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When does the Cas9 nuclease stop?

When does the Cas9 nuclease stop?


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If I understand correctly, the steps of gene editing with CRISPR-Cas9 are roughly as follows

  • Cas9 nuclease and guide RNA form a complex.
  • Cas9+guide RNA complex scans genomic DNA and recognizes the sequences homologous to the guide RNA
  • Induces a double strand break in DNA upstream of the PAM sequence (only breaks when the homologous sequence of the guide RNA is in the vicinity of the PAM).
  • Genetic modification using the mechanism of DNA repair (NHEJ, HDR) after double-strand break.

If so, in the gene editing with CRISPR-Cas9 , gene editing is likely to continue as long as the Cas9 does not lose its activity, am I right?

My question

  • When does the Cas lose its activity?
  • What was the logic of the experiment that identified the timing? / If the matter itself is unexplored, what kind of experiments could be done to find out the timing when the Cas lose its activity?

As this movie shows, if the gene editing occurs successfully, it will indeed introduce a mutation in the target gene.

However, even if it does,-this is just my guess from here on out- it is likely to remain complementary to the target gene, at least upstream of the guide RNA. However, even if the target gene is mutated, there is likely to remain complementary sequence to the guide RNA, at least upstream of the mutation. So, even if one successful gene edit is completed, I think it's possible that gene editing could happen again in the same location. Moreover, even if the targeted site is successfully edited, there is still the possibility of subsequent off-target editing elsewhere. That's why I think the tools exist to stop CAS.


A guide RNA binds to the target gene that has been bitten by CAS. The strand opposite the strand to which the guide RNA was bound has just been cleaved. Quoted from this video.

An idea for an experiment to detect when CAS goes quiet If a single cell colony was created from a gene-edited population and there was genetic variation in the cells born from that colony, then I think that would mean that there would have been Cas9 activity even after the single cell colony was started in culture. Is this idea correct? Could there be a smarter experiment?


Regarding the loss of Cas9 activity, you are already touching on the answer in your question. Cas9 as a nuclease/enzyme is always active, and will continue to cleave double-stranded DNA as long as there are complementary target sites and guide RNAs to guide it there. If the target site is not mutated or completely removed after cleavage, Cas9 can indeed cleave the same site again.

However, even if the target gene is mutated, there is likely to remain complementary sequence to the guide RNA, at least upstream of the mutation.

I'm not sure if I misunderstand the point you are trying to make here, but the introduction of mutations is usually enough to prevent recurrent cleavage of the same site (although studies of off-target effects clearly show that this is not always the case):

Both Streptococcus pyogenes and Staphylococcus aureus Cas9 cleave their target sites between positions -3 and -4, counting from the end of the 20-nt guide sequence (see attached figure below). The double-stranded break can then be repaired either via non-homologous end joining (NHEJ), or via template-driven homologous recombination (HR).

In the former case (NHEJ), double-stranded breaks are sometimes repaired with small errors. These errors usually accumulate near the break site (read about NHEJ to understand why), and such mutations will typically destroy the gRNA recognition site. Note, however, that not all (in fact, very few) NHEJ repair events will result in the introduction of mutations, and "perfectly repaired" breaks both can and will be re-cleaved by Cas9. But since repeated cleavage is lethal to the cell, there is a selective advantage for mutations that prevent re-cleavage of a target site. Also note that the introduction of mutations (and thereby gene editing) via NHEJ is a random process, with no direct control by the researcher.

In the latter case (HR), the DNA template used for repairing the double-stranded break can typically be chosen by the researcher. It can for example be a chemically synthesized DNA fragment or PCR product with at least some sequence homology to regions flanking the cleavage site. But since there is no specific sequence requirement for the region in between the homology regions, this interspacing region can be designed such that it partly or completely replaces the original recognition and/or PAM sequence (violet region in figure below).


Nuclease dead Cas9

Posted on April 1st, 2021 by Dr. Francis Collins

Credit: iStock/Firstsignal

Gene editing has shown great promise as a non-heritable way to treat a wide range of conditions, including many genetic diseases and more recently, even COVID-19. But could a version of the CRISPR gene-editing tool also help deliver long-lasting pain relief without the risk of addiction associated with prescription opioid drugs?

In work recently published in the journal Science Translational Medicine, researchers demonstrated in mice that a modified version of the CRISPR system can be used to “turn off” a gene in critical neurons to block the transmission of pain signals [1]. While much more study is needed and the approach is still far from being tested in people, the findings suggest that this new CRISPR-based strategy could form the basis for a whole new way to manage chronic pain.

This novel approach to treating chronic pain occurred to Ana Moreno, the study’s first author, when she was a Ph.D. student in the NIH-supported lab of Prashant Mali, University of California, San Diego. Mali had been studying a wide range of novel gene- and cell-based therapeutics. While reading up on both, Moreno landed on a paper about a mutation in a gene that encodes a pain-enhancing protein in spinal neurons called NaV1.7.

Moreno read that kids born with a loss-of-function mutation in this gene have a rare condition known as congenital insensitivity to pain (CIP). They literally don’t sense and respond to pain. Although these children often fail to recognize serious injuries because of the absence of pain to alert them, they have no other noticeable physical effects of the condition.

For Moreno, something clicked. What if it were possible to engineer a new kind of treatment—one designed to turn this gene down or fully off and stop people from feeling chronic pain?

Moreno also had an idea about how to do it. She’d been working on repressing or “turning off” genes using a version of CRISPR known as “dead” Cas9 [2]. In CRISPR systems designed to edit DNA, the Cas9 enzyme is often likened to a pair of scissors. Its job is to cut DNA in just the right spot with the help of an RNA guide. However, CRISPR-dead Cas9 no longer has any ability to cut DNA. It simply sticks to its gene target and blocks its expression. Another advantage is that the system won’t lead to any permanent DNA changes, since any treatment based on CRISPR-dead Cas9 might be safely reversed.

After establishing that the technique worked in cells, Moreno and colleagues moved to studies of laboratory mice. They injected viral vectors carrying the CRISPR treatment into mice with different types of chronic pain, including inflammatory and chemotherapy-induced pain.

Moreno and colleagues determined that all the mice showed evidence of durable pain relief. Remarkably, the treatment also lasted for three months or more and, importantly, without any signs of side effects. The researchers are also exploring another approach to do the same thing using a different set of editing tools called zinc finger nucleases (ZFNs).

The researchers say that one of these approaches might one day work for people with a large number of chronic pain conditions that involve transmission of the pain signal through NaV1.7. That includes diabetic polyneuropathy, sciatica, and osteoarthritis. It also could provide relief for patients undergoing chemotherapy, along with those suffering from many other conditions. Moreno and Mali have co-founded the spinoff company Navega Therapeutics, San Diego, CA, to work on the preclinical steps necessary to help move their approach closer to the clinic.

Chronic pain is a devastating public health problem. While opioids are effective for acute pain, they can do more harm than good for many chronic pain conditions, and they are responsible for a nationwide crisis of addiction and drug overdose deaths [3]. We cannot solve any of these problems without finding new ways to treat chronic pain. As we look to the future, it’s hopeful that innovative new therapeutics such as this gene-editing system could one day help to bring much needed relief.

[1] Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice. Moreno AM, Alemán F, Catroli GF, Hunt M, Hu M, Dailamy A, Pla A, Woller SA, Palmer N, Parekh U, McDonald D, Roberts AJ, Goodwill V, Dryden I, Hevner RF, Delay L, Gonçalves Dos Santos G, Yaksh TL, Mali P. Sci Transl Med. 2021 Mar 1013(584):eaay9056.

[2] Nuclease dead Cas9 is a programmable roadblock for DNA replication. Whinn KS, Kaur G, Lewis JS, Schauer GD, Mueller SH, Jergic S, Maynard H, Gan ZY, Naganbabu M, Bruchez MP, O’Donnell ME, Dixon NE, van Oijen AM, Ghodke H. Sci Rep. 2019 Sep 169(1):13292.

[3] Drug Overdose Deaths. Centers for Disease Control and Prevention.

Congenital insensitivity to pain (National Center for Advancing Translational Sciences/NIH)

Opioids (National Institute on Drug Abuse/NIH)

Mali Lab (University of California, San Diego)

NIH Support: National Human Genome Research Institute National Cancer Institute National Institute of General Medical Sciences National Institute of Neurological Disorders and Stroke


DNA Binding and Cleavage

CRISPR/Cas9 systems use a guide RNA with a region complementary to the target DNA to specifically bind their target sequences. However, there is an immediate and inherent issue with this. In order to achieve specificity, longer guide RNAs are beneficial, as each nucleotide in the RNA guide increases the specificity of the nuclease about 4-fold. However, in order for the DNA to melt and accommodate base-pairing to the guide RNA, the longer the RNA guide, the less efficient the nuclease. How can CRISPR/Cas9 systems have such dramatically increased specificity over other nucleases such as TALENS and ZFNS and still maintain roughly the same, if not better, efficiency? (Mali et al. 2013)

The answer is that the CRISPR/Cas9 system uses the Protospacer Adjacent Motif (PAM) binding as a preliminary step in locating the target sequence. As was determined by single molecule fluorescence microscopy, the initial binding of Cas9 to PAM (N-G-G) sequences allows the enzyme to quickly screen for potential target sequences. The enzyme will rapidly detach from DNA that does not have the proper PAM sequence. If the protein finds a potential target with the appropriate PAM, it will to melt the remaining DNA on the target to test whether the remaining target sequence is complementary to its guide sequence. The PAM binding step allows the protein to quickly screen potential targets and avoid melting many non-target sequences in its search for fully complementary sequences to cut. (Sternberg et al. 2014)

In July of 2014, Anders et al. published a crystal structure that led to a model for PAM-dependent target DNA binding, unwinding, and recognition by the Cas9 nuclease. The following images are created based off of figure 4 of the paper, or are images rendered in Pymol (distributed by Schrödinger) using the crystal structure from that paper (obtained from the Protein Data Bank).

Proposed model for PAM-dependent target DNA binding, melting, and recognition by Cas9:

Figure 2: Pam Binding and Phosphate Lock Loop Binding (original image) (crystal image rendered from PDB: 4UN3 Anders et al. 2014.)

The Protospacer Adjacent Motif (PAM) NGG bases of the target DNA strand are shown in yellow. Arginine residues 1333 and 1335 of the PAM Interacting (PI) domain bind to the major groove of the guanine bases in the PAM. A lysine residue in the Phosphate Lock Loop, also in the PI domain, binds the minor groove.

This positions the PAM and target DNA such that serine 1109 in the phosphate lock loop, and two nitrogens of the phosphate lock loop’s backbone, can form hydrogen bonds to the phosphate at position +1 of the PAM. This stabilizes the target DNA such that the first bases of the target sequence (or the protospacer) can melt and rotate upwards towards the guide RNA.

Figure 3: Phosphate Lock Loop Binding and DNA unwinding (original image) (crystal image rendered from. PDB: 4UN3 Anders et al. 2014)

If the target DNA is complementary to the guide RNA strand, the two strands will base pair. This will allow the target DNA to unzip, as the bases flip up and bind the guide RNA. Without the initial PAM binding and stabilization of the +1 phosphate, the guide RNA would very rarely be able to bind the target DNA, and Cas9 would be very inefficient. This illustrates a mechanism that explains why Cas9 is able to have both high efficiency and high specificity, thus making it a powerful genome editing tool.

Figure 4: DNA recognition and cleavage (original image) (crystal image rendered from PDB: 4UN3 Anders et al. 2014.)

Finally, complete annealing of the guide RNA to the target DNA allows the HNH and RuvC nucleases to cleave their respective strands. These nucleases cleave very specifically between the 3rd and 4th nucleotides from the PAM. Again, this specificity of cleavage, as well as the fact that the individual nucleases may be mutated independently and without affecting the ability of Cas9 to bind specific sequences, make the CRISPR/Cas9 system a simultaneously powerful and flexible genome editing tool.


Contents

In a bacterial genome, CRISPR loci contain "spacers" (viral DNA inserted into a CRISPR locus) that in type II adaptive immune systems were created from invading viral or plasmid DNA (called "protospacers"). Upon subsequent invasion, a CRISPR-associated nuclease such as Cas9 attaches to a tracrRNA–crRNA complex, which guides Cas9 to the invading protospacer sequence. But Cas9 will not cleave the protospacer sequence unless there is an adjacent PAM sequence. The spacer in the bacterial CRISPR loci will not contain a PAM sequence, and thus will not be cut by the nuclease, but the protospacer in the invading virus or plasmid will contain the PAM sequence, and thus will be cleaved by the Cas9 nuclease. [4] In genome editing applications, a short oligonucleotide known as a guide RNA (gRNA) is synthesized to perform the function of the tracrRNA–crRNA complex in recognizing gene sequences having a PAM sequence at the 3'-end, thereby "guiding" the nuclease to a specific sequence which the nuclease is capable of cutting. [7] [8]

The canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. [9] Guide RNAs can transport Cas9 to any locus in the genome for gene editing, but no editing can occur at any site other than one at which Cas9 recognizes PAM. The canonical PAM is associated with the Cas9 nuclease of Streptococcus pyogenes (designated SpCas9), whereas different PAMs are associated with the Cas9 proteins of the bacteria Neisseria meningitidis, Treponema denticola, and Streptococcus thermophilus. [10] 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells, but efficiency varies with genome location. [11] Attempts have been made to engineer Cas9s to recognize different PAMs in order to improve the ability of CRISPR-Cas9 to edit genes at any desired genome location. [12]

The Cas9 of Francisella novicida recognizes the canonical PAM sequence 5'-NGG-3', but has been engineered to recognize 5'-YG-3' (where "Y" is a pyrimidine [13] ), thus adding to the range of possible Cas9 targets. [14] The Cpf1 nuclease of Francisella novicida recognizes the PAM 5'-TTTN-3' [15] or 5'-YTN-3'. [16]

Aside from CRISPR-Cas9 and CRISPR-Cpf1, there are doubtless many yet undiscovered nucleases and PAMs. [17]

CRISPR/Cas13a (formerly C2c2 [18] ) from the bacterium Leptotrichia shahii is an RNA-guided CRISPR system that targets sequences in RNA rather than DNA. PAM is not relevant for an RNA-targeting CRISPR, although a guanine flanking the target negatively affects efficacy, and has been designated a "protospacer flanking site" (PFS). [19]

A technology called GUIDE-Seq has been devised to assay off-target cleavages produced by such gene editing. [20] The PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on wild-type alleles. [21]


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Transfect, enrich, screen, and publish—using our GeneArt CRISPR Nuclease Vector Kit. The CRISPR Nuclease system offers a ready-to-use, all-in-one expression vector system with a Cas9 nuclease expression cassette and a guide RNA cloning cassette for fast cloning of a target-specific crRNA. This system allows you to edit and engineer the genomic locus of your choice in a sequence-specific manner from a single plasmid. After relevant targets have been identified with GeneArt CRISPRs, the biologically-relevant mutations can be validated with GeneArt TALs to reduce potential off-targeting.

GeneArt CRISPR Nuclease Vector Kits are reporter vector systems for expression of the functional components needed for CRISPR-Cas genome editing. The kits make it easy to express noncoding guide RNA (including crRNA and tracrRNA), using a plasmid vector that also expresses Cas9 endonuclease.

The GeneArt CRISPR Nuclease Vector with OFP (orange fluorescent protein) for flow cytometry–based sorting of crRNA-expressing cell populations, whereas GeneArt CRISPR Nuclease Vector with CD4 enables bead-based enrichment of crRNA-expressing cells.

GeneArt CRISPR Nuclease Vectors. (A) GeneArt CRISPR Nuclease: OFP Reporter Plasmid map and features of GeneArt CRISPR Nuclease: OFP Reporter. The vector is supplied linearized between nucleotides 6,732 and 6,752, with 5 bp 5´ overhangs on each strand as indicated. (B) GeneArt CRISPR Nuclease: CD4 Enrichment Plasmid map and features of GeneArt CRISPR Nuclease: CD4 Enrichment. The vector is supplied linearized between nucleotides 7,336 and 7,355, with 5 bp 5´ overhangs on each strand as indicated. The linearized GeneArt CRISPR Nuclease Vectors provide a rapid and efficient way to clone double-stranded oligonucleotides encoding a crRNA representing a desired target into an expression cassette that allows sequence-specific targeting of the Cas9 nuclease.

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The Answer

The CRISPR system evolved as an adaptive prokaryotic immune system. The CRISPR system utilizes an RNA-guided endonuclease, Cas9, which is capable of making site-specific cuts at DNA sequences that match unique sequences found between the palindromic repeats in a CRISPR array. In nature, the CRISPR system uses the Cas9 endonuclease to destroy DNA from invading entities, such as bacteriophage.

CRISPR-utilizing organisms can also capture new DNA from invading species and incorporate it into an existing CRISPR array. This ability to incorporate new DNA allows organisms to defend against future infections that harbor the newly incorporated sequence, but also requires that the Cas9 protein be adaptable to countless target DNA sequences. Researchers have harnessed this adaptability of the CRISPR system to create a programable nuclease that can cut a DNA sequences with unparalleled specificity and efficiency.

Additionally, Cas9 has demonstrated considerable versatility. It can efficiently and specifically cut, nick, and bind genomic sequences in prokaryotes, eukaryotes, mammals, and even in human cells and embryos. This versatility has paved the way for a plethora of new advances in genomic engineering and expression control. This website will explore the underlying biochemistry of CRISPR/Cas9, and attempt to explain what, biochemically, makes Cas9 so good at what it does. In short:

CRISPR/Cas9 provides an efficient and flexible way to selectively target and cleave DNA. The Cas9 protein produces double strand breaks through independent RuvC and HNH nuclease domains at a target specified by a guiding RNA molecule. Existing genome-editing techniques, such as Homology-Directed Repair rely on creating double strand breaks. Because the guiding RNA molecule can be modified to target diverse nucleotide sequences, the CRISPR/Cas9 methods provide greater selectivity than other established molecular biology techniques.

Figure: Cartoon Diagram of Cas9 using its 2 nuclease domains to cleave a target DNA molecule at a site specified by the protospacer adjacent motif (NGG) and the 20 nucleotide guide RNA. (original figure)

Moreover, the Cas9 complex targets DNA efficiently through its DNA binding mechanism. Because one nuclease domain can be inactivated without affecting the other, the Cas9 complex can be converted into a nickase. Alternatively, inactivation of both nuclease domains produces a highly specific DNA-binding complex. These different modes of recognition and gene inactivation make CRISPR/Cas9 an exciting opportunity for targeted genome editing.


Acknowledgements

J.v.d.O. is supported by the Netherlands Organization for Scientific Research (NWO) through a TOP grant (714.015.001). J.L is supported by the gravitation program from the Netherlands Organization for Scientific Research (NWO). The work of R.K. is part of the Oncode Institute which is partly financed by the Dutch Cancer Society and was funded by the gravitation program from the NWO. The work of N.G. is supported in part by Stichting Singelswim Utrecht, Stichting FSHD and TKI/Health Holland.


The CRISPR-Cas system in prokaryotes precisely identifies infecting parasitic DNAs and viruses and destroys them. The CRISPR-Cas system has been adapted for facile genome editing, heralding a new age in molecular biology. Jiang et al. show that the Cas9 nuclease adopts a distinct confirmation when it binds to the targeting guide RNA. The guide RNA then assumes a preordered shape. This RNA “seed region” is thus poised to initiate recognition of the DNA target sequence.

Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA “seed” sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition–competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a “seed” mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes.

CRISPR-Cas proteins function in complex with mature CRISPR RNAs (crRNAs) to identify and cleave complementary target sequences in foreign nucleic acids (1). In type II CRISPR systems, the Cas9 enzyme cleaves DNA at sites defined by the 20-nucleotide (nt) guide segment within crRNAs, together with a trans-activating crRNA (tracrRNA) (2) that forms a crRNA:tracrRNA hybrid structure capable of Cas9 association (3). Once assembled on target DNA, the Cas9 HNH and RuvC nuclease domains cleave the double-stranded DNA (dsDNA) sequence within the strands that are complementary and noncomplementary to the guide RNA segment, respectively (3, 4) (Fig. 1A). By engineering a synthetic single-guide RNA (sgRNA) that fuses the crRNA and tracrRNA into a single transcript of 80 to 100 nt (Fig. 1B), Cas9:sgRNA has been harnessed as a two-component programmable system for genome engineering in various organisms (5, 6).

(A) Domain organization of the type II-A Cas9 protein from S. pyogenes (SpyCas9). (B) Secondary structure diagram of sgRNA bearing complementarity to a 20-bp region λ1 DNA. The seed sequence is highlighted in beige. Bars between nucleotide pairs represent canonical Watson-Crick base pairs dots indicate noncanonical base-pairing interactions. The base stacking interaction is indicated by a filled square. (C) Tertiary structure of sgRNA in ribbon representation, with a sigma-A weighted composite-annealed omit 2FobsFcalc electron density map contoured at 1.5σ. (D) Ribbon diagram of SpyCas9-sgRNA complex, color-coded as defined in Fig. 1, A and B. (E) Surface representations of the crystal structure of SpyCas9 in complex with sgRNA (depicted in cartoon) showing the same view as in Fig. 1D and a 180°-rotated view.

The utility of Cas9 for both bacterial immunity and genome engineering applications relies on accurate DNA target selection. Target choice relies on base pairing between the DNA and the 20-nt guide RNA sequence, as well as the presence of a 2– to 4–base pair (bp) protospacer adjacent motif (PAM) proximal to the target site (3, 4). The target complementarity of a “seed” sequence within the guide segment of crRNAs is critical for DNA recognition and cleavage (7, 8). In type II CRISPR systems, Cas9 binds to targets by recognizing a PAM and searching the adjacent DNA for complementarity to the 10- to 12-nt “seed” sequence at the 3′ end of the guide RNA segment (Fig. 1B) (3, 911). Crystal structures of Cas9 bound to sgRNA and a target DNA strand, with or without a partial PAM-containing nontarget strand, show the entire 20-nt guide RNA segment engaged in an A-form helical interaction with the target DNA strand (12, 13). How the “seed” region within the guide RNA specifies DNA binding has remained unknown.

To determine how Cas9 assembles with and positions the guide RNA prior to substrate recognition, we solved the crystal structure of catalytically active Streptococcus pyogenes Cas9 (SpyCas9) in complex with an 85-nt sgRNA at 2.9 Å resolution (Fig. 1 and table S1). The overall structure of the Cas9-sgRNA binary complex, representing the pre–target-bound state of the enzyme, resembles the bilobed architecture of the target DNA–bound state, as observed in electron microscopic studies (14), with the guide segment of the sgRNA positioned in the central channel between the nuclease and helical recognition lobes (Fig. 1, C to E). This structural architecture and guide RNA organization is maintained in the crystal structure of a widely used nuclease-inactive version of Cas9 (D10A/H840A, referred to as dCas9) in complex with sgRNA (fig. S1).

Comparison of SpyCas9 crystal structures representing the protein alone and the RNA-bound and RNA-DNA–bound states of the enzyme reveals the nature of Cas9’s conformational flexibility during sgRNA binding and target DNA recognition (Fig. 2A and figs. S2 and S3). The helical recognition lobe undergoes substantial rearrangements upon sgRNA binding but before DNA association, especially in helical domain 3, which moves as a rigid body by

65 Å into close proximity with the HNH domain (fig. S2D). Superposition of the Cas9-sgRNA pre–target-bound complex onto the target DNA–bound structures reveals further conformational changes, including a modest shift in helical domains 2 and 3, as well as a concomitant displacement of the HNH domain toward the target strand (Fig. 2A and fig. S2, E and F). Together with limited proteolysis data (Fig. 2B and fig. S4), these results show that sgRNA binding drives the major conformational changes within Cas9 (14), although additional structural rearrangements occur upon substrate DNA binding. Interestingly, a guide-target mismatched DNA duplex yields a proteolytic pattern similar to that observed for sgRNA-bound Cas9 (fig. S4B), indicating that Cas9-sgRNA pretarget conformation is competent for PAM recognition because no further conformational change is required prior to target DNA binding.

(A) Structural comparison between Cas9-sgRNA complex (pretarget) and target DNA–bound structure (PDB ID 4UN3) (see also movies S1 and S2). Vector length correlates with the domain motion scale. Black arrows indicate domain movements within Cas9-sgRNA upon target DNA binding. (B) Limited proteolysis to test for large-scale conformational changes of Cas9 upon sgRNA binding and target DNA recognition. (C) Overlay of the Cas9-sgRNA pretarget bound complex with the target DNA–bound structures. For clarity, only the PAM-containing CTD domain is shown. (D) Close-up view of the seed-binding channel in surface representation. (E) Superimposed sgRNAs in the pretarget (beige) and target DNA–bound states (black and orange) with only the guide segments shown for clarity. Helical axis is indicated by dotted line. Dihedral angles (θ) between guide segment nucleobases and those of the A-form RNA-DNA heteroduplex in target DNA–bound structures are shown in parentheses. (F) Schematic showing key interactions of SpyCas9 with the sgRNA seed sequence. The inset highlights the conformational change of Tyr 450 upon target binding.

The single-stranded guide RNA binding triggers ordering of the PAM recognition region of Cas9. In the absence of sgRNA, Cas9’s PAM-interacting C-terminal domain (CTD) is largely disordered (fig. S2A) (14). However, in the Cas9-sgRNA pre–target-bound complex and target DNA–bound structures, the PAM-interaction CTD domain is structured to accommodate the PAM duplex (Fig. 2C). Two critical arginine resides (Arg 1333 and Arg 1335 ) involved in 5′-NGG-3′ PAM recognition (13) are pre-positioned in the Cas9-sgRNA structure to recognize the GG dinucleotide on the nontarget DNA strand. This explains biochemical data indicating that the Cas9-sgRNA complex uses PAM recognition as an obligate step to identify potential DNA target sites (9).

In the Cas9-sgRNA structure, the RNA adopts an L-shaped configuration in which the 5′ guide segment lies in close spatial proximity to stem loop 1 of the sgRNA (Fig. 1C and fig. S5). Similar to the DNA-bound Cas9 complexes, Cas9 in the pre–target-bound state makes extensive hydrogen-bonding contacts and aromatic stacking interactions with the crRNA repeat:tracrRNA anti-repeat duplex and stem loop 1 (fig. S6) (12, 15). In contrast to the sgRNA scaffold (nucleotides G21 to U82) for which clear electron density is observed, we observed unambiguous electron density for only 10 of the 20 nucleotides of the guide RNA segment (nucleotides 11 to 20 Fig. 1, B and C), all of which are located in the seed region. Nucleotides 1 to 10 of the guide RNA segment, although present in the crystals (fig. S1), are disordered. The ordered seed nucleotides (G11 to C20, counting from the 5′ end of the sgRNA) are threaded through the narrow nucleic acid–binding channel formed between the two Cas9 lobes, with their bases facing outward (Fig. 2D and fig. S7). Nucleotides G19, C20, and G11 to U13 are exposed to bulk solvent, whereas nucleotides G14 to C18 are shielded from solvent by helical domain 2. The solvent-exposed PAM-proximal seed nucleotides G19 and C20 are therefore positioned to serve as the nucleation site for initiating target binding. This explains how a 2-bp mismatch immediately adjacent to the PAM in the DNA abolishes Cas9 binding and cleavage activity (9).

The single-stranded guide RNA within the seed region maintains a nearly A-form conformation along the ribose-phosphate backbone (Fig. 2E). To maintain this helical configuration, Cas9 makes extensive hydrogen-bonding interactions with phosphates and 2′-hydroxyl groups of the seed nucleotides (Fig. 2F). Such presentation of the seed sequence in a conformation thermodynamically favorable for helical guide:target duplex formation (16) is reminiscent of the guide RNA positioning observed in eukaryotic Argonaute complexes that recognize transcripts by base pairing with a 6-nt RNA seed sequence (fig. S8, A and B) (1719). This situation is distinct from that observed in the type I CRISPR-Cascade targeting complex, in which the entire crRNA guide region is preordered, rather than just the seed segment (fig. S8C) (2022).

Another similarity between the Cas9-bound sgRNA guide segment and the Argonaute-bound microRNA guide segment is the synchronized tilting of bases at each half-helical turn of the RNA strand. In the Cas9-sgRNA complex, a kink introduced by insertion of Tyr 450 between seed nucleobases A15 and G16 results in coordinated tilting of nucleobases G11 to A15 relative to the same region of the guide RNA in the target-bound state (Fig. 2, E and F, and fig. S8A). Notably, the orientation of Tyr 450 shifts by

120° upon target binding (Fig. 2F). The bases G16 to C20 remain in an untilted orientation that is immediately ready for target DNA base pairing. This nonuniformity in base orientation may account for previous observations showing that the 5-nt sequence of the guide RNA that binds to DNA immediately adjacent to the PAM is the most critical segment for Cas9 binding (23).

Structural and biochemical data suggest that guide RNA binding triggers a large structural rearrangement in Cas9. To test whether the seed segment of the RNA itself contributes to formation of an activated Cas9 conformation, we monitored Cas9-sgRNA assembly with the use of a set of progressively truncated guide RNAs containing 0 to 20 nt of the guide segment (N0 to N20 table S2). Limited proteolysis showed that guide RNA binding confers protection from trypsin digestion only when the guide segment has a length of at least 10 nt of the target recognition sequence (N10) (Fig. 3A and fig. S9). The absence of the guide segment results in moderately decreased Cas9 binding affinity for the RNA (fig. S10). Together, these results indicate that despite forming a stable complex with Cas9 (fig. S11), the crRNA:tracrRNA scaffold region of the sgRNA alone fails to induce the target recognition–competent conformation of Cas9.

(A) SDS–polyacrylamide gel electrophoresis of limited trypsin digestion of SpyCas9 in the presence of truncated guide RNAs. (B) Analytical size-exclusion chromatograms of SpyCas9-sgRNA in the absence or presence of single-stranded target DNA with the indicated number of complementary nucleotides. The dashed line indicates the peak position of stably bound SpyCas9-sgRNA-ssDNA ternary complex eluting from the gel filtration column. (C) Cas9-mediated endonuclease activity time course assays using plasmid and oligonucleotide DNA ( 32 P-labeled on both strands) containing a 20-bp λ1 DNA target sequence and a 5′-TGG-3′ PAM motif. Cn (n = 0, 10, 12, 14, 17, or 20) represents the number of potential guide-target base pairs counted from the PAM end.

To assess the molecular mechanism of Cas9-mediated RNA-DNA hybridization, we first used size exclusion chromatography to evaluate the effects of DNA length on the formation of Cas9-sgRNA-ssDNA (single-stranded DNA) ternary complexes. This analysis showed that target ssDNA length must be at least 10 nt to form a kinetically stable ternary complex with Cas9-sgRNA (Fig. 3B), in good agreement with the requirement for a 10- to 12-bp RNA-DNA heteroduplex to ensure strand propagation observed in Cas9 single-molecule experiments (9, 24). To further explore the importance of the seed region for Cas9-mediated DNA cleavage, we conducted endonuclease activity assays using both plasmid and oligonucleotide DNA substrates and our truncated guide RNAs. The plasmid cleavage assay revealed that the 12-bp seed:DNA heteroduplex is necessary for Cas9-mediated supercoiled plasmid cleavage, which proceeds by nicking first by the RuvC nuclease domain, then by the HNH nuclease domain (Fig. 3C and table S2). These data are consistent with structural observations indicating that the flexible HNH domain can adopt multiple non–catalytically productive states during sgRNA binding and target DNA recognition. In line with previous studies (25), the oligonucleotide cleavage assay showed that the N17 guide RNA displays an almost comparable cleavage rate but much reduced RuvC 3′-5′ exonuclease-trimming activity (3) relative to the N20 guide RNA (Fig. 3C). This trimming activity is more pronounced with the H840A nickase version of Cas9 relative to the D10A nickase version (fig. S12). This observation may explain why the D10A nickase is more efficient than the H840A nickase version of Cas9 when using a double-nicking strategy to enhance genome editing specificity (26).

We propose that the preordered PAM recognition region of the Cas9-sgRNA complex initiates DNA interrogation, followed by base pairing between a short PAM-proximal segment of DNA (1 or 2 bp) and the 3′ end of the seed sequence in the sgRNA (Fig. 4). Conformational changes of Cas9 upon initial DNA binding then accommodate guide RNA strand invasion into and beyond the seed region, triggering additional structural changes necessary for Cas9 to reach a cleavage-competent state. Recent crystal structures of human Argonaute2 bound to a microRNA guide and short RNA target sequences underscore the importance of seed region base pairing for accuracy of target selection (27).

When Cas9 is in the apo state, its PAM-interacting cleft (dotted circle) is largely disordered. In the pretarget state, the PAM-interacting domain and seed sequence from guide RNA are preorganized for PAM recognition, followed by dsDNA melting next to PAM. The nonseed region is disordered and indicated as a dotted line. Base pairing between the seed sequence and the target DNA drives Cas9 into a near-active conformation complete base pairing between the full guide segment and the target DNA strand enables Cas9 to reach a fully active state.

Our results suggest the apparent convergent evolution of a similar mechanism for CRISPR-Cas9. Collectively, our structural and biochemical data show that Cas9 is subject to multilayered regulation during its activation. The preordered RNA seed sequence and protein PAM-interacting cleft enable the Cas9-sgRNA complex to interact productively with potential DNA sequences for target sampling. The inactive conformation of apo Cas9, as well as the additional conformational changes required for the complex to reach its ultimate catalytically active state, could help to avoid spurious DNA cleavage within the host genome and hence minimize off-target effects in Cas9-based genome editing.


Author information

Affiliations

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA

Haiwei Mou, Jordan L. Smith, Lingtao Peng, Chun-Qing Song, Ankur Sheel, Deniz M. Ozata, Erik J. Sontheimer, Melissa J. Moore & Wen Xue

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA

Hao Yin, Qiongqiong Wu, Daniel G. Anderson & Zhiping Weng

Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, 01605, USA

Jill Moore, Xiao-Ou Zhang, Yingxiang Li & Zhiping Weng

Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, People’s Republic of China

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA

Harvard-MIT Division of Health Sciences & Technology, Cambridge, MA, 02139, USA

Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA

Wellstone Muscular Dystrophy Program, Department of Neurology, University of Massachusetts Medical School, Worcester, MA, 01605, USA

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, 01605, USA

Erik J. Sontheimer & Wen Xue

Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA, 01605, USA