Introduction to CRISPR knockout

Clustered regularly interspaced short palindromic repeats (CRISPR) is a genomic phenomenon that gave rise to the prominent CRISPR-Cas9 genomic editing system, which is commonly used in several drug discovery applications. It was initially discovered in bacteria and archaea, which incorporated DNA fragments of bacteriophages into their genome to advance their immune recognition capabilities. In subsequent attacks, bacteria use these sequences to cut segments from the foreign DNA to inactivate bacteriophages. The CRISPR-associated protein 9 (Cas9) aids this mechanism by cutting the recognized DNA.¹

Jennifer Doudna and Emmanuelle Charpentier leveraged this mechanism, engineering a single-guide RNA (sgRNA) that could guide the DNA-cutting process.² Worthy of the Nobel Prize in Chemistry, the CRISPR-Cas9 system laid the foundation for genome engineering. The system was widely used for gene knockouts, where researchers eliminated selected genes to study their functions in disease and develop gene therapy modalities.

The success of CRISPR knockout systems in these applications relies on successful targeting, as off-target knockouts on the human genome can lead to various side effects.

Mechanisms of CRISPR knockout

CRISPR-Cas9 system and components

The sgRNA complex comprises CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA guides Cas9 to its target DNA, while tracrRNA mediates Cas9 activation by stabilizing the complex for efficient target binding.

The Cas9 endonuclease binds the sgRNA complex and, subsequently, the targeted host sequence. Its engagement with the target DNA requires the presence of a downstream protospacer adjacent motif (PAM) in the host genome. This requirement allows CRISPR-Cas9 to define targetable regions. Once at the target, Cas9 cleaves both strands, leading to a double-strand break (DSB) in the host sequence.

DNA repair pathways

The double-stranded break is repaired in the following ways:

• Non-homologous end joining (NHEJ) causes random deletions and insertions at the target site, causing the loss of function in that gene.
• Homologous recombination (HR) is a repair method that incorporates a homologous sequence as a template for accurate gene correction or insertion, minimizing the possibility of mutations.

Key components of a CRISPR knockout experiment

Designing sgRNAs and selecting target genes

The proper design of sgRNA is critical to ensuring target efficiency, maximizing on-target, and minimizing off-target activities.

The workflow for sgRNA design and target gene selection starts by identifying the PAM sequence downstream of the potential target sequence. The researcher should scan the DNA for a 5'-NGG-3' sequence, where N can refer to any base.

Once the PAM region is identified, the 5' start of the sgRNA targeting sequence should ideally be 20 nucleotides upstream. However, the PAM sequence should be excluded during sgRNA construction.

The ideal sgRNA must have a guanine-cytosine (GC) content between 40-60% for maximum stabilization of the DNA-sgRNA complex to mitigate off-target binding.³

Several tools and libraries have been developed to overcome the laborious manual sgRNA selection. Firstly, predesigned gRNAs provide information on on-target and off-target scores for many organisms, including humans, mice, rats, zebrafish, or C. elegans. Custom gRNA design is another option that allows users to evaluate the feasibility of their target sequence.

CRISPR component delivery methods

The success of CRISPR genome editing depends not only on successful sgRNA design but also on the efficient delivery of the system to the target cells. These methods deliver the CRISPR knockout system as a plasmid construct or ribonucleoprotein (RNP) complex.

• Electroporation involves treating the cells with pulses of electric current to increase their permeability so that the CRISPR-Cas9 RNP can infiltrate the cells. This method is beneficial for delivering the RNP with single-stranded donor DNA for homology-directed repair.
• CRISPR components can be directly injected into the cells via microinjection
• Engineered viral vectors can transduce sgRNA and the Cas enzyme genes to the host. However, there is the caveat that the Cas genes can integrate into the host genome and produce undesired changes.
• The RNP or plasmid can be encapsulated in lipid-based nanoparticles (LNP) and delivered via lipofection. LNPs or liposomes reduce the risk of prolonged Cas9 expression, minimizing off-target effects.
• The RNP complex or plasmid can be conjugated to other nanoparticle forms for targeted delivery and release. Gold and zinc nanoparticles demonstrated high delivery efficiency.

Analytical characterization of CRISPR knockout

Genomic screening of knockout clones

Characterizing the genomic modifications after the CRISPR-Cas9 knockout experiments is essential. Genomic screening can help determine the targeting efficiency and functional impact of genomic knockout experiments. Here, knockout clones are screened to identify frameshift mutations, stop codons, indels and targeted disruption of gene function.

• Sanger sequencing and chromatogram analysis: Direct sequencing of the target region
• Next-generation sequencing: High-throughput sequencing of the clones to identify indels, large deletions, and complex mutations.
• Reverse Transcription Quantitative PCR (RT-qPCR): Measurement of the mRNA expression levels of the target gene to confirm loss of gene expression.

Phenotypic screening of knockout effects

In phenotypic screening, the knockout clones' protein expression and cellular functions are assessed.

• Flow cytometry, ELISA assays and Western Blots can screen for the presence or depletion of proteins and provide quantifiable protein expression levels.
• Arrayed CRISPR libraries allow a high-throughput phenotypic screening through viability assays and fluorescent detection of the protein(s) encoded by the target genes.
• Immunofluorescence and immunohistochemistry provide visual confirmation of gene knockout by confirming the absence of the protein.
• Real-time or time-lapse microscopy can visually confirm cell behavior or health disruptions due to absent or disrupted genes and proteins.

Applications of CRISPR knockout

Biological research

CRISPR knockout can serve a myriad of purposes for studying gene function and expression in diseases. By eliminating a set of genes through CRISPR, researchers can uncover their roles in disease onset.

In cancer research, CRISPR screens are commonly used to uncover the genes central to tumorigenic pathways, from proliferation and metastasis to immune evasion. In a 2022 Cell study, researchers performed a high-throughput knockout experiment to identify the genes involved in immunosuppression. The experiment revealed that the loss of function of the TGFβ receptor 2 (Tgfbr2) gene conferred immune resistance and growth advantage to lung tumors.⁴

The knockout approach has also been applied to stem cell biology and developmental studies. A case in point is the 2024 Nature study that focuses on the regulators of aging in neural stem cells. Among the knockout genes, Slc2a4, encoding a glucose transporter, was revealed to decelerate neural stem cell activation and contribute to cognitive decline during aging.⁵

Medical applications

The employment of CRISPR-Cas knockout in gene therapy and personalized medicine continues to thrive, as evidenced by the FDA approval of the first CRISPR-based gene therapy, Casgevy, to treat sickle cell disease. The treatment works by isolating hematopoietic (blood) stem cells from patients, using CRISPR/Cas9 to deactivate the defective BCL11A gene, and transfecting back into patients so that the stem cells can produce functioning hemoglobin.⁶

Several knockout strategies are currently under development for cancer therapy. Immune checkpoint inhibitors, transcription factors and signaling proteins are common CRISPR targets. For instance, preliminary research showed that knocking out the programmed cell death protein 1 gene (PD-1), which inhibits T cell activation, reactivated killer T cells to combat tumor cells.⁷

Industrial biotechnology and synthetic biology

Genetic engineering and synthetic biology are other fields greatly benefiting from CRISPR-Cas9 gene knockout. During the expansion and scaling of industrial cell lines used to produce biotherapeutics, knockout with homologous recombination is employed to disrupt endogenous gene function. This approach helps establish stable and durable clones with high production rates.⁸

Challenges and ethical considerations

Technical challenges

Despite the promising emergence and advancement of CRISPR-Cas9, several technical challenges remain to limit its translation to clinical applications.

One of the key challenges is to prevent cutting at unintended genomic sites due to sgRNA mismatches, ultimately leading to off-target effects. This threat can be resolved through sgRNA optimization or using high-fidelity Cas9 variants. Furthermore, CRISPR-Cas9 exhibits low knockout efficiencies and inefficient editing, highlighting the significance of RNP delivery and donor DNA templates for precise repair.

Another issue may stem from the genomic instability caused by excessive DSBs and indel mutations, which might interfere with innate transcriptional regulation mechanisms. Knockout mutations can be regulated and tailored to the target site using Cas9 nickases (Cas9n) that create single-stranded breaks. Using two Cas9n and two sgRNAs generates staggered breaks mimicking a DSB but are much more target-oriented than the conventional CRISPR-Cas9.

Ethical Considerations in Gene Editing by CRISPR

The challenges and potential risks associated with CRISPR gene knockouts have also led to a debate about their societal impact, possible misuse and safety.⁹ Although it offers potential benefits for the treatment of numerous genetic diseases, the possibility of its use for germline editing sparks concerns about its long-term effects on future generations. In addition, the consequences of off-target editing and unintended mutations are far-reaching, particularly when applied to large clinical trials. Therefore, rigorous safety analysis is necessary before translating CRISPR Cas9 gene knockout to human applications.

Despite its potential in gene therapies, the question of its worldwide accessibility remains. A key concern is whether low-income countries stricken by debilitating genetic disorders will get to reap the fruits of CRISPR-Cas9 knockout tools. Healthcare providers are responsible for implementing policies that promote equitable access to CRISPR gene therapies.

References

1. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 1987;169(12):5429-5433.
2. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816-821.
3. Liu X, Homma A, Sayadi J, Yang S, Ohashi J, Takumi T. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system. Sci Rep 2016;6(1):19675.
4. Dhainaut M, Rose SA, Akturk G, Wroblewska A, Nielsen SR, Park ES, et al. Spatial CRISPR genomics identifies regulators of the tumor microenvironment. Cell 2022;185(7):1223-1239. e20.
5. Ruetz TJ, Pogson AN, Kashiwagi CM, Gagnon SD, Morton B, Sun ED, et al. CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells. Nature 2024;634(8036):1150-1159.
6. Kerwash E, Sajic M, Rantell KR, McBlane JW, Johnston JD, Niewiarowska A, et al. Regulatory Assessment of Casgevy for the Treatment of Transfusion-Dependent β-Thalassemia and Sickle Cell Disease with Recurrent Vaso-Occlusive Crises. Curr Issues Mol Biol 2024;46(8):8209-8225.
7. Tu K, Deng H, Kong L, Wang Y, Yang T, Hu Q, et al. Reshaping tumor immune microenvironment through acidity-responsive nanoparticles featured with CRISPR/Cas9-mediated programmed death-ligand 1 attenuation and chemotherapeutics-induced immunogenic cell death. ACS appl mater inter 2020;12(14):16018-16030.
8. Dalvie NC, Lorgeree T, Biedermann AM, Love KR, Love JC. Simplified gene knockout by CRISPR-Cas9-induced homologous recombination. ACS Synth Biol 2021;11(1):497-501.
9. Piergentili R, Del Rio A, Signore F, Umani Ronchi F, Marinelli E, Zaami S. CRISPR-Cas and its wide-ranging applications: From human genome editing to environmental implications, technical limitations, hazards and bioethical issues. Cells 2021;10(5):969.

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