing experiment. Add coverslips to the wells if needed for microscopy analysis.
3. Seed the cells in each well (e.g., 65,000/well for HEK293T cells). Place the cells back in the cell incubator. Cells can be transformed by lipofection 2-3 days after seeding (50%-70% confluence).

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Prepare dsDNA donor mix for lipofection (on the day of lipofection)
4a. If using a PCR donor: Use 0.8-1.0 µg/µl purified PCR product (diluted in H 2 O, 1 µl per each lipofection experiment). Spin briefly in a bench microcentrifuge at room temperature and keep at room temperature. A final concentration of 0.3-0.4 µM is desired in the 5 µl of Cas9 RNP/donor mix (Fig. 4). For donor DNAs significantly larger or smaller than GFP, consult Basic Protocol 3 to calculate concentrations.
4b. If using a gBlock donor: Add 1 µl H 2 O on the wall of the gBlock tube and spin briefly in a bench microcentrifuge at room temperature. Keep at room temperature.

Prepare the Cas9 RNP mix for lipofection (on the day of lipofection)
5. Prepare the Cas9 RNP mix immediately before starting the lipofection experiment. Store crRNA, tracrRNA, and Cas9 protein on ice.
Optional: Incubate the mix 15 min at 37°C to encourage Cas9 RNP complex formation.
7. Store at room temperature.
9. Prepare the 1× lipofection buffer by diluting 40 µl 5× stock buffer (shake to dissolve any precipitate) in 160 µl of H 2 O in a 1.5-ml microcentrifuge tube. Make a master mix if several experiments are planned.
12. Add the Cas9 RNP/donor/LipoJet mix (from step 11) to the cells in one well of a 6-well plate. Mix by shaking the plate gently and place back in the incubator. If several experiments are planned, steps 10-12 can be performed in parallel.
13. The next day, replace the culture medium of the transformed cells with fresh medium containing Pen/Strep. If necessary, the cells can also be split into several wells using trypsin.
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MAINTENANCE AND PREPARATION OF TISSUE CULTURE CELLS FOR TRANSFORMATION
We recommend the following guidelines for tissue culture handling: r Sterile conditions: It is important to work under sterile conditions when manipulating cell lines and culture reagents. We use antibiotic (Pen/Strep) in the cell culture medium after cell transformation because the Cas9, crRNA, tracrRNA, and donor DNA may not be sterile.
r Cell adherence: HEK293T, DLD1 and U2OS are adherent cells. They can be detached using trypsin treatment. An incubation of 1-2 min followed by pipetting will be enough for HEK293T cells, whereas up to 7-10 min will be necessary for DLD1 and U2OS cells. HEK293T can also be partially detached by routine pipetting, and thus with these cells it is necessary to exercise caution when changing medium.
4. Add 2 ml trypsin-EDTA and wait until the cells become nonadherent.
5. Resuspend the cells and transfer them in a 15-ml Falcon tube containing 2 ml culture medium.
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Current Protocols in Molecular Biology 7. Remove the supernatant and resuspend the cells in 4 ml culture medium with FBS.
8. Transfer 1 ml of the resuspended cells in a 1.5-ml microcentrifuge tube. From the microcentrifuge tube, take 10 µl of resuspended cells and transfer it in a fresh microcentrifuge tube for counting cells.
9. Determine the cell concentration: add 10 µl 0.4% Trypan Blue dye to the 10 µl cell suspension, and count cells using a cell counter.
10. Seed the cells at the desired concentration into the T25 flask (150,000 cells for HEK293T, 200,000 cells for DLD1, 230,000 cells for U2OS). Place the cells back in the cell incubator.

ANALYSIS OF NUCLEOFECTED/LIPOFECTED CELLS
Three days after transformation (Basic Protocol 3 and Alternate Protocol 2), cells can be analyzed by cytometry or microscopy to determine editing efficiency and the expression pattern of the fluorescent reporter fusion. Positive cells can be further sorted and cloned for subsequent analyses, and frozen for long term storage.
16. Wash with PBS/0.25% Triton for 5 min, and then again with PBS.
17. Mount coverslips on microscope slides using antifade mounting medium (containing DAPI). If necessary, remove excess PBS from the coverslip with paper tissue before mounting.
19. Analyze cells for fluorescent reporter expression by fluorescent microscopy. The total number of cells can be determined using the DAPI nuclear dye contained in the mounting medium.
Cell sort using FACS 20. Equilibrate cell culture reagents to room temperature.

Non-transformed cells are used as control.
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Current Protocols in Molecular Biology 22. Prepare two 96-well plates for cell cloning (two plates are generally enough to recover >75 viable clones) with 100 µl of culture medium + Pen/Strep in each well. If the sorted cells are pooled together instead of cloned in plate wells, prepare a 1.5-ml microcentrifuge tube containing 500 ml culture medium with Pen/Strep.

Filter the cells with cell strainers and keep the tubes on ice.
24. Sort the fluorescence-positive cells according to the FACS instructions.
25. Incubate the plates in a cell incubator. After 72 hr, add 125 µl culture medium with Pen/Strep to each well. If the sorted cells have been pooled in a single tube, transfer the contents of the tube into a well of a 6-well plate containing 1.5 ml culture medium with Pen/Strep, and change culture medium 3 days later (again using culture medium with Pen/Strep).
26. Genotype single-cell clones using PCR primers. Use primers from outside the donor sequence to avoid false positives from off-target insertions (Won & Dawid, 2017).

Pooled sorted cells can be analyzed by microscopy or subcloned if necessary.
Cell freezing 27. Grow the cells in a T25 flask to 70%-80% confluence.
28. Equilibrate cell culture reagents to room temperature.
31. Add 100 µl cell suspension to each cryogenic vial and mix by pipetting.
32. Put the tubes in a slow-freezing container and store overnight at −80°C.

Transfer the tubes into a liquid nitrogen tank.
Cell thawing protocol 34. Add 10 ml culture medium to a 15-ml Falcon tube and warm to 37°C.
35. Transfer the cryogenic vials from the liquid nitrogen storage and warm them in your hand. As soon as the culture medium is liquid, transfer the cells to the Falcon tube containing warm culture medium. Spin down the cells for 3 min at 350 × g (1426 rpm with A-4-62-MTP rotor), room temperature, and resuspend in 2 ml culture medium.
36. Transfer the 2 ml cells into a well of a 6-well plate. The following day, change the culture medium (Pen/Strep can be included if needed).

REAGENTS AND SOLUTIONS
Use gloves and clean labware throughout to avoid contamination.

Background Information
The genome engineering method presented here is supported by a prior study from our group that examined how linear donor DNAs engage with the cell's machinery for HDR to repair Cas9-induced double-strand breaks (Paix, Folkmann, Goldman, et al., 2017). Our findings suggest that donor DNAs are not integrated at Cas9-induced DNA breaks, but are used as templates for repair synthesis.
The repair process involves several steps (Fig. 2), including: 1. Resection of the cleavage site (by 5 exonucleases to expose single-stranded 3 ends on either side of the break), 2. Pairing of the resected ends with the donor DNA, 3. Repair synthesis that extends the 3 resected ends using the donor as a template, 4. Template switching which allows the newly synthesized strand to pair with the resected end on the other side of the lesion, 5. Fill-in synthesis and ligation.
Two key features of the repair process inform the design of the donor DNA. First, annealing of the donor DNA to the resected ends at the locus can be achieved with relatively short homology arms on the donor DNA. Homology arms that are 35 nucleotides (nt) long are sufficient for efficient pairing and initiation of repair synthesis (Paix, Folkmann, Goldman, et al., 2017). Second, frequent template switching during repair synthesis requires the donor DNA to be designed in such a way as to minimize any homology between the edit and the cleavage site, as template switching in this region will prevent incorporation of the edit (Paix, Folkmann, Goldman, et al., 2017). For this reason, it is important to minimize the distance between the edit and the Cas9 cleavage site (<10 bp is ideal). Recoding of the sequence between the edit and the Cas9 cleavage site also can be used to help reduce premature template switching (Basic Protocol 2 and Fig. 3). Even with recoding, however, we have found that distances greater than 10 bp do not yield high editing efficiencies (Paix, Folkmann, Goldman, et al., 2017).
It is important to keep in mind that most fluorescent reporter insertions obtained by this protocol will target only one allele. The other allele (or alleles depending on the cell type) may be unaffected or may contain small insertions and/or deletions created by a Cas9 cleavage event that was repaired by nonhomologous end joining (NHEJ; Paix, Folkmann, Goldman, et al., 2017). For this reason, it is important when deriving clonal populations to sequence both alleles to ensure there are no unwanted mutations.
In regard to reagent costs, the main cost is associated with the custom crRNA (ß$100 U.S.). Donor DNA costs range between ß$25 for PCR donors and ß$100 for gBlock donors. The cost of Cas9 protein is negligible when Cas9 in produced in house, and the cost associated with the tracrRNA is a few dollars per experiment. The cost of other disposable reagents amounts to $50-200 per experiment depending of the delivery method used and the types of cell analyses performed. The suppliers referenced in this protocol were the ones used to develop this genome engineering method; alternative suppliers may be used as preferred.

Critical Parameters and Troubleshooting
No edits or very low editing efficiency may be due to the following.
Low crRNA efficiency. We recommend a cr-RNA with 50% GC content and a 3 G in the spacer sequence that will cleave within a 10-bp window of the desired fluorescent reporter insertion site (Doench et al., 2014;Gagnon et al., 2014;Paix, Folkmann, Goldman, et al., 2017;Paquet et al., 2016).
Precipitation of Cas9 in the Cas9 RNP/donor mix. We recommend adding each reagent in the order specified in Basic Protocol 3 and Alternate Protocol 2 to avoid precipitation of Cas9.

Current Protocols in Molecular Biology
Overgrown cell culture. Avoid using cells that have reached confluence. We recommend using cells at no more than 75% confluence for transformation.
Gene is not expressed or expressed at a very low level. Because the identification of the edits depends on fluorescent reporter expression, make sure that the gene of interest is expressed in the cell type that you will use for your editing experiment. The Protein Atlas database provides useful expression information for a number of commonly used tissue culture cells (https://www.proteinatlas.org/; Uhlen et al., 2015). Keep in mind that fluorescent reporter fusions with low, diffuse expression may be difficult to detect above background in a cytometer. We recommend direct observation of the transformed cells by microscopy to avoid missing low-expression fusions.
Fusion is not stable. In some cases, the fusion may interfere with protein stability or protein dynamics (Nagasaki et al., 2017). A flexible linker sequence (Chen, Zaro, & Shen, 2013) can be added to the insert in order to improve folding of the tagged protein. A different insertion site can also be tested.

Understanding Results
When targeting a site within 10 bp of the Cas9 cleavage site, depending of the locus targeted, the delivery method, and the cell type used, researchers can expect editing efficiencies ranging from 0.5% to 20% (Tables 1 and  2 and Supporting Information, Table S1). This range is suitable for localization studies in nonclonal populations, and for isolation of edited clonal populations from sorted single cells.
We recommend including a positive control when using the protocol for the first time.
Positive control for PCR donor. Use the Lamin A/C crRNA (crRNA AP1629; Supporting Information, Table S3), eGFP forward and reverse primers (primer AP1618 and AP1619; Supporting Information, Table S2) and GFP plasmid pAP1698 (available at AddGene; Fig. 7). Perform a PCR as described in Basic Protocol 2 using an annealing temperature of 61.5°C and an elongation step of 45 sec. Purify on a MinElute column and measure the concentration with a NanoDrop (expected concentration should be >1000 ng/µl). Use 7 µg for nucleofection or 1 µg for lipofection. Expected efficiency is ß15% for nucleofection in HEK293T cells ( Fig. 4 and Table 1).

Time Considerations
Gathering of reagents. 5-7 days in advance of the nucleofection or lipofection, users should design and order the crRNA, and the PCR primers or gBlock required for the donor. The other reagents can be ordered in bulk (tracrRNA and Cas9) or synthesized in the lab (Cas9) and used for several experiments. Tissue culture cells are passaged 2-4 days before transformation.
Cell transformation and analyses. Only 0.5-2 hr of hands-on work is needed is for transformation and plating of the edited cells. Cytometer and microscopy analyses are performed 2-3 days later.