CRISPR Genome Editing: From Molecular Principles to Therapeutic Applications

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CRISPR Genome Editing: From Molecular Principles to Therapeutic Applications

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CRISPR Genome Editing: From Molecular Principles to Therapeutic Applications

Gene editing is a technology that induces genome insertion, deletion, or base replacement by precisely modifying the genome sequence.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is currently the most widely used genome editing technology. It utilizes Cas proteins with nucleic acid endonuclease function, such as Cas9 and Cas12a, for site-specific DNA recognition and cleavage.

With the approval of the first CRISPR/Cas9 gene editing therapy, Casgevy (exagamglogene autotemcel, exa-cel), by the FDA in December 2023, CRISPR genome editing technology has truly moved from science to application.

So what is CRISPR genome editing technology? What are its wide-ranging applications? What challenges does it face? Let’s explore them one by one.

01 Molecular Principles of CRISPR Genome Editing

So far, gene editing technology has mainly gone through the following three generations:

• First generation: Zinc Finger Nucleases (ZFNs)
• Second generation: Transcription Activator-Like Effector Nucleases (TALENs)
• Third generation: CRISPR/Cas system gene editing technology

When the CRISPR/Cas system combines with the target, the nuclease catalyzes DNA cleavage, resulting in DNA double-strand breaks (DSBs), and gene modification (editing) is introduced through cellular DNA repair pathways.

Cellular DNA repair pathways mainly include: 1. End-joining pathways leading to short insertions or deletions (indels); 2. Homology-directed repair (HDR) using an exogenous DNA repair template for precise modifications.

The typical Cas9 protein derived from Streptococcus pyogenes is the first Cas nuclease to be re-used for genome editing, and it is still the most widely used gene editor due to its inherent high activity and specificity.

Cas12a is a Cas nuclease derived from the type V CRISPR-Cas system, discovered years after Cas9, and is also re-used for genome editing.

Cas9 consists of a dual-guide RNA composed of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) or single-guide RNA (sgRNA), while Cas12a only has crRNA.

The recognition of target DNA depends on the complementarity of the spacer sequence with the guide RNA and the presence of a protospacer adjacent motif (PAM) adjacent to the original spacer region. Cas9 recognizes NGG PAM, while Cas12a requires TTTV PAM.

02 Limitations and Solutions of CRISPR Genome Editing

The re-use of the CRISPR-Cas system as a simple and efficient programmable gene editing tool has greatly promoted basic and applied research in many fields, laying the foundation for the development of targeted gene therapy and various biotechnological applications.

However, the functional characteristics of highly evolved biological defense systems differ from the expected functions of precise genome editing tools. Therefore, the application potential of the first-generation CRISPR-based gene editing tools is limited by several key factors, mainly including specificity, targeting range, the need for reliance on endogenous DSB repair mechanisms to achieve genome editing, and the delivery of CRISPR components being restricted by delivery vectors, target cells, or organ tissues.

To address these limitations, researchers have adopted specific technical solutions:

• Specificity: By developing high-fidelity nuclease variants, chemically modified guide RNAs, and controlled expression of genome editors, the off-target activity of genome editors has been addressed.
• Targeting range: The NGG PAM sequence requirement of SpCas9 limits the range of genome sites that can be targeted. This problem is addressed by using Cas9 engineered variants with alternative or relaxed PAM requirements, other naturally derived Cas9 orthologs with alternative PAM requirements, and Cas12a enzymes.
• Control of editing outcomes: Various methods have been developed, including asymmetric or retained HDR repair templates, cell cycle synchronization, and NHEJ inhibitors, to enhance HDR efficiency and suppress the formation of indels through end-joining pathways. Second-generation techniques (such as Base or Prime editing) can introduce precise modifications independently of HDR.
• Delivery: Stably transporting genome editor components into cells requires vectors, and the technology of vectors has made great progress. Electroporation/nucleofection, lipid nanoparticles, and viral vectors facilitate the cellular delivery of genome editor components.

03 Development of CRISPR Editing Technology

Due to the limitations of CRISPR editing technology, especially concerns about the genetic toxicity of DSBs and the necessity of addressing the low efficiency of HDR, further development of second-generation CRISPR technology has been promoted. This technology mediates genome editing without relying on DSB formation and HDR, such as Base editors (BEs), Prime Editors (PEs), transcriptional regulation CRISPRi and CRISPRa, and RNA editing technology.

3.1 Base Editors (BEs)

Base editors use the fusion technology of Cas9 nickase (nCas9) and a nucleobase-modifying enzyme, composed of programmable DNA-binding proteins such as dCas nucleases or Transcription Activator-Like Effector (TALE) repeat arrays, fused with a deaminase, to convert one base to another base without the need for a double-strand break.

This method is particularly effective for introducing specific point mutations (A-to-G or C-to-T, as well as A-to-C or C-to-G), enabling precise gene correction or introduction of stop codons for precise gene knockout.

3.2 Prime Editor (PE)

Prime Editor (PE) has a prime editor (PE) protein, usually a nickase Cas9 (nCas9) fused with a reverse transcriptase (RT), and a guide RNA for Prime Editor (pegRNA), which contains single guide RNAs (sgRNAs) with a primer binding site (PBS) and a RT template at its 3′-end. It targets editing sites without introducing double-strand breaks (DSBs) and donor DNA templates, enabling the insertion, deletion, and replacement of sequences of up to several dozen nucleotides at the target site.

3.3 Transcriptional Regulation CRISPRi and CRISPRa

CRISPR technology is not only suitable for genome editing but also for transient manipulation of gene expression, such as CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) transcriptional regulation techniques, which control gene transcription by fusing deactivated Cas9 (dCas9) with transcriptional regulation domains (such as VP64 or KRAB) fused to the gene promoter through RNA guidance.

Cas9’s catalytic inactivation mutants were initially used to target gene promoters in bacteria and spatially block RNA polymerase, thereby inhibiting RNA transcription.

In eukaryotic cells, to inhibit gene expression, the nucleic acid inactivated Cas9 can be fused to various transcriptional and epigenetic modulators, such as the KRAB transcriptional silencer protein domain, and targeted to regions of active transcription genes.

The resulting method is called CRISPRi, which can effectively knock down gene expression, serving as an alternative to RNA interference based on small interfering RNA (siRNA).

CRISPRa is achieved by fusing dCas9 to an activation domain, such as VP64 and its derivatives. Similarly, the nucleic acid inactivated Cas9 protein fusion can be used to activate the expression of specific genes by directly recruiting transcriptional activators or regulating chromatin states.

3.4 RNA Editing Technology

Unlike genome editing, RNA editing technology utilizes the Cas13 nuclease targeted to RNA for targeted transcript degradation (when catalytically active) or for transcript editing (when catalytically inactive and fused to an adenosine deaminase).

04 Applications of CRISPR Editing Technology in Basic Research and Human Medicine

CRISPR technology has changed gene research by allowing scientists to simulate pathogenic mutations in various experimental models, create large-scale whole-genome screening methods, and develop synthetic gene recording devices for studying normal development and disease progression.

The technological advances of CRISPR genome editing in basic research and new therapies have brought many potentially transformative applications for human health (Figure 5), mainly including the following:

a. CRISPR-induced gene knockout or mutation: CRISPR nucleases, base editors, and primer editors help produce specific gene changes, including gene knockout, knockdown, and targeted mutations in cultured cell lines and animals, thus modeling human genetic disease mutations.

b. CRISPR screening: These applications involve high-throughput gene function analysis using a guide RNA library. Cells transduced with these libraries undergo selection-based assays, and next-generation sequencing is used to analyze the enrichment or depletion of guide RNAs in cell populations, identifying genes involved in specific biological processes. CRISPR screening can be performed using CRISPR nucleases, CRISPRi/a transcriptional regulators, as well as base and primer editors.

c. Ex vivo therapeutic gene editing: These therapeutic approaches involve editing cells from patients or healthy donors in a controlled laboratory environment, and then reintroducing the modified cells (re) into patients.

d. In vivo therapeutic genome editing: In contrast to ex vivo strategies, in vivo therapeutic genome editing involves directly applying CRISPR genome editors to affected tissues in the body through local or systemic delivery, targeting specific organs or tissues, often using methods based on lipid nanoparticles or viral vectors.

Many proof-of-concept preclinical studies have successfully demonstrated in vivo therapeutic genome editing, particularly efforts to restore dystrophin protein expression by muscle-directed delivery of AAV9 vector carrying Cas9 and guide RNA in a Duchenne muscular dystrophy animal model, and by AAV9-mediated delivery of CBE to the brain in a mouse model to rescue spinal muscular atrophy.

05 Emerging Technologies in Genome Editing

Over the past decade, the limitations of current CRISPR technology have become increasingly apparent, and new methods and strategies continue to be developed and refined to address these limitations and improve the efficacy and versatility of CRISPR-based genome editing.

5.1 Compact RNA-Guided Nucleases

These emerging third-generation tools and techniques (Figure 6) include recently discovered compact RNA-guided nucleases, which are applicable to DSB-based editing and can also be used for RNA-guided DNA binding platforms of other genome editors modes (such as BE and PE).

5.2 CRISPR-Guided Recombinases and Transposons

The development of CRISPR-guided recombinases and transposons shows potential, and new methods for editing RNA transcripts in genome editing technologies, such as DNA polymerase editors, engineered CRISPR integrases, target-induced reverse transcription, epigenetic editors, and artificial intelligence in gene editing, etc.

• DNA Polymerase Editors: This technology combines Cas9 nickase with DNA polymerase and a single-stranded DNA template, such as using the HUH endonuclease. A key difference from prime editing (PE) is that it uses DNA polymerase instead of reverse transcriptase and delivers the DNA template in a reverse manner.
• CRISPR-related transposons: These naturally occurring mobile genetic elements utilize the CRISPR effector complex to bind to transposase proteins for RNA-guided transposition, inserting long DNA sequences into specific genome sites.
• Engineered CRISPR integrases: These technologies are based on fusing guide editing with site-specific serine recombinases. Guided editing initially introduces the recombinase att site at the target DNA location, followed by recombinase-mediated insertion of large DNA payloads.
• Target-induced reverse transcription: This process involves fusing Cas9 nickase with a reverse transcriptase derived from a non-long terminal repeat (LTR) retrotransposon and RNA fusion of the transposon. It results in the insertion of targeted DNA into the genome by generating a primer for reverse transcription associated with the transposon-related RNA.
• Epigenetic Editors: By fusing a deactivated dCas9 with DNA methyltransferase and histone modification enzymes, targeted chromatin modifications can be made at specific genome locations, achieving heritable inhibition of gene expression (CRISPR off) without changing the underlying DNA sequence. Gene reactivation (CRISPR on) involves using Cas9 fused with DNA demethylase and transcriptional activator domain to target the suppressed gene.
• Artificial intelligence in gene editing: Significant progress has been made in protein and guide design from scratch, as well as in computational predictions of off-target sites and editing outcomes.

In Conclusion

Genome editing technology has undergone several generations of development, and currently, the CRISPR/Cas system is the most widely used. Although CRISPR genome editing has limitations, its future is bright. It not only has the potential to drive research breakthroughs and revolutionize human medicine but also has the potential to strengthen agriculture and address ecological issues, thus laying the foundation for a healthier and more sustainable future for future generations.

CRISPR Genome Editing: From Molecular Principles to Therapeutic Applications

References: CRISPR Genome Editing: From Molecular Principles to Therapeutic Applications

1.Wood, A.J., Lo, T.W., Zeitler, B., Pickle, C.S., Ralston, E.J., Lee, A.H., Amora, R., Miller, J.C., Leung, E., Meng, X., et al. (2011). Targeted Genome Editing Across Species Using ZFNs and TALENs. Science 333, 307.

2.Doudna, J.A., and Charpentier, E. (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096.

3.Martin Pacesa, Oana Pelea, and Martin Jinek, Past, present, and future of CRISPR genome editing technologies, Cell 187, February 29, 2024

4.Marek Marzec, Goetz Hensel, Prime Editing: Game Changer for Modifying Plant Genomes, Trends Plant Sci. 2020 Aug;25(8):722-724.

(source:internet, reference only)

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