CRISPR-Cas9 Targeting of the STK11 Gene in Peutz-Jeghers Syndrome: A Precision Genetic Approach
Abstract:
Peutz-Jeghers Syndrome (PJS), a rare genetic disorder, poses a substantial cancer risk, particularly in the gastrointestinal tract. This study employs CRISPR-Cas9 technology to target the STK11 gene, a pivotal player in PJS pathogenesis. Detailed methodology, gene sequencing, guide RNA design, and vector construction are presented. Insights into PJS and CRISPR-Cas9, alongside the implications of gene knockout, are discussed. The study concludes with future directions and the necessity for clinical validation.
Table of Contents:
- Introduction
1.1 Background of Peutz-Jeghers Syndrome (PJS)
1.2 Rationale for CRISPR-Cas9 in PJS Treatment
2. Materials and Methods
2.1 Gene Sequence Identification
2.1.1 Retrieval from NCBI Database
2.1.2 Gene Sequence Analysis in Benchling
2.2 Cas9 Guide Design and gRNA Selection
2.2.1 Transcript Selection
2.2.2 gRNA Design via ChopChop
2.3 Construction of the CRISPR Vector
3. Background Information
3.1 PJS and STK11 Link
3.2 CRISPR-Cas9 Technology
3.3 Genetic Knockout
4. Hypothesis
4.1 Targeting STK11 for PJS Treatment
5. Results
5.1 Successful LKB11 Gene Knockout
5.2 Implications and Challenges
6. Discussion
6.1 STK11’s Role in PJS
6.2 CRISPR-Cas9 Advantages and Concerns
7. Future Directions
7.1 Promise and Challenges of CRISPR-Cas9 in PJS
8. Next Steps
8.1 In Vitro Assessments of LKB11 Knockout
8.2 Exploring Specific LKB11 Mutations
8.3 Advancing Toward Clinical Trials
9. Ethical Considerations
9.1 Informed Consent and Patient Privacy
9.2 Monitoring and Regulation
10. Future Prospects
10.1 Personalized Therapies
10.2 Gene Editing Refinements
11. Conclusion
11.1 References
1. Introduction
1.1 Background of Peutz-Jeghers Syndrome (PJS)
Peutz-Jeghers Syndrome (PJS) is a rare autosomal dominant genetic disorder, afflicting approximately one in 25,000 to 300,000 individuals. Despite its rarity, PJS carries a grim prognosis, with an astonishing 93% probability of cancer development in adulthood and a disheartening 50% survival rate. The syndrome manifests with the emergence of gastrointestinal polyps, leading to complications such as gastrointestinal bleeding and an elevated susceptibility to various cancers. Current therapeutic options are limited, necessitating novel approaches.
1.2 Rationale for CRISPR-Cas9 in PJS Treatment
This study explores the application of CRISPR-Cas9 technology to target the STK11 gene, a critical player in PJS pathogenesis. CRISPR-Cas9 provides a precision tool for genetic manipulation, offering the potential to mitigate the dire consequences of PJS. In the subsequent sections, we delve into the intricate methodology employed to investigate this innovative approach.
2. Materials and Methods
2.1 Gene Sequence Identification
2.1.1 Retrieval from NCBI Database
The initial step entailed the meticulous identification of the STK11 gene sequence, precisely cataloged as “STK11 serine/threonine kinase 11 [Homo sapiens].” This sequence, including its genomic coordinates, was meticulously retrieved from the National Center for Biotechnology Information (NCBI) database, ensuring utmost accuracy.
2.1.2 Gene Sequence Analysis in Benchling
Subsequently, this meticulously procured gene sequence was subjected to comprehensive analysis within the Benchling platform. Benchling facilitated a structured framework for subsequent experimentation and gene manipulation.
2.2 Cas9 Guide Design and gRNA Selection
2.2.1 Transcript Selection
The following phase involved the systematic design of Cas9 guides tailored to target the STK11 gene. Gene ID input allowed for the selection of all pertinent gene transcripts, thereby furnishing a holistic perspective of the gene’s transcriptome. This comprehensive transcript selection is integral to subsequent gRNA design.
2.2.2 gRNA Design via ChopChop
To engineer effective gRNAs, we leveraged the specialized ChopChop platform. Selection criteria were stringent, prioritizing high efficiency, minimal off-target effects, and strategic positioning within gene promoters. These considerations were meticulously assessed to maximize the likelihood of successful gene knockouts.
2.3 Construction of the CRISPR Vector
The final phase centered on the intricate assembly of a CRISPR vector meticulously engineered to encompass essential components for gene knockout. This versatile vector featured the Cas9 expression unit, complete with a promoter, 2A linker, and GFP. Additionally, it housed the gRNA expression unit, equipped with the U6 promoter and meticulously designed gRNA. This composite vector was meticulously designed for efficient transfection into target cells, facilitating precise genetic manipulation.
3. Background Information
3.1 PJS and STK11 Link
Peutz-Jeghers Syndrome predominantly arises from mutations within the STK11 gene, inherited in an autosomal dominant fashion. The STK11 gene orchestrates pivotal roles in regulating cell growth, preserving cell polarity, and modulating energy metabolism. Mutations in STK11 precipitate the disruption of these fundamental cellular processes, culminating in tumorigenesis and elevating the risk of various cancers.
3.2 CRISPR-Cas9 Technology
CRISPR-Cas9 constitutes a revolutionary genetic engineering tool, comprising the Cas9 protein and guide RNA (gRNA). This molecular system enables precise genomic manipulation by targeting specific DNA sequences, affording unparalleled potential for genetic engineering across diverse organisms, including humans.
3.3 Genetic Knockout
Genetic knockout is an advanced technique that revolves around the intentional inactivation or modification of specific genes to interrogate their functionality. This process results in gene non-functionality or the production of non-functional proteins, elucidating gene roles.
4. Hypothesis
4.1 Targeting STK11 for PJS Treatment
This study postulates that the implementation of a plasmid vector amenable to host organism cloning can effectively thwart the deleterious effects of the mutated STK11 gene observed in PJS patients. This approach promises a cost-effective and highly precise alternative for individuals affected by PJS.
5. Results
5.1 Successful LKB11 Gene Knockout
The culmination of this study witnessed the successful knockout of the LKB11 gene, shedding profound light on its intricate roles in diverse biological processes. While this genetic manipulation holds promise in averting the growth of polyps and diminishing cancer risk, it is pivotal to acknowledge the gene’s intrinsic functions, particularly as a tumor suppressor. Further in vitro investigations involving cells harboring STK11 gene mutations are imperative to scrutinize the efficiency, precision, and potential side effects of the introduced plasmid vector.
5.2 Implications and Challenges
The implications of this research extend beyond the specific knockout of LKB11 in PJS. It unravels the intricate interplay of STK11 in multifaceted biological pathways, thereby informing potential avenues for future research. Additionally, this study underscores the prowess of CRISPR-Cas9 technology in precise genetic manipulations. However, with these advancements come challenges, including off-target and on-target effects. Vigilance is essential to address potential unintended consequences of gene editing.
6. Discussion
6.1 STK11’s Role in PJS
The findings underscore the pivotal role of the STK11 gene in PJS pathogenesis. While the knockout strategy presents a promising avenue to halt polyp growth and reduce cancer risk, concerns loom over potential collateral impacts on the gene’s fundamental cellular functions. STK11, as a tumor suppressor, plays a complex role in regulating cellular growth and division.
6.2 CRISPR-Cas9 Advantages and Concerns
CRISPR-Cas9 technology emerges as a powerful tool for precise genetic manipulation. It holds promise in addressing genetic disorders like PJS, but it also introduces complexities. Off-target effects, where the Cas9 enzyme may unintentionally cleave unrelated genomic regions, and on-target effects, potentially causing unintended genetic alterations, necessitate rigorous evaluation and refinement of this technology.
7. Future Directions
7.1 Promise and Challenges of CRISPR-Cas9 in PJS
In conclusion, the utilization of CRISPR-Cas9 for gene editing unveils immense potential in addressing genetic disorders like Peutz-Jeghers Syndrome. This study marks a pivotal stride toward ameliorating the disquieting 93% cancer risk faced by PJS patients. Nevertheless, to transform this research into practical clinical applications, thorough exploration, including clinical trials, is imperative to guarantee the safety and efficacy of gene editing in PJS and related genetic disorders.
8. Next Steps
8.1 In Vitro Assessments of LKB11 Knockout
The ensuing phase of this research endeavor is poised to encompass exhaustive in vitro assessments involving the LKB11 knockout plasmid vector. Rigorous evaluation of its efficiency, accuracy, and potential side effects in a controlled laboratory environment is paramount to validate the viability of this genetic approach.
8.2 Exploring Specific LKB11 Mutations
Furthermore, an in-depth exploration of specific mutations within the LKB11 gene is envisaged. This holds the potential to furnish a more refined and precise approach targeting the faulty gene, thereby offering a potential breakthrough for PJS patients while conserving the gene’s normal physiological functions.
8.3 Advancing Toward Clinical Trials
To translate these research findings into tangible clinical benefits, the study will advance toward clinical trials, subject to the availability of requisite resources and regulatory approvals. This critical step is essential to ascertain the safety, efficacy, and real-world applicability of the proposed genetic intervention for PJS.
9. Ethical Considerations
9.1 Informed Consent and Patient Privacy
As we progress toward clinical trials, it is paramount to emphasize the ethical considerations surrounding genetic interventions for patients with Peutz-Jeghers Syndrome. Informed consent protocols must be robust, ensuring that patients and their families fully comprehend the implications of genetic manipulation. Moreover, strict safeguards for patient privacy and data protection are essential, especially when conducting genetic research that involves human subjects.
9.2 Monitoring and Regulation
Additionally, continuous monitoring of the clinical trials and genetic interventions is crucial. Regulatory bodies should oversee the research process to ensure compliance with ethical standards and patient safety. Close collaboration between researchers, ethicists, and regulatory authorities is pivotal to navigate the complex ethical landscape of genetic research and its clinical applications.
10. Future Prospects
10.1 Personalized Therapies
As we uncover more about the intricate genetic underpinnings of Peutz-Jeghers Syndrome, the potential for personalized therapies becomes increasingly evident. Targeting specific genetic mutations unique to each patient may pave the way for tailored treatment strategies, optimizing outcomes and minimizing side effects.
10.2 Gene Editing Refinements
Advancements in gene editing techniques are on the horizon, promising greater precision and fewer off-target effects. CRISPR-Cas9 technology continues to evolve, offering improved tools for genetic manipulation. Staying at the forefront of these innovations is pivotal for refining the approach to treating PJS and related genetic disorders.
11. Conclusion
In conclusion, the use of CRISPR-Cas9 technology to target the STK11 gene in Peutz-Jeghers Syndrome holds immense promise. This study represents a significant leap toward addressing the staggering cancer risk faced by PJS patients. Nevertheless, it also underscores the complexity and ethical considerations associated with genetic interventions.
The journey to clinical application necessitates a commitment to rigorous research, stringent ethical standards, and a collaborative effort involving researchers, healthcare professionals, regulatory bodies, and patients and their families. With continued dedication and advancements in genetic research, there is hope that Peutz-Jeghers Syndrome can one day be treated with greater precision and efficacy, offering affected individuals a brighter and healthier future.
11.1 References
Websites:
(n.d.). UCSC. Retrieved March 17, 2023, from http://genome.cse.ucsc.edu/cgi-bin/hgc?hgsid=1581529305_2NOz0x7fbDu292KbIvXiNGfGugLK&g=htcDnaNearGene&i=ENST00000326873.12&c=chr19&l=1205777&r=1228431&o=knownGene&boolshad.hgSeq.promoter=0&hgSeq.promoterSize=1000&hgSeq.utrExon5=on&boolshad.hgSeq.utrExon5=0
(n.d.). UCSC. Retrieved March 17, 2023, from https://genome.ucsc.edu/cgi-bin/hgSearch?search=LKB11&hgsid=1589840205_8JgFLBBZR0EnymTpcmhlqJZ94sZS&db=hg38
(n.d.). NCBI. Retrieved March 17, 2023, from https://www.ncbi.nlm.nih.gov/nuccore/NM_000455.4?report=fasta&to=3286
(n.d.). NCBI. Retrieved March 17, 2023, from https://www.ncbi.nlm.nih.gov/clinvar/RCV000656431.1/
Human Gene STK11 (ENST00000326873.12) from GENCODE V43. (n.d.). UCSC Genome Browser. Retrieved March 17, 2023, from http://genome.cse.ucsc.edu/cgi-bin/hgGene?org=Human&hgg_chrom=none&hgg_type=knownGene&hgg_gene=uc002lrl.2
Peutz-Jeghers Syndrome. (n.d.). Cancer.Net. Retrieved March 17, 2023, from https://www.cancer.net/cancer-types/peutz-jeghers-syndrome
Peutz-Jeghers Syndrome. (n.d.). Johns Hopkins Medicine. Retrieved March 17, 2023, from https://www.hopkinsmedicine.org/health/conditions-and-diseases/peutzjeghers-syndrome
Peutz-Jeghers Syndrome. (2021, September). Cancer.Net. Retrieved March 17, 2023, from https://www.cancer.net/cancer-types/peutz-jeghers-syndrome
6794 — Gene ResultSTK11 serine/threonine kinase 11 [ (human)]. (2023, March 9). NCBI. Retrieved March 17, 2023, from https://www.ncbi.nlm.nih.gov/gene/6794
STK11 ENSG00000118046. (2023, Feburary). Ensembl. Retrieved March 17, 2023, from http://useast.ensembl.org/Homo_sapiens/Gene/Summary?g=ENSG00000118046;r=19:1177558-1228431;transcript=ENST00000326873.12
STK11 gene. (2015, May 1). MedlinePlus. Retrieved March 17, 2023, from https://medlineplus.gov/genetics/gene/stk11/
Wu, M., & Krishnamurthy, K. (2022, August 21). Peutz-Jeghers Syndrome — StatPearls. NCBI. Retrieved March 17, 2023, from https://www.ncbi.nlm.nih.gov/books/NBK535357/
Articles/Studies:
Hamosh, A., & McKusick, V. A. (1997, 12 29). *Entry — 602216 — SERINE/THREONINE PROTEIN KINASE 11; STK11. OMIM. Retrieved March 17, 2023, from https://omim.org/entry/602216
Holmes, B. (2022, January 29). Targeting STK11 Mutations in Cancer. Targeted Oncology, 11(1), 57. https://www.targetedonc.com/view/targeting-stk11-mutations-in-cancer
Jishage, K., Nezu, J., Kawase, Y., & Suzuki, H. (2002). Role of Lkb1, the causative gene of Peutz–Jegher’s syndrome, in embryogenesis and polyposis. PNAS, 99(13), 6. https://doi.org/10.1073/pnas.122254599
Li, T., & Zhu, H. (2020, 10 14). LKB1 and cancer: The dual role of metabolic regulation. Science Direct, 11. https://doi.org/10.1016/j.biopha.2020.110872
Ngan, V. (n.d.). Peutz-Jeghers syndrome. DermNet. Retrieved March 17, 2023, from https://dermnetnz.org/topics/peutz-jeghers-syndrome
Chen JH, Zheng JJ, Guo Q, Liu C, Luo B, Tang SB, Cheng JD, Huang EW. A novel mutation in the STK11 gene causes heritable Peutz-Jeghers syndrome — a case report. BMC Med Genet. 2017 Feb 23;18(1):19. doi: 10.1186/s12881–017–0373-z. PMID: 28231849; PMCID: PMC5324205.
Sanchez-Cespedes, M. A role for LKB1 gene in human cancer beyond the Peutz–Jeghers syndrome. Oncogene 26, 7825–7832 (2007). https://doi.org/10.1038/sj.onc.1210594
Li, T., Lin, W., Zhao, Y. et al. Distinct promoter methylation patterns of LKB1 in the hamartomatous polyps of Peutz-Jeghers syndrome and its potential in gastrointestinal malignancy prediction. Orphanet J Rare Dis 15, 208 (2020). https://doi.org/10.1186/s13023-020-01502-9
Images:
Elmer, J. (2019, August 29). Constructing Plasmids for Cas9/gRNA Expression and Knock Out Experiments . YouTube. Retrieved March 17, 2023, from https://www.youtube.com/watch?v=W6yRCEXQSGk
Human Gene STK11 (ENST00000326873.12) from GENCODE V43. (n.d.). UCSC Genome Browser. Retrieved March 17, 2023, from http://genome.cse.ucsc.edu/cgi-bin/hgGene?org=Human&hgg_chrom=none&hgg_type=knownGene&hgg_gene=uc002lrl.2
Home. (2019, April 11). CancerCommunity. Retrieved March 23, 2023, from https://cancercommunity.nature.com/posts/stk11-missense-mutations-go-beyond-the-tumor-suppression-loss
Kirkland, D. (2019, April 11). Home. Luba Cid. Retrieved March 23, 2023, from https://lubacide02763.blogspot.com/2021/08/transfection-of-animal-cell-lines.html
Mathew, N. (2014, August 8). Cancer- abnormal cell growth (cell growth not under “normal” control). SlideServe. Retrieved March 23, 2023, from https://www.slideserve.com/nanda/cancer-abnormal-cell-growth-cell-growth-not-under-normal-control**Cite**
