TITLE: Generation of knock-in and knock-out CRISPR/Cas9 editing in mammalian cells
AUTHORS: Michael Hanna, Pietro De Camilli

[DESCRIPTION]
This protocol is to help with the generation of knock-in and knock-out genomically edited mammalian cells lines using CRISPR/Cas9.

[BEFORE_START]
Knock-out mammalian cell lines
General overview of generating clonal knock-out mammalian cells lines using CRISPR/Cas9

a. The purpose of this part of the protocol is to explain the methods of generating a clonal cell line that has been edited at the endogenous locus to knock-out a gene of interest. In particular, this part of the protocol focuses on using hTERT-RPE1 cells (https://www.atcc.org/products/crl-4000) as they are chromosomally stable, are generally easy amenable to CRISPR/Cas9 genomic editing, reliably diploid, and have a flat nature ideal for imaging of organelles. However, this protocol has been applied to other cell types, but subtleties in protocols are not discussed here.

b. This protocol for generating knock-out cells uses a single guide RNA (gRNA) sequence usually directed at an early exon (e.g., exon 2). Importantly, this protocol utilizes the well-established PX458 plasmid (Addgene # 48138) to express the gRNA and Cas9 protein with a cleavable GFP that serves as a reporter for cloning.

c. All imaging and further cell culture techniques can be found here: DOI of other protocol


Knock-in mammalian cell lines
General overview of generating clonal knock-in mammalian cells lines using CRISPR/Cas9

a. The purpose of this part of the protocol is to explain the methods of generating a clonal cell line that has been edited at the endogenous locus to knock-in a fluorescent protein tag in gene of interest. In particular, this part of the protocol focuses on using HeLa cells (https://www.atcc.org/products/ccl-2?geo_country=au) as they are easy to transfect with DNA and amenable to genomic editing. However, this protocol has been successfully applied to other cell types, but subtleties in protocols are not discussed here.

b. This protocol for generating knock-in cells uses a single guide RNA (gRNA) sequence usually directed toward the start ATG codon, but also at internal sites. Importantly, this protocol utilizes the now established homology independent targeted integration (HITI) ORANGE system and accompanying plasmid (Addgene # 131471) to express the gRNA, Cas9 protein, and sequence of the fluorescent protein tag to be inserted at the endogenous locus. A more detailed protocol and explanation about the engineering can be found here: 10.1371/journal.pbio.3000665

[STEPS]
SECTION: Knock-out mammalian cell lines - Choosing a gRNA for editing endogenous locus of gene of interest
1. All gRNA sequences used in these protocols were identified using the publicly available CRISPR/Cas9
gRNA design program CHOPCHOP (https://chopchop.cbu.uib.no/). For the generation of SHIP164
knock-out cells, a gRNA directed toward exon 2 was selected based on the predicted efficiency score
and low off-target sites. For the exact sequences used, please see Table X in our publication.
SECTION: Knock-out mammalian cell lines - Cloning the gRNA into PX458
2. Linearize PX458 plasmid by using the restriction enzyme BbsI (New England Biolabs) following the
manufacturers guidelines. Run the linearized plasmid on an agarose gel (e.g., 1% supplemented
with Midori Green or Ethidium Bromide for visualization) and purified using a NucleoSpin Gel and
PCR Clean-up kit (Takara)
SECTION: Knock-out mammalian cell lines - Cloning the gRNA into PX458
3. Synthesize a single stranded oligonucleotide (ssOligo) containing the desired 20bp gRNA sequence by IDT. The ssOligo contained 25bp of homology at each end that corresponds with the flanking sequence of the linearized PX458 plasmid.
SECTION: Knock-out mammalian cell lines - Cloning the gRNA into PX458
4. Ligate the ssOligo into the purified linearized PX458 plasmid using NEB HiFi assembly mixture following manufacturers protocol (https://www.neb.com/-/media/nebus/files/applicationnotes/ appnote_bridging_dsdna_with_ssdna_oligo_and_nebuilder_hifi_dna_assembly_to_create_sgrna-cas9_expression_vector.pdf?rev=8537d5c90b07406b85b9f2221e2410f6).
SECTION: Knock-out mammalian cell lines - Transfection of PX458-gRNA into RPE1 cells
5. Plate 1.0x105 wild-type RPE1 cells in to one well of a 6-well plate.
SECTION: Knock-out mammalian cell lines - Transfection of PX458-gRNA into RPE1 cells
6. Replace 1440 minlater, the complete media with media without antibiotics.

SECTION: Knock-out mammalian cell lines - Transfection of PX458-gRNA into RPE1 cells
7. Transfect cells with 1 µg   using FugeneHD (Promega) following the manufacturers protocol.
SECTION: Knock-out mammalian cell lines - Transfection of PX458-gRNA into RPE1 cells
8. Incubate cells with the transfection reagent for480 min - 1440 min be sorting.

SECTION: Knock-out mammalian cell lines - FAC sorting of PX458-gRNA transfected RPE1 cells
9. Remove RPE1 cells transfected for 1440 min with the PX458-gRNA plasmid from the 6-well dish
using standard trypsinization.
SECTION: Knock-out mammalian cell lines - FAC sorting of PX458-gRNA transfected RPE1 cells
10. Spun down the cells at 500 g for 5 min to pellet them, and aspirate the media off.

SECTION: Knock-out mammalian cell lines - FAC sorting of PX458-gRNA transfected RPE1 cells
11. Resuspend the cell pellet in 500 µL of Room temperature  sorting buffer.
SECTION: Knock-out mammalian cell lines - FAC sorting of PX458-gRNA transfected RPE1 cells
12. Based on the presence of GFP signal compared with empty-PX458 control cells, a gate was created to
sort individual cells into wells of a 96-well plate.
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
13. Once cells in 96-wells have grown into a discernable colony (visualized by brightfield microscope), move colonies to 24-well plates to expand them.
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
14. When the colonies have grown to near confluency (~80%) of the 24-well, there are enough cells to then
collect for genomic DNA purification and to also maintain on a new 24-well plate.
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
15. Performe genomic DNA of clonal cells using the DNeasy purification kit (Qiagen 69056).
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
16. Use purified genomic DNA to amplify a 250-300bp region surrounding the predicted gRNA cut
site using Taq polymerase.
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
17. Run the resulting PCR product on an agarose gel to confirm correct size and then purified as
described above.
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
18. Ligate the purified PCR product into the pTOPO-TA sequencing vector following the manufacturers protocol.
(https://www.thermofisher.com/order/catalog/product/450071?ICID=search-450071#/450071?ICID=search-450071)
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
19. Pick enough colonies of the resulting transformation to sequence ~20 clones for each clonal cell line being characterized. Typically, 5-8 cell clones were characterized at a time.
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
20. Keep only clonal cell lines where every sequence confirmed an indel that would result in a premature stopcodon.
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
21. Expand the selected clonal cell lines, and aliquots frozen down for safe keeping.
SECTION: Knock-out mammalian cell lines - Identification and verification of clonal knock-out RPE1 cells
22. Confirm confirmation of protein expression levels consistent with a knock-out by immunoblotting using a specific antibody against the protein of interest compared against polyclonal empty-PX458 cells.
SECTION: Knock-in mammalian cell lines - Choosing a gRNA for editing endogenous locus of gene of interest
23. Choose gRNA sequences as described above.
SECTION: Knock-in mammalian cell lines - Choosing a gRNA for editing endogenous locus of gene of interest
24. One important difference is finding a gRNA based on the ideal location for inserting your fluorescent
protein tag.
SECTION: Knock-in mammalian cell lines -  Cloning the pORANGE plasmid for fluorescent protein tag knock-in
25. Clone the gRNA sequence into pORANGE as described above for the gRNA cloning into PX458.
SECTION: Knock-in mammalian cell lines -  Cloning the pORANGE plasmid for fluorescent protein tag knock-in
26. Once the gRNA sequence insertion into pORANGE was confirmed by sequencing, linearize the plasmid using HindIII/XhoI.
SECTION: Knock-in mammalian cell lines -  Cloning the pORANGE plasmid for fluorescent protein tag knock-in
27. Purify linearized plasmid as described above.
SECTION: Knock-in mammalian cell lines -  Cloning the pORANGE plasmid for fluorescent protein tag knock-in
28. Ligate the fluorescent protein DNA amplified by PCR (typically mNeonGreen or mScarlet-i) with flanking
reverse complement gRNA + PAM sequences, Gly-Ser linkers, and corresponding cut sites into the linearized pORANGE plasmid using standing cloning techniques.
SECTION: Knock-in mammalian cell lines - Transfection of pORANGE into HeLa cells
29. Plate 5.0x104 wild-type HeLa cells in to one well of a 6-well plate.
SECTION: Knock-in mammalian cell lines - Transfection of pORANGE into HeLa cells
30. Replace 1440 min later, the complete media with media without antibiotics.

SECTION: Knock-in mammalian cell lines - Transfection of pORANGE into HeLa cells
31. Transfect cells with 500 ng  using FugeneHD (Promega)
following the manufacturers protocol.
SECTION: Knock-in mammalian cell lines - Transfection of pORANGE into HeLa cells
32. Incubate cells with the transfection reagent for 480 min-1440 min.

SECTION: Knock-in mammalian cell lines - Transfection of pORANGE into HeLa cells
33. Exchange the media after transfection with complete media and cells were allowed to grow to
near confluency (2-3 days) before being split 1:5 onto another well of a 6-well plate.
SECTION: Knock-in mammalian cell lines - Transfection of pORANGE into HeLa cells
34. A key component of the knock-in process is to wait at least five days before continuing on as the
genomic editing is a slow process.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
35. Split HeLa cells after five days post-transfection and plate ~1.0x104 cells on a 35mm glass bottom dish (Mattek).
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
36. Cells were allowed to grow to near confluency on the dish (~3-4 days).
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
37. Image the dish using a confocal microscope to identify cells positive for the fluorescent marker.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
38. The reason behind plating the glass bottom dish so low initially is to allow colonies to grow, which
makes finding cells that are positive for the fluorescent marker easier.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
39. If there are cells positively identified, then split the cells on the dish and serially dilute for
subcloning.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
40. Subcloning begins with plating 10 cells per well in a 96-well glass bottom plate.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
41. Allow cells in the well to grow to ~50-60% confluency before imaging of wells using a confocal microscope.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
42. Split wells with positively identified cells—for the fluorescent marker— to a well of a 24-well plate and allowed to expand.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
43. Split the cells once the 24-well is near confluency, serially dilute, and plate at one cell per well of a 96-well glass bottom plate.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
44. Allow again, cell colonies to expand until they are easily visualized by a brightfield microscope.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
45. Image colony using a confocal microscope to identify the fluorescent protein marker.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
46. Split clones positively identified for the fluorescent protein marker to a new 24-well and allow to expand.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
47. Split colonies once the well is nearly confluent, and some will be used for purifying the genomic
DNA as described above.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
48. Use primers directed against the region of the genome surrounding the site of endogenous editing to amplify by PCR this area and run on an agarose gel as described above.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
49. Purify the DNA based on the size of the PCR band and ligate into TOPO-TA sequencing vector.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
50. Keep only clonal cell lines where every sequence confirmed the correct knock-in sequence.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
51. Expand the selected clonal cell lines and aliquots frozen down for safe keeping.
SECTION: Knock-in mammalian cell lines -  Identification, cloning, and verification of clonal knock-in HeLa cell lines.
52. Confirm confirmation of protein expression levels consistent with a knock-in by immunoblotting using a specific antibody against the protein of interest and fluorescent protein tag compared against polyclonal empty-pORANGE cells.