3.2.5: Enzymes for Genetic modifications

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

​​​​​​Restriction Endonucleases: Recognition, Mechanism, and Specificity

• Explain how Type II restriction enzymes recognize palindromic sequences, distinguish between sticky-end and blunt-end cleavage, and describe how host methylation protects against self-digestion.
• Describe the catalytic mechanism of phosphodiester bond cleavage by Type II restriction enzymes, including the roles of Mg²⁺ as a Lewis acid, a general base to activate the nucleophilic water, and a general acid to stabilize the 3'-O⁻ leaving group.
• Interpret the stereochemical evidence (inversion at phosphorus) that argues against a covalent enzyme-DNA intermediate in EcoRI and EcoRV, and explain how this distinguishes the restriction enzyme mechanism from that of serine proteases.

CRISPR-Cas9: Structure, Conformational Dynamics, and Target Recognition

• Describe the domain architecture of Cas9 (NUC lobe with HNH and RuvC nuclease domains, REC lobe, bridge helix), identify the key catalytic residues in each nuclease domain, and explain how each domain contributes to blunt-end cleavage of target DNA.
• Explain the ordered assembly of the Cas9 ternary complex — including why sgRNA binds before target DNA — and describe the conformational changes in the REC lobe that activate the enzyme and position the catalytic residues.
• Analyze how PAM sequence recognition by Arg1333 and Arg1335 triggers local DNA unwinding and heteroduplex formation with the sgRNA, and explain why the absence of a PAM sequence prevents off-target cleavage.

CRISPR-Cas9 as a Gene Editing Tool: Applications and Limitations

• Compare CRISPR-Cas9 to earlier gene editing platforms (zinc finger nucleases, TALENs) and explain the mechanistic and practical advantages of using a linear RNA recognition element over an engineered DNA-binding protein.
• Evaluate strategies for delivering CRISPR-Cas9 components into eukaryotic cells (electroporation, AAV, lipid nanoparticles), and explain why transient expression systems are preferred over continuous expression for minimizing off-target effects.
• Explain how PAM sequence frequency in the human genome, PAM mutations (e.g., D1135E), and Cas9 orthologs from other bacteria collectively expand the range of targetable genomic sequences for therapeutic applications.

It is difficult to read newspapers and news magazines without encountering the CRISPR-Cas9 gene editing system, which has the potential to make gene editing routine in disease diagnosis, treatment, and cure, as well as in genetic modification of organisms to improve their quality and quantity for food and natural product production. In this section, we will explore the mechanism of restriction enzymes that made gene cloning possible, as well as the CRISPR-Cas gene-editing system.

Restriction Endonucleases

A restriction enzyme, restriction endonuclease, or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are a class within the broader endonuclease group. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site or if the recognition and cleavage sites are separate. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e., each strand) of the DNA double helix. Here, we will focus on the Type II restriction enzymes routinely used in molecular biology and biotechnology applications.

As with other restriction enzymes, Type II Restriction Enzymes occur exclusively in unicellular microbial life forms––mainly bacteria and archaea (prokaryotes)––and are thought to protect these cells from viruses and other infectious DNA molecules. Inside a prokaryote, restriction enzymes selectively cut foreign DNA in a process called restriction digestion; meanwhile, a modification enzyme (a methyltransferase) protects host DNA by modifying it and blocking cleavage. Together, these two processes form the restriction-modification system.

The first Type II Restriction Enzyme discovered was HindII from the bacterium Haemophilus influenzae Rd. The event was described by Hamilton Smith (Figure 7.23) in his Nobel lecture, delivered on 8 December 1978:

‘"In one such experiment we happened to use labeled DNA from phage P22, a bacterial virus I had worked with for several years before coming to Hopkins. To our surprise, we could not recover the foreign DNA from the cells. With Meselson’s recent report in our minds, we immediately suspected that it might be undergoing restriction, and our experience with viscometry told us that this would be a good assay for such an activity. The following day, two viscometers were set up, one containing P22 DNA and the other Haemophilus DNA. Cell extract was added to each, and we began quickly taking measurements. As the experiment progressed, we became increasingly excited as the viscosity of the Haemophilus DNA held steady while the P22 DNA viscosity fell. We were confident that we had discovered a new and highly active restriction enzyme. Furthermore, it appeared to require only Mg²⁺ as a cofactor, suggesting that it would prove to be a simpler enzyme than that from E. coli K or B.

After several false starts and many tedious hours with our laborious but sensitive viscometer assay, Wilcox and I succeeded in obtaining a purified preparation of the restriction enzyme. We next used sucrose gradient centrifugation to show that the purified enzyme selectively degraded duplex, but not single-stranded, P22 DNA to fragments averaging around 100 bp in length, while Haemophilus DNA present in the same reaction mixture was untouched. No free nucleotides were released during the reaction, nor could we detect any nicks in the DNA products. Thus, the enzyme was clearly an endonuclease that produced double-strand breaks and was specific for foreign DNA. Since the final (limit) digestion products of foreign DNA remained large, it seemed to us that cleavage must be site-specific. This proved to be the case, and we were able to demonstrate it directly by sequencing the termini of the cleavage fragments.’"

Restriction enzymes are named according to the taxonomy of the organism in which they were discovered. The first letter of the enzyme refers to the organism's genus, and the second and third to the species. This is followed by letters and/or numbers identifying the isolate. Roman numerals specify different enzymes from the same organism. For example, the enzyme ‘HindIII’ was discovered in Haemophilus influenzae, serotype d, and is distinct from the HindI and HindII endonucleases also present within this bacterium. The DNA-methyltransferases (MTases) accompanying restriction enzymes are named similarly, and given the prefix ‘M.’. When there is more than one MTase, they are prefixed ‘M1.’, ‘M2.’, etc, if they are separate proteins, or ‘M1∼M2.’ when joined.

Restriction Enzymes that recognize the same DNA sequence, regardless of where they cut, are termed ‘isoschizomers’ (iso = equal; skhizo = split). Isoschizomers that cut the same sequence at different positions are called ‘neoschizomers’ (neo = new). Isoschizomers that cut at the same position are frequently, but not always, evolutionarily drifted versions of the same enzyme (e.g., BamHI and OkrAI). Neoschizomers, by contrast, are often evolutionarily unrelated enzymes (e.g., EcoRII and MvaI).

Type II Restriction Enzymes are a conglomeration of many different proteins that, by definition, have the common ability to cleave duplex DNA at a fixed position within, or close to, their recognition sequence. This cleavage generates reproducible DNA fragments and predictable gel electrophoresis patterns, making these enzymes invaluable reagents for laboratory DNA manipulation and investigation. Almost all Type II Restriction Enzymes require divalent cations, usually Mg²⁺, as essential components of their catalytic sites. Ca²⁺, by contrast, often inhibits Type II Restriction Enzymes.

The recognition sequences of Type II Restriction Enzymes are palindromic, with two possible types of palindromic sequences. The mirror-like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backward on a single DNA strand, as in GTAATG. An inverted repeat palindrome is also a sequence that reads the same forward and backward, but the forward and backward sequences are found on complementary DNA strands (i.e., in double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG). Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes. The cleavage position within the palindromic sequence can vary by enzyme and can produce either single-stranded overhanging sequences (sticky ends) or blunt-ended DNA products. Figure\(\PageIndex{1}\) below shows examples of staggered and blunt-end cuts by restriction enzymes.

 

Figure \(\PageIndex{1}\): Staggered and blunt end cut sequences by EcoR1 and Sma1

The host can use methylation to protect its own genome from cleavage. For example, the methylation of the EcoRI recognition sequence by the M.EcoRI methyltransferase (MTase) changes the sequence from GAATTC to GAm6ATTC (m6A = N6-methyladenine). This modification completely protects the sequence from EcoRI cleavage.

Type II Restriction Enzymes initially bind non-specifically to the DNA and proceed to slide down the DNA, scanning for recognition sequences as shown in Figure (\PageIndex{2}\):. Upon binding the correct palindromic sequence, the enzyme associates with the metal cofactor and mediates catalytic cleavage of the DNA via strain distortion and catalysis by approximation.

Figure (\PageIndex{2}\): Schematic illustration of the steps involved in DNA recognition and cleavage by REases . Pingoud et al., Nucleic Acids Research, Volume 42, Issue 12, 8 July 2014, Pages 7489–7527, https://doi.org/10.1093/nar/gku447.   Creative Commons CC BY license

Figure (\PageIndex{3}\) below shows a space-filling model of EcoRV DNA recognition and cleavage.  The protein loosely binds DNA and scans for its cognate sequence. Once it finds the sequence, EcoRV kinks the DNA at a 50° angle and cleaves at the cognate sequence.

Figure (\PageIndex{3}\) below shows a space-filling model of EcoRV DNA recognition and cleavage.  Thomas Splettstoesser.  Wikimedia Commons.  https://commons.wikimedia.org/wiki/F...eaving_DNA.png

One of the most important questions about a hydrolase's catalytic mechanism is whether hydrolysis involves a covalent intermediate, as is typical for the proteases described previously. This can be decided by analyzing the stereochemical course of the reaction. This was done first for EcoRI and later for EcoRV. Both enzymes were found to cleave the phosphodiester bond with inversion of the chiral center at the phosphorus, which argues against the formation of a covalent enzyme–DNA intermediate. Thus, it is proposed that cleavage involves the direct nucleophilic attack of the substrate by a water molecule, as shown in Figure (\PageIndex{4}\) below.

Figure (\PageIndex{4}\): A General Mechanism for DNA Cleavage by EcoRI and EcoRV. An activated water molecule attacks the phosphorus in the phosphodiester bond to be cleaved, proceeding with inversion of configuration. X, Y, and Z are a general base, a Lewis acid, and a general acid, respectively. Figure adapted from: Pingoud, A., Wilson, G.G., and Wende, W. (2014) Nuc Acids Res 42(12):7489-7527.

Type II restriction enzymes typically form a homodimer when binding to DNA, as shown in the crystal structure of BglII in Figure 7.26B. BglII catalyzes phosphodiester bond cleavage at the DNA backbone through a phosphoryl transfer to water. Studies of restriction-enzyme mechanisms have revealed several general features that seem true in almost all cases. However, each enzyme likely uses some variation of this general mechanism (Figure 7.25). This mechanism requires a base to generate hydroxide from water, which acts as the nucleophile and attacks the phosphorus in the phosphodiester bond. Also required is a Lewis acid to stabilize the extra negative charge of the pentacoordinate transition-state phosphorus, as well as a general acid or metal ion that stabilizes the leaving group (3’-O⁻). Some Type II Restriction Enzymes (such as EcoRV and BamHI) require two divalent metal cofactors. In contrast, other enzymes require only one divalent metal cofactor (such as EcoRI and BglII).

Structural studies of endonucleases have revealed a similar active-site architecture, with residues following the weak consensus sequence Glu/Asp-(X)_9-20-Glu/Asp/Ser-X-Lys/Glu. BglII's active site is similar to other endonucleases, following the Asp-(X)₉-Glu-X-Gln sequence. In its active site, a divalent metal cation, most likely Mg²⁺, interacts with Asp-84, Val-94, a phosphoryl oxygen, and three water molecules. One of these water molecules can act as a nucleophile because of its proximity to the scissile phosphoryl group. The nucleophilic water molecule is positioned to attack the phosphoryl group via a hydrogen bond with the side-chain amide oxygen of Gln-95 and contact with the metal cation. Interaction with the metal cation effectively lowers its pK_a, promoting the water's nucleophilicity as shown in Panel A of Figure (\PageIndex{5}\) below (from Pingoud, A., Wilson, G.G., and Wende, W. (2014) Nuc Acids Res 42(12):7489-7527). During hydrolysis, the divalent cation can stabilize the 3'-O^- leaving group and coordinate proton abstraction from one of the coordinated water molecules

Figure (\PageIndex{5}\):  BglII catalyzes phosphodiester bond cleavage at the DNA backbone through a phosphoryl transfer to water.  Gwilliams10 .  Wikipedia.  https://en.wikipedia.org/wiki/BglII

Figure (\PageIndex{6}\) shows the coordination of the Mg²⁺ cofactor within the active site of the BglII enzyme. Figures from G Williams

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Figure (\PageIndex{6}\):  BglII active site containing residues that coordinate to a metal ion and water molecules including the nucleophilic water that breaks the scissile phosphodiester bond at the recognition site..  By Gwilliams10 - Own work, Public Domain, https://commons.wikimedia.org/w/inde...?curid=5459257

CRISPR-Cas 9

The CRISPR (clustered regularly interspaced short palindromic repeats) operon was initially discovered as part of the adaptive immune system of bacteria and archaea, which must defend themselves against viruses (bacteriophages) and unwanted plasmids transferred from other bacteria. It would be ideal for bacteria to recognize previous exposure to viruses and their nucleic acids as the basis of their immunological memory system. Given that viral DNA tends to integrate into the host genome (allowing later transcription and translation of viral genes during new virus production), immunological memory could be based on that integrated viral DNA. Without going into detail, viral DNA can be integrated between two direct repeats in the bacterial genome. DNA from different viruses from previous exposures is also incorporated in the same fashion. One integration site is the CRISPR operon. The DNA of the CRISPR operon contains both protein-coding and noncoding regions, which are transcribed and processed to form at least three RNA molecules, as shown in Figure (\PageIndex{7}\) below.

• a coding Cas 9 mRNA; this is translated to produce the Cas 9 (CRISPR-associated protein);
• a noncoding cr-RNA (CRISPR RNA)
• a noncoding tracr-RNA (trans-activating CRISPR RNA)

Figure (\PageIndex{7}\): DNA of the CRISPR operon

The two mature noncoding RNAs eventually associate to form a binary complex. In eukaryotic CRISPR-Cas9 gene-editing applications, the two noncoding RNAs are covalently combined into a single large synthetic guide RNA (sgRNA), described later in this section. The Cas9 protein is an endonuclease that cleaves both strands of bound target dsDNA in a blunt-end fashion at specific sequences. This occurs after the DNA binds to two arginines (1333 and 1335) in Cas9 through a short (3-5+ bases) recognition protospacer adjacent motif (PAM) located three base pairs from the cleavage site. The DNA must also bind in a complementary and specific fashion to the protein-bound noncoding crRNA and tracrRNA (or a single sgRNA molecule for gene-editing applications). Binding and cleavage of target DNA render the invading bacteriophage inactive.

Basic research into the bacterial CRISPR system has led to revolutionary, explosive eukaryotic applications of this gene-editing system. The hope is that CRISPR technology will give us a precise and incredibly cheap way to do gene therapy in diseased cells and organisms. Given its role in transforming our ability to edit the genome and potentially cure genetically based diseases, we will explain its mechanism.

We have discussed the structure and function of many proteins. Protein enzymes are key to life as they catalyze almost all biological reactions. Most key enzymes are regulated. Cas9 activity must be carefully controlled. Think of the consequences if the enzyme cleaves promiscuously at off-site targets! This section will help you understand several critical features of this enzyme:

1. How does the enzyme find its correct target site, a 20-nucleotide DNA sequence, and a proximal PAM site, among all the possible alternative sites? Think of how many PAM sequences must be in the host DNA genome!
2. How can the enzyme be "turned" on when it finds its target site and remains off when free, but more importantly, when bound off-site?

First, we will discuss the apo-form of the enzyme without bound substrate and RNA.

Apo- and Holo-Cas9

This section will focus on the Type II-A Cas9 from Streptococcus pyogenes (SpyCas9 or SpCas9). Cas9 is an endonuclease that cleaves both strands of DNA 3 base pairs from a DNA motif, NCC/NGG, called PAM. It has two distinct lobes. The nuclease lobe (NUC), amino acids 1-56 and 718-1368, has two different nuclease domains for the two cleavages. The recognition, or receptor, lobe (REC), amino acids 94-717, interacts with RNA molecules. It also has an arginine-rich bridge helix (57-93).

The enzyme has two catalytic nuclease domains:

• HNH-like nuclease domain cleaves the "target" DNA strand, which is complementary to the RNA that confers specificity to the enzyme. The key catalytic residues are His840 and Asn854. It also contains a Mg ion;
• The Ruv-like domain cleaves the complementary "non-target" strand, with key active-site residues Asp 10, Glu 762, Asp 986, and His 983. It also contains a bound Mn ion. The two lobes are separated by two linkers, amino acids 712-717, and an arginine-rich bridge (basic helix - BH), amino acids 628-658.

The overall structure of the apoenzyme (without bound RNA and DNA, pdb id 4cmp) is shown in Figure (\PageIndex{8}\) below, which shows the NUC domain (light blue) with the two catalytic domains (HNH and Ruv), the REC domain (orange), and the BH helix (red).

Figure (\PageIndex{8}\): Apoenyzme Cas9 (without bound RNA and DNA (4cmp)

A close-up view showing the two catalytic sites is shown in Figure (\PageIndex{9}\) below.

Figure (\PageIndex{9}\): Two catalytic sites in Cas9

Figure \(\PageIndex{10}\) shows an interactive iCn3D model of Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA (4OO8) (long load time). The Cas9 enzyme is shown as a gray transparent surface with an underlying cartoon rendering. The DNA is shown as colored sticks. The RNA is shown as a cyan cartoon.

Figure \(\PageIndex{10}\): Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA (4OO8). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...RjzJBFVt5qRjS7 (long load time)

A comparison of the crystal structure of the apo-Cas9 and the ternary Cas9: sgRNA:DNA target strand complex shows a significant conformational change on binding nucleic acids. The structure of the holoenzyme (ternary complex) is shown in Figure (\PageIndex{11}\) below.

Figure (\PageIndex{11}\): Structure of the holoenzyme Cas9 (ternary complex)with bound guide RNA and DNA

The extent of the conformation change between apo- and holo-Cas9 enzymes can be seen by examining the distance between D435 and E 944/945 in Figure (\PageIndex{12}\) below. We describe the importance of this change later.

 

Figure (\PageIndex{12}\): Distance between D435 and E 944/945 going from the apo-Cas9 (left) to the holo-Cas9 (right) enzyme

Figure (\PageIndex{13}\) below shows the pathway from the transcription of the relevant CRISPR genes (coding and noncoding) to the assembly of the ternary complex and the blunt-end cut of the target DNA strand three nucleotides from the PAM sequence.

Figure (\PageIndex{13}\): Pathway from the transcription of the relevant CRISPR genes (coding and noncoding) to the assembly of the ternary complex and the blunt-end cut of the target DNA strand three nucleotides from the PAM sequence.

Figure (\PageIndex{14}\) below shows an expanded view of the ternary complex.

Figure (\PageIndex{14}\): Expanded view of the ternary complex of Cas9 with guide RNA and DNA

 

Mechanism of DNA binding and cleavage

The above figures do not speak to the mechanism of the binding processes that form the ternary complex. Kinetic and structural studies have been conducted to elucidate the mechanism of binding and cleavage and address the following questions:

• Which binds first, the RNA or DNA?
• What are the consequences of the profound conformational changes on the formation of the ternary complex?

The specificity of target DNA binding depends both on enzyme:PAM DNA and enzyme:sgRNA (or tracr- and crRNA) interactions. It should seem improbable that the trinucleotide PAM DNA sequence (NGG in S. pyogenes), which interacts with a pair of arginines (R 1333, R 1335) through H-bonding, as shown in the images above, and other local sites in Cas 9 would provide the sole or even the majority of the binding interactions. Figure (\PageIndex{15}\) below shows the Args:PAM interaction (pdb code 4un3)

Figure (\PageIndex{15}\): Args:PAM interaction in holo-Cas9 (4un3)

Hence, RNA most likely binds first. Indeed, it does, with the tracrRNA implicated in the recruitment of Cas9 and the crRNA providing specificity for target DNA binding. The resulting Cas9:RNA binary complex could then search the relevant DNA genome. This could include bacteriophage DNA during viral infection or eukaryotic DNA if the CRISPR DNA operon with genes for Cas9 and an sgRNA was transfected into a eukaryotic cell. After RNA binding, the enzyme changes conformation and allows loose DNA binding through Cas 9: PAM interactions.

Studies have shown that the apo form can also bind DNA, but only loosely and indiscriminately. It dissociates quickly, and binding is affected by generic polyanions such as the glycosaminoglycan heparin, which indicates its nonspecific nature. Once bound, the complex surveys both off-target and target DNA. If a target DNA contained a PAM sequence, the complex would undergo another conformational change to position the HNH and Ruv nuclease catalytic residues and locally unwind the duplex DNA to make the blunt-end cuts.

Cas 9 binding to the PAM site would promote better interaction of the unwound DNA and the bound RNA. If no PAM were present, no catalytically effective Cas 9:target DNA would form. This prevents off-site cleavage. These allosteric changes and controls are vital to endonuclease function. Here are some findings that support this proposed mechanism:

• The conformation of apo Cas9 is catalytically inactive;
• On binding RNA to form a binary complex, Cas9 undergoes a dramatic conformational change, mostly in the REC lobe. However, when it binds DNA nonspecifically, the conformational changes are much smaller. This suggests that most conformational changes occur before DNA binding. In a way, RNA acts as an allosteric activator of the enzyme (and is the major source of binding specificity for target DNA). Conformational changes can be determined directly by comparing crystal structures or by spectral techniques such as fluorescence resonance energy transfer (FRET) between two attached fluorophores.
• Cas9: RNA interactions lead to ordering of the region of the RNA that interacts with the DNA PAM sequence and adjacent deoxynucleotides (a "seed sequence"), allowing the Cas9:RNA complex to scan and interact with potential DNA targets with PAM sequences;
• Once a PAM site is found, conformational changes lead to unwinding of the dsDNA, which allows heteroduplex formation between the crRNA and the target DNA strand;
• Since Cas 9 recognizes a variety of DNA target sequences (but of course, only a specific PAM sequence), the binding of the target sequence depends on the geometry, not the sequence, of the target DNA;
• Since binding of off-target DNA to the Cas9:RNA complex occurs but with very infrequent cleavage, binding and cleavage are very distinct steps;
• On specific DNA binding, the HNH catalytic site moves near the sessile DNA bond site. Crystal structures show that the active-site His is not close enough to facilitate cleavage, suggesting that binding a second metal ion (see below) may be necessary. Molecular dynamics studies show that the HNH domain is "remarkably plastic."

Figure (\PageIndex{16}\) below shows an animation that illustrates the relative conformational changes going from the apo Cas9 to the binary Cas9:sgRNA complex to the ternary Cas9: sgRNA: target DNA complex. The NUC catalytic domain is shown in light blue, the REC (receptor or RNA-binding domain) in orange, sgRNA in red, and the target DNA in green. Note again that upon binding RNA to form a binary complex, Cas9 undergoes a dramatic conformational change, mostly in the REC lobe. The pdb protein sequences shown were aligned using PDB Efold.

Figure (\PageIndex{16}\): Animation illustrating conformational changes going from the apo Cas9 to the binary Cas 9:sgRNA complex to the ternary Cas 9: sgRNA: target DNA complex

A potential abbreviated catalytic mechanism for the Ruv nuclease domain is shown in Figure (\PageIndex{17}\) below. The red arrows indicate the second set of electron movements. His 983 acts as a general base to abstract a proton from the water, making it a more potent nucleophile. An intermediate trigonal bipyramidal phospho-intermediate is formed, and the preceding transition state is stabilized by the proximal Mg²⁺ ion (an example of electrostatic or metal ion catalysis). Magnesium is positioned through its interaction with the negatively charged carboxyl groups of Asp 10, Glu 762, and Asp 986.

Figure (\PageIndex{17}\): Abbreviated catalytic mechanism for the Ruv nuclease domain of Cas9

A second metal ion might be recruited to the Ruv site to facilitate further DNA cleavage. The HNH catalytic site has a structure (beta-beta-alpha) and a conserved His in common with a class of nucleases that require one metal ion. In contrast, the Ruv catalytic site lacks this common secondary structural motif. It has a critical histidine, a common feature in endonucleases that use two metal ions.

CRISPR and Eukaryotic Gene Editing

How could blunt-end cutting of both DNA strands by Cas9 lead to the holy grail of specific eukaryotic gene editing with no off-site effects? Cutting the DNA genome seems like a bad idea. It is potentially so bad that many DNA repair mechanisms have evolved to fix the cut. These include homologous recombination. If corrective DNA is supplied, along with the components of the CRISPR system, a cell could effectively add the corrective DNA after the double-stranded cut and repair a deleterious mutation. Consult a molecular biology textbook for more insight into homologous recombination.

Mutations in the PAM sequence prevent Cas9 nuclease activity. Hence, the NGG PAM sequence is vital for the above interactions and activities. This would seem to limit the utility of CRISPR-Cas9 in eukaryotic gene editing until one realizes that the GG dinucleotide occurs at a 5.2% frequency in the human genome, corresponding to over 160 million occurrences. Even then, it might not occur in a desired gene target. Cas9 nucleases from other bacteria extend the range of activity of the CRISPR/Cas system as they interact with other PAM sequences (NNAGAA and NGGNG for S. thermophilus and NGGNG for N. meningitidis). Likewise, mutations in the S. pyogenes PAM (NGG) have been made. A D1135E mutation retains but increases the specificity for the normal NGG PAM site. D1135V, R1335Q, and T1337R mutations alter the optimal PAM recognition site to NGAN or NGNG.

CRISPR editing can be easily used to knock out specific genes. In addition, if cells are transfected with a plasmid with many target sequences, the system can edit multiple genes in one experiment. This would be very useful in studies of diseases linked to multiple genes. Since the cost of CRISPR reagents (plasmids, RNAs) is so inexpensive, and the specificity of editing is so high, the great excitement about CRISPR use for gene editing in human disease and for modification of plant and fungal genomes is warranted.

Other systems have been developed to bind to and cleave a target DNA sequence. They typically contain a protein that binds to a specific DNA target and an associated endonuclease that cleaves within the target DNA site. Typical prokaryotic restriction enzymes bind to and cut at a specific nucleotide sequence (for example, EcoR1 cleaves at G/AATTC palindromic sequences) to form sticky ends. The protein itself binds to this DNA recognition site. Other examples are based on the structure of known transcription factors. Libraries of genetically engineered proteins with Zn finger DNA-binding domains (designed for specific DNA target sequences) fused to endonucleases have been created. Other examples are proteins called TALENs (transcription activator-like effector nucleases). These are fusion proteins containing a TAL effector DNA-binding domain and a nuclease. In each case, a 3D-folded protein serves as the specific target-DNA recognition molecule. Think how much easier it is to make a 1D-DNA recognition element, a simple linear RNA sequence, which would adopt the correct 3D structure on binding its complementary target.

One major problem in using CRISPR for gene editing remains: how to get the CRISPR components into the right cells in an organism. In effect, it's the same problem faced by small drug designers, only the components are much larger. Ex vivo applications, in which diseased cells are removed from the body, repaired by CRISPR, and then reinjected, are likely to be more successful. In these cases, electroporation would allow the uptake of Cas9 and the sgRNA. In vivo therapy has included adeno-associated viruses that encapsulate Cas9 and sgRNA genes. This technique, used for other gene delivery systems, can be tolerated immunologically. However, this system allows for continual gene expression, which is undesirable for gene editing. After an initial "fix" of a mutant gene, continued expression of the CRISPR-Cas9 genes would increase the chances of off-target cutting. A more recent approach is to deliver the mRNA in artificial lipid nanoparticles that can be taken into cells. Once inside the cell, the mRNA is translated into protein and sgRNA, and gene editing can occur before the RNA and protein are degraded.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

Restriction endonucleases are bacterial enzymes that protect prokaryotic cells from foreign DNA by cleaving it at specific palindromic recognition sequences. Type II restriction enzymes, the most widely used in molecular biology, require Mg²⁺ as a cofactor and cleave duplex DNA at fixed positions within or near their recognition sequences, producing either sticky (staggered) or blunt ends. The host protects its own DNA from cleavage through methylation of the same recognition sequences by accompanying methyltransferases. Type II enzymes scan DNA nonspecifically until they encounter their cognate palindromic sequence, at which point they induce a conformational change — exemplified by the ~50° kinking of DNA by EcoRV — and carry out hydrolysis. Stereochemical analysis showing inversion of configuration at the phosphorus center establishes that cleavage proceeds through direct nucleophilic attack by an activated water molecule, without a covalent enzyme-DNA intermediate. The Mg²⁺ ion plays multiple catalytic roles: activating the nucleophilic water by lowering its pKa, stabilizing the pentacoordinate transition state, and facilitating departure of the 3'-O⁻ leaving group.

The CRISPR-Cas9 system represents a prokaryotic adaptive immune mechanism in which sequence records of past viral infections, stored as spacer sequences within the CRISPR operon, are transcribed into crRNA and tracrRNA molecules that guide the Cas9 endonuclease to complementary foreign DNA sequences for destruction. Cas9 contains two distinct lobes — a nuclease lobe (NUC) harboring the HNH and RuvC catalytic domains, and a recognition lobe (REC) that interacts with the guide RNA — connected by an arginine-rich bridge helix. The enzyme is catalytically inactive in its apo form. Binding of the sgRNA to the REC lobe induces a dramatic conformational change that primes the enzyme to scan DNA for a short protospacer adjacent motif (PAM) sequence. PAM recognition by two arginine residues triggers further conformational changes, local DNA unwinding, and heteroduplex formation between the crRNA and the target strand, ultimately positioning the HNH and RuvC catalytic residues to make a blunt-end double-strand cut three base pairs from the PAM site. This multi-step, allosterically controlled activation mechanism ensures that cleavage is highly specific and that off-target cutting is minimized.

The ease with which a simple linear RNA guide sequence can be designed to direct Cas9 to virtually any genomic locus has made CRISPR-Cas9 transformative for eukaryotic gene editing, surpassing earlier platforms such as zinc finger nucleases and TALENs in simplicity, cost, and programmability. The double-strand break introduced by Cas9 can be exploited to knock out genes or, when a corrective DNA template is supplied, to repair disease-causing mutations through homologous recombination. The enormous frequency of PAM sequences in the human genome, combined with the availability of Cas9 orthologs with alternative PAM preferences and engineered PAM variants with altered or enhanced specificity, greatly expands the range of targetable sequences. Ongoing challenges include efficient and safe delivery of CRISPR components into target cells in vivo, with lipid nanoparticle delivery of mRNA currently emerging as a preferred approach for therapeutic applications because it limits transient expression and reduces the risk of off-target cleavage.

7.4 References:

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