CRISPR/Cas9
Cas9 Mechanism
The key step in editing an organism’s genome is selective targeting of a specific sequence of DNA. Two biological macromolecules, the Cas9 protein and guide RNA, interact to form a complex that can identify target sequences with high selectivity.
The Cas9 protein is responsible for locating and cleaving target DNA, both in natural and in artificial CRISPR/Cas systems. The Cas9 protein has six domains, REC I, REC II, Bridge Helix, PAM Interacting, HNH and RuvC (Figure 1) (Jinek et al. 2014; Nishimasu et al. 2014).
The Rec I domain is the largest and is responsible for binding guide RNA. The role of the REC II domain is not yet well understood. The arginine-rich bridge helix is crucial for initiating cleavage activity upon binding of target DNA (Nishimasu et al. 2014). The PAM-Interacting domain confers PAM specificity and is therefore responsible for initiating binding to target DNA (Anders et al. 2014; Jinek et al. 2014; Nishimasu et al. 2014; Sternberg et al. 2014). The HNH and RuvC domains are nuclease domains that cut single-stranded DNA. They are highly homologous to HNH and RuvC domains found in other proteins (Jinek et al. 2014; Nishimasu et al. 2014).
Figure 1: Cas9 Protein. The Cas9 protein is comprised of six domains: Rec I, Rec II, Bridge Helix, RuvC, HNH, and PAM Interacting. Domains are shown in schematic, crystal, and map form. (original figure) (crystal image rendered from PDB: 4CMP Jinek et al. 2014.)
The Cas9 protein remains inactive in the absence of guide RNA (Jinek et al. 2014). In engineered CRISPR systems, guide RNA is comprised of a single strand of RNA that forms a T-shape comprised of one tetraloop and two or three stem loops (Figure 2) (Jinek et al. 2012; Nishimasu et al. 2014). The guide RNA is engineered to have a 5′ end that is complimentary to the target DNA sequence.
Figure 2: Engineered Guide RNA. Engineered guide RNA is a single strand of RNA. It forms one tetraloop and two or three stem loops (three shown). Target complimentary region is shown in red. (original figure) (crystal image rendered from PDB: 4UN3 Anders et al. 2014.)
This artificial guide RNA binds to the Cas9 protein and, upon binding, induces a conformational change in the protein (Figure 3). The conformational change converts the inactive protein into its active form. The mechanism of the conformational change is not completely understood, but Jinek and colleagues hypothesize that steric interactions or weak binding between protein side chains and RNA bases may induce the change (Jinek et al. 2014).
Figure 3: Activation of Cas9 protein by guide RNA binding. Binding of the guide RNA induces a conformational change in the Cas9 protein. The conformational change causes activation of the Cas9 nuclease activity (Jinek et al. 2014). (original figure) (crystal image rendered from PDB: 4UN3 Anders et al. 2014)
Once the Cas9 protein is activated, it stochastically searches for target DNA by binding with sequences that match its protospacer adjacent motif (PAM) sequence (Sternberg et al. 2014). A PAM is a two- or three-base sequence located within one nucleotide downstream of the region complementary to the guide RNA. PAMs have been identified in all CRISPR systems, and the specific nucleotides that define PAMs are specific to the particular category of CRISPR system (Mojica et al. 2009). The PAM in Streptococcus pyogenes is 5′-NGG-3′ (Jinek et al. 2012). When the Cas9 protein finds a potential target sequence with the appropriate PAM, the protein will melt the bases immediately upstream of the PAM and pair them with the complementary region on the guide RNA (Sternberg et al. 2014). If the complementary region and the target region pair properly, the RuvC and HNH nuclease domains will cut the target DNA after the third nucleotide base upstream of the PAM (Anders et al. 2014) (Figure 4).
Figure 4: Target DNA binding and cleavage by Cas9. 1) Cas9 scans potential target DNA for the appropriate PAM (yellow stars). (2) When the protein finds the PAM, the protein:guide RNA complex will melt the bases immediately upstream of the PAM and pair them with the target complimentary region on the guide RNA (Sternberg et al. 2014). (3) If the complimentary region and the target region pair properly, the RuvC and HNH domains will cut the target DNA after the third nucleotide base upstream of the PAM. (original figure) (crystal images: Lower left and right rendered from PDB: 4UN3 Anders et al. 2014; Upper left rendered from PDB: 4CMP Jinek et al. 2014)
Hi, I’d like to use (adapt) these figures for my thesis introduction – how should I cite you? Thanks!
Cavanagh & Garrity, “CRISPR Mechanism”, CRISPR/Cas9, Tufts University, 2014.
https://sites.tufts.edu/crispr/ (Date of Access)
We’re very glad you appreciated the images! However you want to cite them is probably fine, if you change them you can say “adapted from” at the beginning. Good Luck!
This entire explanation of the CRISPR/Cas9 system is fantastic. I have linked it to my learning management system as required reading for my undergraduate Genetics class – and plan on using (and citing) your figures in my lecture. Thank you so much for this great work!
Hi,
We are revising the Cas9 entry in the Wikipedia. You could of course also do this too.
If you put your diagrams (e.g. Fig 1) up, we could incorporate them into the entries (possibly with some modification). I think they would make a great addition.
Cheers
Chris
Thanks for the explanation, It’s really clear and helpful.
First: Thank you for the helpful information.
Secondly: I would like to cite from the content of this page, who would be the authors, if I may ask. (first name and last name)
Thank you in advance
Excellent explanation,very simple ansd soo precise.
Thank you so much for this article. It’s easily one of the best explanations of CRISPR Cas9 on the internet
Thanks for the good explanation and diagramatic representation of CRISPR mechanism. It was helpful. Thank you 🙂
Very nicely explained. Thanks a lot!
Hello,
Thank you for the detailed explanation. I’ve one query though. If PAM sequences are specifically found only in invading bacteriophages, then how is the CRISPR system applied in other organisms which do not have PAM sequences? Like in mice etc??
Hi,
Great explanation! I was wondering- is the gRNA sequence removed from the target region after it is annealed to initiate cleavage? That is, is the annealing transient?
Thank you for these simplified explanation.
Great Explaination. It would be more helpful for the newbies if you can include some animations/videos about the CRISPR/Cas system in it.
what are the full names of the 6 domains, thanks
I am working on the CRISPR to edit the Virulunce gene on Fowladenovirus but can i use the Cas 9 protien for S.pyogens
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The description of CRISPR/Cas 9, based on this model is super cool and very easy to understand. I can explain this model to any one now, I have been finding it difficult to understand it, but now I know and better person
does CRISPR return to normal after cleaving the DNA? Does it remain in its cleaved formation?
great article, so clear and descriptive, can you guys post other articles on crispr relating to eliminating death
I think the spelling in the illustration of the gRNA is ‘complementary’ as opposed to ‘complimentary’.
But that’s trivia, this is a great article.
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Normally, Cas9 from different species recognize different PAM.
Do they cut DNA at the same site (3-4 nucleotide upstream from PAM)?
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Does anyone know the molecular mechanism of crispr AS AN ORGANIC CHEMIST WRITE A CHEMICAL MECHANISM?
thank u for your comprehensive demonstration, but i also wonder how CRISPR system activates genes, could u show me a further illustration if that doesn’t bother u?
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Does the Cas9 protein injected into a body “find” the target sequence in every cell in the body? If so how? Does it duplicate itself?
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The key step in editing an organism’s genome is selective targeting of a specific sequence of DNA. Two biological macromolecules, the Cas9 protein and guide RNA, interact to form a complex that can identify target sequences with high selectivity.
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Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
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