CRISPR-Cas 9 in Genome Editing

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Read our scientific technical answers to your top 3 frequently asked questions (FAQs) on CRISPR-Cas9:

1. CRISPR-Cas9: What's the Buzz About?
2. Why Use CRISPR-Cas9 Antibodies?
3. Any Publications citing Novus’ CRISPR-Cas9 Antibodies?

CRISPR-Cas9: What's the Buzz About?

CRISPR-Cas9 is an RNA-guided DNA endonuclease enzyme (mwt: 158.4 kDa), which is now widely used in molecular biology for the induction of double strand breaks in DNA. CRISPR-Cas9 technology can be used to induce insertion or deletion mutations, specific sequence replacements or insertions, and large deletions or genomic rearrangements at any desired genomic location. The genes encoding CRISPR-Cas are essential to the bacterial adaptive immune system, enabling bacteria to mount an immune response against invading genetic material. The best characterized CRISPR-associated nucleases are the Cas9 proteins from Streptococcus pyogenes (S. pyogenes).

Why Use CRISPR-Cas9 Antibodies?

Antibodies serve as great tool when working with Cas9 transfectants, and the top four reasons for using CRISPR-Cas9 antibodies are given below:

i. Transfection Efficiency

Verifying the success of CRISPR-Cas9 transfection is the primary reason to use CRISPR-Cas9 antibodies. Comparing the expression of Cas9 in transfected versus mock transfected cells is important to assess the efficiency of transfection. With the help of CRISPR-Cas9 antibodies, one can use Western blot and/or immunostaining to verify transfection efficiency.

CRISPR-Cas9 Antibody (6G12) [NBP2-52398] Hela cells or Hela cells expressing Flag-tagged S. pyogenes Cas9 under the control of the PTight (Tet-ON) promoter were treated for 24h with 1ug/ul Doxycycline, fixed and permeabilized with Methanol/Acetone and blocked in 2% BSA in PBS for 2 h at RT. Cells were stained with 6G12 hybridoma supernatant (diluted 1:10) at 4°C o/n, followed by incubation with anti-mouse AF488 coupled secondary antibody for 1 h at RT. Nuclei were counter-stained with Hoechst 33342.

ii. CRISPR-Cas9 Expression

The levels as well as the duration of Cas9 expression are highly critical for CRISPR-Cas9 based genome editing. In stable clones, high expression levels of Cas9 may result in non-specific activity. This may be controlled through isolating multiple clones and screening them for Cas9 expression levels through Western blot analysis. For transient transfections, a chronic or prolonged expression of Cas9 may lead to the development of more off-target mutations. Transient nature of CRISPR-Cas9’s expression may be checked through Western blot analysis of the transfected samples collected at various time points.

CRISPR-Cas9 Antibody (6G12) [NBP2-52398] Hela cells or Hela cells expressing Flag-tagged S. pyogenes’ Cas9 under the control of the PTight (Tet-ON) promoter were treated for 24h with 1ug/ul Doxycycline and lysed under native conditions. 30ug of whole cell lysate per lane was separated by 7.5% SDS-PAGE, transfered to nitrocellulose membrane and incubated with 6G12 hybridoma supernatant (diluted 1:100) at 4˚C O/N. After washing, the membranes were incubated with secondary HRP-conjugated antibody and bands were visualized by ECL and exposure of X-ray films. Pre-stained marker bands were visualized with Blue Marker Antibody (NBP2-33376). Note the specific band of CRISPR-Cas9 at 158.4 kDa position.

iii. Subcellular Localization

CRISPR-Cas9 must translocate to the nuclei of transfected cells for executing its effects on DNA. Nuclear localization of Cas9 may be verified through ICC/IF or IHC staining with CRISPR-Cas9 antibodies.

CRISPR-Cas9 Antibody (7A9-3A3) [NBP2-36440] ICC/IF analysis of CRISPR-Cas9 transfected HEK293 cells using CRISPR-Cas9 antibody (clone 7A9-3A3). Red staining represents CRISPR-Cas9 positivity while DAPI stained nuclei are visible in blue color. This image is from a verified end user and was submitted via a product review.

iv. Target Binding of CRISPR-Cas9

CRISPR based genome editing has two main elements: a synthetic RNA called “guide RNA” (gRNA) and a non-specific CRISPR-Cas9 enzyme. Structurally, the gRNA is composed of a “scaffold” sequence which is essential for Cas9 enzyme’s binding to gRNA and a user-designated “spacer” or “targeting” sequence which defines the genomic target to be manipulated. ChIP grade CRISPR-Cas9 antibodies are an excellent tool for testing the binding specificity of Cas9 enzyme using a gRNA of your choice and the primers for targeted/non-targeted region.

CRISPR-Cas9 Antibody (6G12) [NBP2-52398] NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9 (Flag tagged), and a GFP-targeting gRNA were fixed with formaldehyde, harvested and sonicated to get 200-500bp DNA fragments. 50ug chromatin was incubated over night at 4°C with the indicated antibodies (200ul hybridoma supernatants of clones 7A9 and 6G12; 5ug Flag antibody) followed by incubation with protein G beads for 3h at 4°C. After washing chromatin was eluted from the beads and crosslinking was reversed over night at 65°C. After a proteinase K digestion step, DNA was separated using phenol/chloroform/isoamyl alcohol, precipitated with ethanol/sodium acetate and dissolved in water. For qPCR, primers either targeting the GFP gene or as negative control non-targeted regions (Ppap2c +7122 and Prkcd +24069 from transcription start) were used.

Any Publications citing Novus’ CRISPR-Cas9 Antibodies?

1. Choi JG, Dang Y, Abraham S et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther. 2016 Apr 07 [PMID:27052803] (antibody use: ICC/IF)
2. Chu VT, Weber T, Graf R et al. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 2016 Jan 16 [PMID:26772810] (antibody use: WB)
3. Chiou SH, Winters IP, Wang J et al. Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev 2015 Jul 15 [PMID:26178787] (antibody use: WB)