CRISPR Advancements for Human Health

Abstract

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a powerful gene editing technology that is revolutionizing biomedical research and clinical medicine. The CRISPR system allows scientists to rewrite the genetic code in virtually any organism. This review provides a comprehensive overview of CRISPR and its clinical applications. We first introduce the CRISPR system and explain how it works as a gene editing tool. We then highlight current and potential clinical uses of CRISPR in areas such as genetic disorders, infectious diseases, cancer, and regenerative medicine. Challenges that need to be addressed for the successful translation of CRISPR to the clinic are also discussed. Overall, CRISPR holds great promise to advance precision medicine, but ongoing research is still required to optimize delivery, efficacy, and safety.

Introduction

The CRISPR system is comprised of a CRISPR-associated (Cas) endonuclease along with a single guide RNA (sgRNA) designed to target a specific DNA sequence. Cas nucleases are enzymes that can bind and create double-stranded breaks in DNA. sgRNAs contain a scaffold structure that complexes to the Cas protein and also includes a uniquely engineered segment that can be designed to direct the Cas protein to a specific DNA sequence of interest. CRISPR technology enables precise targeting of nearly any genomic location simply by altering the nucleotide sequence of the sgRNA. This targeted approach can help to correct disease-causing mutations or suppress genes linked to the onset of diseases.¹ CRISPR has been adapted for deleting gene function (knockout), adding new gene function (knock-in), activation or repression of endogenous genes, and genomic diagnostic screening techniques.

Advanced CRISPR approaches such as base editing and prime editing use modified Cas enzymes which can induce precise single nucleotide changes in the genome without creating double-strand DNA breaks.² CRISPR can also be used to activate genes (CRISPRa) or inactivate genes (CRISPRi) by targeting modified sgRNA/Cas complexes to the gene’s promoter region, recruiting transcription factors for increased gene expression or repressors for decreasing gene expression.³

While CRISPR-Cas technology has demonstrated immense potential as a genome editing tool, its use in clinical applications is still in the early stages. As of January 2024, only 89 clinical trials employing CRISPR are currently underway, highlighting that much work remains to translate this technology into approved gene therapies.⁴ Notably, unintended alterations in DNA can occur through the utilization of CRISPR, and the long-term consequences of these modifications on patient health remain uncertain. However, given the considerable benefits that CRISPR offers, it is plausible to anticipate that these challenges will be overcome in the foreseeable future.

Figure 1.

Traditional CRISPR-Cas9 genome editing. The sgRNA contains a scaffold structure that complexes to the Cas9 protein and also includes a uniquely engineered segment that can be designed to direct the Cas9 protein to a specific DNA sequence of interest. Once bound to the DNA at the target location, the Cas9 protein creates a double-stranded break in the DNA. The cell naturally heals this double-stranded break using one of two main repair mechanisms. Non-homologous end joining (NHEJ) is the most common mechanism typically resulting in indel formation. Homology-directed repair (HDR) is a less efficient repair mechanism that allows for introduction of exogenous DNA through the use of a donor DNA repair template.

CRISPR Modifications and Advancements

CRISPR-based genome editing technologies have rapidly progressed, enabling more precise and versatile modifications to DNA sequences. Since wildtype Streptococcus pyogenes Cas9 (SpCas9) nucleases cause double-stranded DNA breaks that can result in unwanted nucleotide insertions or deletions (indels), scientists have developed modified Cas9 proteins such as a nickase Cas9 (nCas9) and a catalytically dead Cas9 (dCas9). nCas9 only produces a single-stranded “nick” in the DNA rather than cleaving both strands. dCas9 is able to bind DNA but does not create a break in the DNA at all. By fusing either nCas9 or dCas9 to DNA modifying enzymes, scientists can alter specific nucleotides without the risk of introducing indels.

Base editors, comprised of modified nCas9 or dCas9 fused to a deaminase, precisely convert C-to-T or A-to-G nucleotide substitutions without creating a double-stranded break in the DNA.⁵ One of the limitations of base editing is that the system cannot be used to alter every possible nucleotide combination. A more recent advancement, prime editing, has further broadened the range of editable genetic sites. Prime editing involves a fusion of nCas9 to an engineered reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA guides the nCas9 to the region of interest, and also includes a segment that consists of the desired nucleotide sequence for repairing the DNA after the single-stranded nick has been generated.⁶ Since prime editing is designed to fix single base pair changes and other small mutations, it is predicted to be capable of treating up to 89% of genetic mutations in humans. Whereas the other 11% of human mutations include larger disruptions such as having too many copies of a gene or when an entire gene is missing.^7,8

As an alternative to modifying DNA, an RNA editing system has also been developed. This system utilizes a modified CRISPR effector protein, Cas13b, combined with an adenosine deaminase domain to edit adenosine-to-inosine accurately and effectively in RNA molecules. This approach also holds promise for correcting many mutations that contribute to specific human diseases.⁹ Compared to DNA-based methods, editing RNA offers a safer therapeutic profile due to the transient nature of RNA edits.

While delivering these complexes remains a challenge, Cas molecules can be introduced into the cell as a synthetic gene sequence using adeno-associated virus (AAV), where the viral vectors carry the genetic instructions for the Cas protein. However, the large size of wildtype SpCas9 poses limitations for delivery with AAV vectors. To overcome this, researchers have engineered a compact CRISPR enzyme called Axidibacillus sulfuroxidans Cas12f (AsCas12f) that has comparable gene editing activity to the wild type SpCas9, while being one-third the size of commonly used Cas9. This modified AsCas12f was successfully delivered in vitro in mice using AAV, demonstrating its potential for more efficient CRISPR-based genetic therapies.¹⁰

Together, these engineered CRISPR systems and new approaches mark significant advances in precision genome editing capabilities, including more specific on-target edits, the ability to modify a broader range of sites, and enhanced delivery options like AAV-compatible AsCas12f. Continued engineering of compact yet efficient Cas nucleases and novel effector fusions will help drive the next generation of precise, versatile and deliverable genome editing tools.

Delivery of CRISPR Reagents

One of the key challenges for using CRISPR in the clinic is ensuring safe and effective delivery of the CRISPR components to the targeted tissues and minimizing off-target effects. New viral vector delivery systems like AAV are being engineered to overcome this obstacle. Extensive in vitro and in vivo animal model testing is being done to evaluate on-target editing and safety.

Multiple approaches are being explored to deliver CRISPR-Cas components into cells within the body for therapeutic editing applications. These can be categorized as viral vector-based delivery strategies or non-viral carrier-based methods.

Viral vectors may integrate into host cells and cause problems such as mutations, carcinogenesis, and immune responses.¹¹ However, recombinant AAVs are non-replicating and tend to remain episomal without integrating into the genome, except in rare cases. These advantages allow their use for packaging CRISPR reagents into the AAV genome for cell editing.¹² AAVs can also provide tissue-specific tropism due to the cell-specific serotypes of AAVs, have reduced immunogenicity compared to other viral vectors, and enable transient gene expression.¹³ However, challenges remain regarding efficiency and safety.

Non-viral delivery methods include the use of nanoparticles or liposomes to encapsulate ribonucleoprotein complexes.^14–16 Nanoparticles made from engineered lipids or polymers can improve cell uptake, increase editing potency beyond what is achievable by protein alone, expand delivery beyond the liver, and potentially reduce immunogenicity compared to viral approaches.¹⁷ Another non-viral method uses messenger RNA (similar to RNA vaccines) to encode the CRISPR components, achieving transient and controlled expression without needing to deliver DNA.¹⁸

Lastly, the CRISPR system can be used without delivering the CRSIPR components directly into the body. Ex vivo therapy involves removing cells from the patient, editing them outside the body, and then returning them to treat genetic disorders like sickle cell anemia or enhance immune responses for cancers.

Figure 2.

Expanding CRISPR toolbox. CRISPR-based genome editing technologies are rapidly progressing. CRISPRa and CRISPRi can be used to recruit transcription factors for increased gene expression or repressors for decreasing gene expression. Base editors have been designed to convert C-to-T or A-to-G nucleotide substitutions without creating a double-stranded break in the DNA. Prime editing includes a small, engineered segment of new sequence that is used for precisely repairing the DNA after a single-stranded nick has been generated. Continued advancement of CRISPR-based tools will help drive the next generation of precise, versatile and deliverable genome editing tools.

Potential Clinical Applications

Genetic Disorders

The introduction of CRISPR technology brings a hopeful new phase in treating genetic diseases. This powerful gene-editing tool offers amazing potential to directly address the root causes of many different genetic disorders. Recent clinical trials have demonstrated the potential of CRISPR-Cas gene editing to treat blood disorders like sickle cell disease (SCD) and beta-thalassemia. In a phase 1/2 study, individuals with transfusion-dependent beta-thalassemia received autologous CD34⁺ hematopoietic stem and progenitor cells modified using CRISPR-Cas9 to disrupt the BCL11A erythroid enhancer and induce fetal hemoglobin production. Participants experienced significant and sustained increases in fetal hemoglobin levels after receiving the gene-edited cells.¹⁹ The FDA recently approved the first CRISPR therapy for sickle cell anemia.²⁰ Additionally, strategies to rectify the sickle cell mutation in the beta-globin gene or compensate by overexpressing gamma-globin are actively under investigation.²¹ Cystic fibrosis patients stand to benefit from CRISPR interventions aimed at correcting mutations in the CFTR gene, such as the prevalent F508del allele, and restoring proper CFTR channel function.²² In the case of Duchenne muscular dystrophy, approaches involving exon skipping or direct correction of dystrophin mutations through CRISPR hold the potential to restore dystrophin protein production in muscle cells.²³ Leber congenital amaurosis, which is primarily attributed to CEP290 mutations, is being targeted with CRISPR strategies that seek to repair specific disease-causing mutations.²⁴ Hemophilia, characterized by deficiencies in Factor VIII or Factor IX, is being addressed through CRISPR editing of liver cells to restore normal blood clotting mechanisms. A novel dual gene editing strategy using CRISPR has been shown to provide long-term phenotypic correction in a mouse model of hemophilia B, offering a promising new approach for the treatment of this disease.²⁵ Studies in Huntington’s Disease pig models have shown that disease treatment can be optimized by lowering mutant huntingtin protein levels via the targeting of expanded CAG repeats in the HTT gene.²⁶ Additionally, CRISPR shows promise in difficult to treat mitochondrial diseases by modifying the gene expression through targeted knock-in or directly editing mutations in the mitochondrial DNA.²⁷

Infectious Diseases

The CRISPR-Cas system has the potential to aid in diagnosing diseases, preventing their spread, and contributing to potential treatments. CRISPR-based systems have shown tremendous potential for ultrasensitive detection of pathogens. Novel assays couple isothermal amplification with CRISPR mediated DNA cleavage for single-copy detection of viral or bacterial nucleic acids within 1–2 hours.²⁸ These assays can be readily adapted to detect emerging outbreak pathogens. CRISPR can also improve sequencing-based detection of rare antimicrobial resistance genes by removing abundant human gene sequences or blocking background DNA to enrich pathogen signals.²⁹ CRISPR has shown the potential to prevent or cure various viral and bacterial diseases. HIV requires CCR5 receptors to enter the cell. Knocking out the CCR5 gene results in cell resistance to HIV and the absence of HIV infection in patients. In humanized mouse models, CRISPR delivered via AAV to knock out the receptor CCR5 prevented HIV infection.³⁰ In clinical trials, CRISPR-based gene therapy is being utilized to treat HIV in humans. This approach targets the viral DNA of HIV that is integrated in the host genome, aiming to disrupt its ability to replicate and maintain infection.³¹ CRISPR can be used to treat infectious diseases by targeting the pathogenic nucleic acids. For instance, the Cas13d nuclease was able to target and destroy SARS-CoV-2 RNA in vitro and also reduce influenza viral load in respiratory epithelial cells.³² CRISPR has been used to successfully targeted human papillomavirus genes E7 and E9 in cell lines, which could help treat HPV-associated cancers.³³ Additionally, modified CRISPR nucleases such as Cas3 paired with phages have been able to target antibiotic-resistant bacteria to overcome infections.³⁴

Cancer

CRISPR technology is showing significant promise in cancer therapy. It is being actively used in several clinical trials, which focus on various strategies of knocking out oncogenes, correcting mutations in tumor suppressor genes, and enhancing the efficacy of T-cell therapies. These trials are exploring CRISPR’s potential in different types of cancer and its ability to improve immune system responses against cancer cells. For example, in vitro studies have shown that CRISPR can be used to inactivate oncogenic KRAS, which is mutated in many cancers like lung and pancreatic cancer.³⁵ Researchers have also shown that CRISPR can be employed to correct mutations in tumor suppressor genes, particularly the TP53 gene.³⁶

In conjunction with chimeric antigen receptor (CAR) T-cell therapy to target cancer antigens, CRISPR mediated gene editing technology has allowed for the potential to target a wide range of cancers. CRISPR-Cas9 gene editing has been utilized to knock out the immune checkpoint receptor PD-1 in tumor-specific T-cells.^37–39 Disruption of the PD-1 signaling axis in PD-1 knockout T-cells enhanced anti-tumor immunity, as evidenced by increased tumor infiltration and cytotoxicity of PD-1 knockout T-cells compared to control T-cells in murine tumor models.^40,41

In addition to engineering patient’s own T-cells (autologous T-cells), there is increasing interest in using T-cells from healthy donors (allogeneic T-cells) as an off-the-shelf cell therapy product.⁴² Gene-edited allogeneic T-cells, with mechanisms to reduce graft-vs-host rejection, have shown promise as a strategy to broaden access to engineered T-cell therapies.⁴³ Recent studies have demonstrated the feasibility of disrupting genes such as PD-1 and TCR using CRISPR-Cas9 in allogeneic T-cells before adoptive transfer into patients.^44,45 Allogeneic CRISPR-edited T-cell therapies are now being evaluated in early-phase clinical trials, with the goals of maintaining anti-tumor potency while minimizing the risk of graft-vs-host disease.⁴⁶

Regenerative Medicine

CRISPR technology is revolutionizing regenerative medicine, especially stem cell-based treatments, by addressing immune rejection challenges. This is primarily achieved by editing the major histocompatibility complex-human leukocyte antigen (MHC-HLA) genes in donor cells to match those of the recipient, creating immune-tolerant cells.⁴⁷ This breakthrough significantly reduces the need for immunosuppressive drugs, mitigating their adverse effects and long-term risks. A notable application is in diabetes treatment, where CRISPR-edited stem cells could offer a durable cure without continuous immunosuppression.⁴⁸

Ethical Considerations

CRISPR-based genome editing technologies have revolutionized the field of genetic modification, allowing for precise and targeted modifications to the genome. However, these technologies also raise significant safety and ethical concerns, specifically off-target effects and potential germline editing. Off-target effects refer to unintended editing at sites other than the intended target locus. Studies have shown that CRISPR nucleases can induce mutations at off-target sites that differ from the target sequence by a few nucleotides.⁴⁹ These off-target effects can potentially lead to unpredictable and deleterious mutations. To address this issue, various strategies are being developed to improve targeting specificity, predict potential off-target sites using computational algorithms, and analyze off-target effects through whole genome sequencing.⁵⁰ Human germline editing, which involves modifying the genetic material of embryos or germline cells, is highly controversial. Concerns include the potential for these heritable modifications to have unknown and irreversible effects on future generations.⁵¹ While the intention behind germline editing is to eliminate genetic disease traits, there is a lack of consensus on whether it should be permitted given the current limitations in terms of knowledge and technical capabilities. The ongoing ethical debates revolve around arguments supporting parental reproductive freedom and the prevention of heritable diseases, versus concerns about permanently altering the human genome in ways that could have far-reaching unknown consequences. Moving forward, it is crucial to minimize off-target effects and restrict applications to somatic cells until safety and consensus can be firmly established regarding human germline modifications. Careful regulations and transparent public discourse are essential to govern the responsible advancement of CRISPR-based genome editing technologies.

Conclusion

CRISPR-based genome editing technologies offer unprecedented capabilities for precise genetic modification. However, the ethical concerns surrounding off-target effects and human germline editing necessitate careful consideration and regulation. Efforts are being made to improve delivery techniques and targeting specificity minimizing off-target activity, while the debate regarding human germline editing continues.