What is the Cas9 enzyme and how do biohackers use it?

Understanding the Cas9 Enzyme in CRISPR Systems

The Cas9 enzyme is the life-blood of the CRISPR-Cas9 gene editing system that has changed how scientists manipulate genes. You need to understand its unique structure and origin to learn about its importance.

Cas9 vs other nucleases: What makes it unique?

The Cas9 nuclease works differently from traditional restriction enzymes that recognize specific DNA sequences. It provides unmatched flexibility through RNA guidance. This 160-kDa protein has two significant lobes – the recognition (REC) lobe binds guide RNA and the nuclease (NUC) lobe cuts DNA. The NUC lobe contains RuvC and HNH domains that cleave opposing DNA strands and create precise double-strand breaks.

Cas9’s programmability makes it special compared to other nucleases. Earlier gene editing technologies like zinc fingers and TALENs needed complex protein engineering to recognize new targets. Cas9 can be redirected by changing a 20-nucleotide sequence in its guide RNA. This feature makes Cas9 more flexible, adaptable, and easier to use.

Cas9 is different from other CRISPR-associated proteins in several key ways:

• Target recognition: Cas9 needs a specific protospacer adjacent motif (PAM) – typically NGG for the commonly used SpCas9 – downstream of its target site
• Cutting pattern: Cas9 generates blunt-ended DNA breaks, unlike Cas12a (formerly Cpf1) which creates staggered cuts
• RNA requirements: Cas9 forms a ribonuclease complex with just a single guide RNA, while some systems use complex multi-protein complexes

Scientists have engineered many Cas9 variants with expanded capabilities. These include variants with altered PAM specificities (NGAG, NGCG) and improved specificity that minimize off-target effects.

The role of Cas9 in bacterial immune defense

Cas9 naturally works as a vital component of bacterial adaptive immunity against viral invasion. Bacteria capture small fragments of viral DNA and integrate them into their genome between repetitive sequences called CRISPR arrays. These fragments work as a genetic memory of previous infections.

Bacteria transcribe these stored sequences into CRISPR RNAs (crRNAs) that guide Cas9 to matching viral DNA during later viral attacks. The process needs both crRNA and a trans-activating CRISPR RNA (tracrRNA) that form a guide RNA complex together. This RNA complex helps Cas9 scan DNA until it finds a matching PAM sequence.

Cas9 checks the adjacent DNA sequence for complementarity with its guide RNA after finding a PAM. The enzyme changes its shape upon finding a match. This activates its nuclease domains that cleave both strands of viral DNA and neutralize the threat.

Cas9’s role goes beyond simple defense in bacterial pathogenicity. To cite an instance, see Francisella novicida, where Cas9 works with small RNAs to repress bacterial lipoprotein expression. This helps the pathogen avoid immune detection by preventing Toll-like receptor 2 activation. Cas9 also downregulates the CovR/S system that controls virulence factors in Streptococcus pyogenes.

Scientists have used their knowledge of these natural functions to adapt Cas9 for precise genetic modifications that are transforming biotechnology and medicine.

How Cas9 Works in CRISPR Gene Editing?

The Cas9 enzyme’s molecular machinery works through a precise sequence of events that makes shared genetic modifications possible. Scientists who want to use CRISPR-Cas9 gene editing technology need to know how this mechanism works.

Guide RNA and PAM sequence recognition

The Cas9 enzyme cuts DNA only after target recognition. This process needs two key components: a guide RNA and a protospacer adjacent motif (PAM). The commonly used Streptococcus pyogenes Cas9 (SpCas9) needs a PAM sequence 5′-NGG-3′, where N can be any nucleotide.

The targeting process follows a specific order:

1. Cas9 first scans DNA to find the PAM sequence, which is a vital gatekeeper
2. After finding the PAM, Cas9 starts separating DNA strands
3. The “seed sequence” (10 bases next to the PAM) pairs up with the target DNA
4. The guide RNA pairs completely with the target strand to form the R-loop
5. This shape change turns on the nuclease domains that cut DNA

Note that Cas9 won’t check if the guide RNA matches the target DNA without a proper PAM—whatever the match quality. Bacteria use this requirement to protect themselves from cutting their own CRISPR arrays.

Double-strand breaks and DNA repair pathways

Cas9 uses two different nuclease domains to create a double-strand break (DSB). The HNH domain cuts the target strand that matches the guide RNA. The RuvC domain cuts the non-target strand. Studies show Cas9 mostly creates blunt-ended cuts (61.57% of cases) and sometimes makes staggered cuts with 5′ ssDNA overhangs (35.04% of cases).

The cell starts repair mechanisms after Cas9 creates a DSB. These mechanisms decide the final editing result. Four main repair pathways compete to fix the break:

Non-Homologous End Joining (NHEJ): This pathway joins broken ends directly with minimal processing. It often adds or removes small pieces of DNA (indels). NHEJ usually results in gene knockouts.

Homology-Directed Repair (HDR): This high-accuracy pathway uses a template like donor DNA to guide precise repair. HDR makes specific genetic changes but happens less often than NHEJ.

Microhomology-Mediated End Joining (MMEJ): This pathway uses short matching sequences (5-25 base pairs) near the break to guide repair. It creates predictable deletions.

Single-Strand Annealing (SSA): This method uses larger matching regions and can remove big stretches of DNA between repeats.

The cell cycle stage, DNA end structure, and local chromatin environment determine which pathway gets used. In spite of that, NHEJ usually wins out, which makes precise editing harder.

Cas9 nuclease vs restriction enzyme EcoRI

Cas9 and restriction enzymes like EcoRI both cut DNA, but they work quite differently:

EcoRI recognizes and cuts at one specific sequence. Cas9’s ability to be programmed through guide RNA design makes it nowhere near as limited for genome editing. This key difference explains why CRISPR-Cas9 has become scientists’ favorite tool for precise genetic changes.

Applications of Cas9 in Medicine and Research

The Cas9 enzyme’s remarkable precision has sparked a medical revolution that turns theoretical gene editing into real treatments for conditions once thought incurable. CRISPR-Cas9 now helps solve some of humanity’s biggest challenges in medicine, agriculture, and environmental sciences.

CRISPR-Cas9 in treating genetic diseases

CASGEVY, the world’s first CRISPR therapy achieved a historic milestone with regulatory approval. This groundbreaking treatment targets sickle cell disease and beta-thalassemia by activating fetal hemoglobin production through the BCL11A gene. Patients now have access to a one-time treatment that could potentially cure their condition.

CRISPR therapies show promise in treating several other genetic diseases:

• Cystic fibrosis: Patient cells had their CFTR mutations fixed without damage to other parts of their genetic code. Vertex Pharmaceuticals now develops a CRISPR-based treatment for this condition.

• Duchenne muscular dystrophy: Scientists created a smart approach targeting 12 mutation hotspots that covers most of the roughly 3,000 different mutations behind this disease.

• Huntington’s disease: University of Illinois Urbana-Champaign researchers used CRISPR/Cas13 to target and cut mRNA that codes for mutant proteins. This silences problematic genes without permanent DNA changes.

Base editing and prime editing advancements

New technologies beyond traditional CRISPR-Cas9 offer even more precise genetic modifications:

Base editing lets scientists directly convert one DNA base to another without creating double-strand breaks. Scientists developed this technique in 2016 by combining CRISPR/dCas9 with rat-derived cytidine deaminase to convert C to U base. Their work continued to replace A-T base pairs with C-G base pairs.

Prime editing, which emerged in 2019, marks another breakthrough. The system works with:

1. A reverse transcriptase fused with Cas9n (H840A)
2. A pegRNA containing both a primer binding site and reverse transcription template

Scientists can now make all 12 possible base substitutions plus precise insertions and deletions. Prime editing cuts just one DNA strand and uses pegRNA as a template for the desired edit. This method could help fix about 90% of known disease-causing mutations.

Cas9 in agricultural and environmental biotech

CRISPR-Cas9 agricultural applications focus on:

• Crop improvement: The technology makes precise genetic changes to boost yield, quality, and stress tolerance. Scientists have created herbicide-resistant rice using dual base editors.

• Disease resistance: CRISPR-Cas9 has created virus-resistant cucumbers and fungus-resistant rice.

• Accelerated domestication: Scientists succeeded in domesticating wild tomatoes from scratch by editing specific domestication-related loci.

Environmental applications of CRISPR-Cas9 include:

• Biofuel development: The technology helps make biofuel production more efficient, which could reduce our dependence on fossil fuels.

• Phytoremediation: Scientists modify plants to boost their absorption and accumulation of environmental pollutants, especially in areas with heavy metal contamination.

How Biohackers Use Cas9 Outside the Lab

The Cas9 enzyme has moved beyond laboratory walls and into the hands of citizen scientists and biohackers who conduct genetic experiments outside traditional institutions. This spread of CRISPR technology opens up exciting possibilities but also brings serious concerns.

DIY CRISPR kits and availability

You can now buy DIY CRISPR kits online for as little as $159. Amateur experimenters can modify genes right at home. The Odin, a company started by biohacker and former NASA scientist Josiah Zayner, sells kits with all the parts needed for simple gene editing experiments. A typical bacterial engineering kit has:

• Cas9 plasmid, guide RNA, and template DNA
• Non-pathogenic E. coli bacteria
• Growth media and antibiotics
• Simple lab equipment (pipettes, tubes, petri plates)

These kits let users modify bacteria and yeast, though the technology could work more broadly. Advanced kits for human tissue culture experiments cost about $800, showing different levels of sophistication biohackers can access.

Notable biohacking experiments with Cas9

Some biohackers have gone beyond microorganisms to experiment on themselves. Josiah Zayner made headlines as the first person to attempt editing his own genes using CRISPR. He targeted his myostatin gene that controls muscle growth and injected CRISPR components into his forearm to increase muscle mass.

Among other biohackers, one tried DIY gene therapy for HIV, but his viral load went up instead. Both men showed their procedures live online, which drew attention to biohacking despite their failed experiments.

Risks of unsupervised gene editing

Unsupervised CRISPR experiments bring several risks. The FDA opposes DIY gene therapy kits, but regulation remains tough since people can legally buy components for “research purposes”. Self-administration makes monitoring or control nearly impossible.

Scientists worry about off-target effects damaging genes that control tumor growth. Zayner argues these risks exist in theory, but well-designed guide RNAs keep the probability low.

As biohacking becomes more common, experts fear dangerous applications might go underground if pushed from public view. This fear drives the ongoing debate about balancing state-of-the-art advances with proper safeguards for this powerful technology.

Ethical and Legal Considerations of Cas9 Use

CRISPR-Cas9 technology’s global expansion has created serious ethical challenges alongside its scientific breakthroughs. Scientists, regulators, and society must address the responsibilities that come with gene modification power, especially for changes that future generations will inherit.

Germline editing and global regulations

Global regulations on germline editing show dramatic variations. Approximately 40 countries ban or discourage germline editing research because of ethical and safety concerns. These include 15 Western European nations that have strict bans in place. Canada has made human germline editing a crime with fines up to 500,000 Canadian dollars and 10-year jail terms. China took a different path with less oversight, which led to controversial experiments.

The scientific community took action against premature clinical use through international efforts. Chinese researcher He Jiankui’s 2018 announcement about editing embryos that resulted in twin girls with modified CCR5 genes sparked immediate response. Many scientists backed a pause on clinical uses of human germline editing that weren’t ready.

Off-target effects and safety concerns

The biggest problem with Cas9 enzyme use is its risk of unwanted genetic changes. Off-target effects happen when Cas9 modifies unintended genomic sites. These unexpected cuts could create adverse outcomes. Research shows Cas9 can handle up to three mismatches between guide RNA and genomic DNA. Studies indicate these off-target mutations affect exon regions more than predicted, which could cause widespread genomic damage.

Scientists use several methods to find these unwanted edits:

• Digenome-seq: A highly sensitive technique that finds indels at just 0.1% frequency
• BLESS and BLISS: Technologies that capture DNA breaks right when samples are fixed
• Computational prediction tools: Software that predicts possible off-target sites

The debate over open-source gene editing

CRISPR-Cas9 technology’s open access creates opportunities and risks. Supporters say responsible access leads to breakthrough treatments. Restricting access might force experiments underground where nobody can monitor them.

Critics worry about “genetic upper and lower classes” forming if people use the technology for enhancement instead of treatment. Bioethicists say this concern grows as CRISPR becomes more accessible.

The challenge lies in finding the right balance between progress and safety. Robert Truog, director of the Center for Bioethics at Harvard Medical School, puts it clearly: “This conversation is not about the fundamental merits of germline gene editing, which in the long run will almost certainly be highly beneficial. Instead, it’s about the oversight of science”.

Conclusion

Cas9 enzyme stands as one of the most transformative breakthroughs in modern biotechnology. This piece explains how these molecular scissors work as the core component of CRISPR systems. They cut DNA at specific locations with RNA molecules as guides. Cas9’s programmability makes it far more versatile than traditional restriction enzymes, which has made genetic modification more available than before.

The potential of Cas9 technology to treat previously incurable genetic diseases is remarkable. CASGEVY’s FDA approval for sickle cell disease and beta-thalassemia marks a historic milestone. Research now targets conditions like cystic fibrosis and Duchenne muscular dystrophy. Base editing and prime editing techniques have refined genetic modification capabilities. These advances could address all but one of these known disease-causing mutations.

Serious ethical questions still need answers. DIY CRISPR kits make science more democratic but raise safety concerns about uncontrolled experiments. Researchers face challenges with off-target effects since unwanted genetic changes could lead to collateral damage. Different countries have varying regulations on germline editing. This reflects deep social divisions about changing heritable genetic traits permanently.

Cas9’s story represents both scientific achievement and ethical dilemma. This technology’s evolution requires society to balance new ideas with proper safeguards. Cas9’s future depends on scientific breakthroughs and agreement about responsible use among research, medical, agricultural, and biohacking communities.