8.6: Enzymes for Genetic modifications

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Learning Goals (ChatGPT o3-mini, 2/1/25)

1. Describe the Mechanism of Restriction Endonucleases:

   • Explain how Type II restriction enzymes recognize specific palindromic sequences in double-stranded DNA and catalyze double-strand breaks, generating predictable fragment sizes.

2. Understand the Restriction-Modification System:

   • Illustrate how bacteria use restriction enzymes together with DNA-methyltransferases to differentiate between self and non-self DNA, thereby protecting their genomes from viral infection.

3. Analyze the Catalytic Mechanism of Type II Restriction Enzymes:

   • Describe the role of divalent metal ions (e.g., Mg²⁺) in activating water for nucleophilic attack on the phosphodiester bond and discuss the stereochemical evidence (inversion of configuration) that supports a direct hydrolytic mechanism.

4. Interpret Enzyme Specificity and Nomenclature:

   • Discuss how restriction enzymes are named based on their organism of origin and how isoschizomers and neoschizomers differ in their cleavage patterns and evolutionary origins.

5. Compare and Contrast Gene Editing Technologies:

   • Compare traditional restriction enzyme-based cloning methods with modern CRISPR-Cas9 gene editing, emphasizing their mechanisms, applications, and limitations.

6. Explain the Structure and Function of CRISPR-Cas9:

   • Describe the architecture of Cas9, including its two catalytic nuclease domains (HNH and RuvC) and the roles of the guide RNA (crRNA and tracrRNA, or the synthetic sgRNA) in conferring target specificity.

7. Understand the Allosteric Regulation of Cas9:

   • Analyze how the binding of guide RNA triggers conformational changes in Cas9 that prime the enzyme for DNA binding and cleavage, and explain the role of the protospacer adjacent motif (PAM) in target recognition and cleavage specificity.

8. Evaluate the Applications and Challenges of CRISPR Gene Editing:

   • Assess how CRISPR-Cas9 is used in disease diagnosis, therapy, and genetic modifications, and discuss strategies for minimizing off-target effects and ensuring efficient delivery into target cells.

9. Integrate Concepts in Protein Structure and Enzyme Mechanism:

   • Relate the structural features of both restriction enzymes and Cas9 to their catalytic mechanisms, emphasizing how protein conformation, cofactor binding, and specific amino acid residues contribute to their function.

These goals are intended to guide your study and critical analysis of gene editing systems, helping you connect fundamental enzyme mechanisms with their revolutionary applications in biotechnology and medicine.

It is difficult to read newspapers and newsmagazines without encountering the CRISPR-Cas9 gene editing system that 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 chapter 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 one class of the broader endonuclease group of enzymes. 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, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks 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 case and we were able to demonstrate it directly by sequencing the termini of the cleavage fragments.’"

Figure (\PageIndex{35}\): Hamilton Smith and Daniel Nathans at the Nobel Prize press conference, 12 October 1978 (reproduced with permission from Susie Fitzhugh). Original Repository: Alan Mason Chesney Medical Archives, Daniel Nathans Collection. Image from: Pingoud, A., Wilson, G.G., and Wende, W. (2014) Nuc Acids Res 42(12):7489-7527.

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 genus of the organism and the second and third to the species. This is followed by letters and/or numbers identifying the isolate. Roman numerals are used to 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, on the other hand, 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, properties that have made 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⁺, on the other hand, often acts as an inhibitor of 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 strand of DNA, as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backward, but the forward and backward sequences are found in complementary DNA strands (i.e., of 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 position of cleavage within the palindromic sequence can vary depending on the enzyme and can produce either single-stranded overhanging sequences (sticky ends) or blunt-ended DNA products. Table \(\PageIndex{8}\) below shows examples of staggered and blunt end cuts by restriction enzymes.

EcoR1
Sma1

Table \(\PageIndex{8}\): 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 with the DNA and proceed to slide down the DNA scanning for recognition sequences as shown in Figure (\PageIndex{36}\):. Upon binding to the correct palindromic sequence, the enzyme associates with the metal cofactor and mediates catalytic cleavage of the DNA using the mechanism of strain distortion and catalysis by approximation.

Figure (\PageIndex{36}\): DNA Recognition and Cleavage by Type II Restriction Endonucleases. (A) Pictorial view of an EcoRV dimer scanning nonspecifically along the DNA until a specific binding site is recognized. This causes coupling with the metal cofactor and strain distortion of the DNA. Hydrolysis of the phosphodiester bond is mediated and the DNA cleavage products are released from the enzyme. (B) shows a space-filling model of EcoRV DNA recognition and cleavage. Figure (A) from Pingoud, A., Wilson, G.G., and Wende, W. (2014) Nuc Acids Res 42(12):7489-7527. and Figure (B) from Thomas Splettstoesser

One of the most important questions regarding the catalytic mechanism of a hydrolase 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{37}\) below.

Figure (\PageIndex{37}\): A General Mechanism for DNA Cleavage by EcoRI and EcoRV. An activated water molecule attacks the phosphorous in-line with the phosphodiester bond to be cleaved, which proceeds with an 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 with 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 on the mechanism of restriction enzymes have revealed several general features that seem true in almost all cases. However, the actual mechanism for each enzyme is most likely some variation of this general mechanism (Figure 7.25). This mechanism requires a base to generate the hydroxide ion from water, acting as the nucleophile and attacking 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⁻). Two divalent metal cofactors are required in some Type II Restriction Enzymes (such as in EcoRV and BamHI). In contrast, other enzymes only require one divalent metal cofactor (such as in EcoRI and BglII).

Structural studies of endonucleases have revealed a similar architecture for the active site with the 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 (Figure 7.26A). The nucleophilic water molecule is positioned for attack onto the phosphoryl group by a hydrogen bond with the side chain amide oxygen of Gln-95 and its 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{38}\) 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{38}\): Proposed Reaction Mechanism for the Type II Restriction Endonuclease, BglII. (A) Schematic diagram of the catalytic mechanism demonstrating the utility of Mg2+ ions and polar amino acid residues within the active site to activate and position a water molecule for nucleophilic attack on the phosphodiester bond of the DNA substrate. (B) Crystal structure of the BglII dimer with double-stranded DNA and (C) Coordination of the Mg2+ cofactor within the active site of the BglII enzyme. Figures from G Williams

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 both 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 the tendency of viral DNA to integrate into the host genome (which allows later transcription and translations of the viral genes in the process of new virus production), immunological memory could be based on that viral integrated 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 site of integration 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{24}\) 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{24}\): DNA of the CRISPR operon

The two mature noncoding RNAs eventually associate to form a binary complex. When using CRISPR-Cas 9 in eukaryotic gene editing applications, the two noncoding RNAs are covalently combined into one large synthetic guide RNA (sg-RNA), described later in this section. The Cas 9 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 cr+tracr-RNAs (or a single sg-RNA molecule for gene editing applications). Binding and cleavage of target DNA would render DNA from an invading bacteriophage inactive.

Basic research into the bacterial CRISPR system has led to revolutionary and 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. The activity of Cas 9 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-Cas 9

This section will focus on the Type II-A Cas9 from Streptococcus pyogenes (SpyCas9 or SpCas9). Cas 9 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 the RNA molecules. There is also 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 His 840 and Asn 854. It also contains a Mg ion;
• 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{25}\) 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{25}\): Apoenyzme Cas9 (without bound RNA and DNA (4cmp)

A close up view showing the two catalytic sites is shown in Figure (\PageIndex{26}\) below.

Figure (\PageIndex{26}\): Two catalytic sites in Cas9

Figure \(\PageIndex{27}\) 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 \(\PageIndex{27}\): 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-Cas 9 and the ternary Cas 9: 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{28}\) below.

Figure (\PageIndex{28}\): Structure of the holoenzyme Cas9 (ternary complex)with bound guide RNA and DNA

The extent of the conformation change between apo- and holo-Cas 9 enzymes can be seen by examining the distance between D435 and E 944/945 in Figure (\PageIndex{29}\) below. The importance of this change will be described later.

Figure (\PageIndex{29}\): Distance between D435 and E 944/945 going from the apo-Cas9 (left) to the holo-Cas 9 (right) enzyme

Figure (\PageIndex{30}\) 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{30}\): 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{31}\) below shows an expanded view of the ternary complex.

Figure (\PageIndex{31}\): 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{32}\) below shows the Args:PAM interaction (pdb code 4un3)

Figure (\PageIndex{32}\): Args:PAM interaction in holo-Cas9 (4un3)

Hence it is most likely that RNA binds first. Indeed, it does with the tracRNA implicated in the recruitment of Cas and the crRNA providing specificity for target DNA binding. The resulting Cas9:RNA binary complex could then search the relevant DNA genome. That would include the DNA of the bacteriophage in viral infection or eukaryotic DNA if the CRISPR DNA operon with the genes for Cas 9 and a sg-RNA was transfected into the 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 it does so 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, both off-target and target DNAs would then be surveyed. 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 was present, no catalytically effective Cas 9:target DNA would form. This prevents off-site cleavage. These allosteric changes and controls are vital to the function of the endonuclease. Here are some findings that support this proposed mechanism:

• the conformation of apo Cas 9 is catalytically inactive;
• on binding RNA to form a binary complex, Cas 9 undergoes a dramatic conformational change, mostly in the REC lobe. However, on binding DNA in a nonspecific fashion, the conformational changes are much smaller. This suggests that most changes in conformation occur before DNA binding. In a way, RNA acts as an allosteric activator of the enzyme (as well as the major source of binding specificity to target DNA). Conformational changes can be determined directly by comparison of crystal structures or spectral techniques such as fluorescence resonance energy transfer (FRET) between two different attached fluorophores.
• Cas 9: 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 Cas 9: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 Cas 9: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 sufficiently close 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{33}\) below shows an animation that illustrates the relative conformational changes going from the apo Cas 9 to the binary Cas 9:sgRNA complex to the ternary Cas 9: 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 on binding RNA to form a binary complex, Cas 9 undergoes a dramatic conformational change, mostly in the REC lobe. The pdb protein sequences shown were aligned using pdbEfold.

Figure (\PageIndex{33}\): Animation illustrating conformational changes going from the apo Cas 9 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{34}\) 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). The magnesium is positioned through its interaction with negatively charged carboxyl groups of Asp 10, Glu 762, and Asp 986.

Figure (\PageIndex{33}\): Abbreviated catalytic mechanism for the Ruv nuclease domain of Cas9

A second metal ion might be recruited to the Ruv site to further facilitate the cleavage of the DNA. The HNH catalytic site has a structure (beta-beta-alpha) and conserved His in common with a class of nucleases that require one metal ion. In contrast, the Ruv catalytic site does not have this common secondary structural motif. It has a critical histidine, which are both common features found in endonucleases that use two metal ions.

CRISPR and Eukaryotic Gene Editing

How could blunt-end cutting of both DNA strands by Cas 9 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, as well as 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-Cas 9 in eukaryotic gene editing until one realizes that the GG dinucleotide has a 5.2% frequency of occurrence in the human genome, corresponding to over 160 million occurrences. Even then, it might not occur in a desired gene target. Cas 9 nuclease from other bacteria extends 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. meningidtis). Likewise, mutations in the S. pyogenes PAM (NGG) have been made as well. 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, Eco R1 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 is 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 must be solved: how to get the CRISPR components in the correct 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, when diseased cells are removed from the body, repaired by CRISPR, and then reinjected, are likely to have more success. In these cases, electroporation would allow the uptake of Cas 9 and the sg-RNA. In vivo therapy has included adeno-associated viruses in which Cas 9 and sg RNA genes could be encapsulated. 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-Cas 9 genes would increase the chances for off-target cutting. A more recent approach is to deliver the mRNA in artificial lipid nanoparticles that can be taken into cells. Once free and translated into protein and sg RNA inside the cell, gene editing can occur before the RNA and protein are degraded.

Summary

Chapter Summary: Restriction Enzymes and CRISPR-Cas9 Gene Editing

This chapter explores two revolutionary tools that have transformed molecular biology and biotechnology: the classical restriction endonucleases and the modern CRISPR-Cas9 gene editing system.

It begins by examining the fundamental principles of restriction enzymes, particularly the widely used Type II enzymes. These enzymes recognize specific palindromic DNA sequences and cleave both strands at precise sites, generating reproducible fragment patterns essential for gene cloning and analysis. The chapter details the restriction-modification system in prokaryotes, where bacteria and archaea protect their own genomes via DNA methylation while using these enzymes to degrade foreign DNA. Emphasis is placed on the catalytic mechanism—where divalent metal ions, usually Mg²⁺, activate a water molecule to directly hydrolyze the phosphodiester bond with inversion of configuration—and on how enzyme nomenclature and classifications (including isoschizomers and neoschizomers) reflect evolutionary relationships and cleavage patterns.

The discussion then transitions to the CRISPR-Cas9 system, which has rapidly gained attention due to its unprecedented precision and versatility in gene editing. Originating as part of the adaptive immune system in bacteria, CRISPR-Cas9 utilizes a guide RNA (either as separate crRNA and tracrRNA or as a fused single guide RNA) to direct the Cas9 endonuclease to a target DNA sequence adjacent to a protospacer adjacent motif (PAM). Detailed structural insights reveal that Cas9 consists of distinct lobes—the nuclease (NUC) lobe housing the HNH and RuvC catalytic domains, and the recognition (REC) lobe responsible for RNA binding. Binding of the guide RNA triggers significant conformational changes that “activate” Cas9, enabling it to specifically bind and cleave target DNA with blunt-end cuts.

Furthermore, the chapter outlines the critical factors that govern Cas9 specificity and activity, including the allosteric changes induced upon PAM binding and the kinetic distinction between nonspecific DNA association and target recognition. It also addresses practical considerations in applying CRISPR-Cas9 for eukaryotic gene editing, such as delivery strategies (e.g., viral vectors, lipid nanoparticles) and the importance of minimizing off-target effects.

In summary, this chapter integrates the biochemical principles underlying restriction enzymes and CRISPR-Cas9, illustrating how understanding enzyme mechanisms, structure-function relationships, and regulatory features has not only advanced basic molecular biology but also paved the way for transformative applications in disease diagnosis, treatment, and genetic engineering.

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