Research Using CRISPR

How to Create Knockout Cells Using CRISPR

This tutorial is part 1 of 4 in the series “How to create knockout cells using CRISPR”. Use Benchling’s free molecular biology tools to plan your own CRISPR experiment and design your own gRNAs here.

When generating knockouts, CRISPR is the easiest, cheapest and fastest genome editing technique1, 2, 3. Here we provide a comprehensive, step-by-step tutorial to help you design your first CRISPR knockout experiment. Our series will cover how you can:

  1. Plan your knockout experiments using CRISPR
  2. Design gRNAs to target your gene of interest
  3. Synthesize desired gRNAs
  4. Deliver gRNAs and Cas9 to your target cells
Mechanism to generate an insertion/deletion (indel) knockout using CRISPR

Before we get into the experimental details, let’s review how CRISPR works: CRISPR consists of a guide RNA (gRNA) and a DNA endonuclease, Cas9. The gRNA, which you can design yourself, determines where insertions or deletions (indels) will occur. Once the gRNA and Cas9 are expressed in cells, the gRNA will direct Cas9 to bind to the target sequence and introduce a double-strand break. The cell can repair the break with either non-homologous end joining (NHEJ) or homologous directed repair (HDR).

NHEJ is the most active repair mechanism but often leads to indels near the target sequence. If the indel occurs within the open reading frame, it may introduce a frameshift that causes a premature stop codon, eliminating the gene function. You can then use mismatch cleavage assays to identify which cells contain indels at your gene of interest. By using the cell’s imperfect repair mechanism, CRISPR allows you to simply construct cell lines with indel knockouts at your genomic region of choice.

CRISPR mechanism

What should you decide before starting your experiments?

Before you start, you need to determine:

  • Which cell line to work with: Is your cell line hard to transfect (e.g. Jurkat, primary cell lines) or post-mitotic (e.g. neurons, hepatocyte)? In this case, you might have a higher delivery efficiency using viral vectors, rather than chemical transfection. However, viral production can be difficult and time-consuming. If you are new to the protocol, it could take a month to optimize the procedure.
  • Which Cas9 to use: Streptococcus pyogenes Cas9 (SpCas9) is the most common Cas9 for genome engineering. But you might still want to consider other variants if:
    • You want to use AAV to deliver CRISPR: The maximal cargo size for AAV is around 4.5 kb and just SpCas9 itself is 4.2 kb. If you need to add control elements or selection markers to your CRISPR, then a smaller Cas9, such as Staphylococcus aureus Cas9 (SaCas9)4 or Streptococcus thermophilus Cas9 (St1Cas9)5 might work better for you.
    • Your target genomic region does not have the SpCas9 protospacer adjacent motif (PAM) sequence, 5’-NGG-3’: The target sequence has to be immediately upstream of the PAM sequence to ensure successful Cas9 binding. There are other Cas9 variants with different PAM sequences (Table 1). If your target sequence is AT-rich, Cpf1, another RNA-guided DNA endonuclease, could be a good choice for you6.
    • You need to reduce further unwanted off-target mutations: Researchers have modified SpCas9 to increase its specificity; examples include SpCas9 D1135E7, eSpCas98, and SpCas9-HF19. These variants are new and have not been as widely used as wild type SpCas9. But the research backing them is sound. They might be worth a try.

Table 1. Cas9 variants and PAM sequences

Cas9 variant PAM sequence Note
Neisseria meningitidis (NM)5 NNNNGATT
SpCas9 NGG
eSpCas98 NGG enhanced specificity
SpCas9-HF19 NGG enhanced specificity
SpCas9 D1135E7 NGG enhanced specificity
Staphylococcus aureus Cas9 (SaCas9)10 NNGRRT or NNGRR(N)
(R = A or G)
~1 kb shorter than SpCas9; efficiency similar to SpCas9
Streptococcus thermophilusCas9 (St1Cas9)5 NNAGAAW
(W = A or T)
~1 kb shorter than SpCas9; efficiency similar to SpCas9
Treponema denticola (TD)5 NAAAAC
What’s next?

OK. Now you have decided the cell line and Cas9 for our experiments. In the next blog post, we will show you how to start designing gRNAs.

Want to use Benchling’s CRISPR tools at your company?

Request a Demo

  1. Jinek, Martin et al.A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity.Science 337.6096 (2012): 816-821. ^
  2. Cong, Le et al.Multiplex genome engineering using CRISPR/Cas systems.Science 339.6121 (2013): 819-823. ^
  3. Mali, Prashant et al.RNA-guided human genome engineering via Cas9.Science 339.6121 (2013): 823-826. ^
  4. Ran, F Ann et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature 520.7546 (2015): 186-191. ^
  5. Esvelt, Kevin M et al. “Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.Nature methods 10.11 (2013): 1116-1121. ^
  6. Zetsche, Bernd et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell 163.3 (2015): 759-771. ^
  7. Kleinstiver, Benjamin P et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities.Nature (2015). ^
  8. Slaymaker, Ian M et al.Rationally engineered Cas9 nucleases with improved specificity.Science 351.6268 (2016): 84-88. ^
  9. Kleinstiver, Benjamin P et al.High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects.Nature (2016). ^
  10. Ran, F Ann et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature 520.7546 (2015): 186-191. ^



  1. Hi,
    There is something missing in your CRISPR story. Where are the two last points of your tutorial? Hope you will update the blog.
    Thank you anyway.

    1. Thanks Stefania! We’ve momentarily deprioritized parts 5 and 6, since all of the relevant parties are quite occupied with other work. However, we do hope to finish the tutorial someday soon!

Leave a Reply