Introduction to Cas9 and its significance

Cas9 is an RNA-guided DNA nuclease at the heart of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. This enzyme can be imagined as the molecular scissors in CRISPR systems, cutting double-stranded DNA at locations specified by guide RNA sequences. This prominent gene was adapted from Streptococcus pyogenes, a bacterial species that cuts snippets of viral DNA to disrupt the pathogenic functions of bacteriophages.¹ This mechanism was translated into gene editing applications by Doudna and Charpentier et al., which allowed its widespread use in pharmaceuticals, agriculture and industrial microbiology.²

This article explores the unique structure and mechanisms of Cas9 nuclease, its role in the CRISPR-Cas9 mechanism and advancements in achieving targeted gene editing.

Structure and function of the Cas9 protein

The Cas9 nuclease comprises a bilobed architecture of a recognition lobe (REC) and nuclease lobe (NUC), each with significant functional domains.

The REC lobe facilitates the binding between the guide RNA (gRNA) and the target DNA through its bridge helix and REC 1, 2 and 3 domains. While the bridge helix aids the connection between the lobes and gRNA recognition, the REC domains bind the gRNA and stabilize the gRNA-Cas9 complex.³

The NUC lobe contains the HNH and RuvC domains that act as DNA scissors, cutting the target and non-target DNA strands, respectively. Another component is the PAM-interacting domain (PI), which recognizes the protospacer adjacent motif (PAM) sequence near the target sequence, serving as a checkpoint to confirm the eligibility of the target DNA for cutting.³

The domains in these two lobes cooperate to enable gRNA to direct Cas9 nuclease towards its complementary DNA sequence, where the PAM sequence brings about conformational changes that activate the NUC domains to initiate cutting.

An additional element is required to help the CRISPR system translocate to the nucleus. In research applications, this is achieved by fusing short amino acid sequences called nuclear localization signals (NLS) into Cas9 to permit its nuclear import.

Mechanism of CRISPR-Cas9 genome editing

Role of guide RNAs

In bacterial CRISPR systems, gRNA consists of a CRISPR RNA (crRNA) containing ~20 nucleotides complementary to the target DNA and a trans-activating CRISPR RNA (tracrRNA) that stabilizes the crRNA-target DNA binding. The two components are fused in eukaryotic gene editing applications to form a synthetic single-guide RNA (sgRNA).

DNA target recognition via sgRNA and PAM sequence

As the complementary sgRNA directs Cas9 nuclease to the target DNA, it scans the genome to find the PAM sequence, typically 5'-NGG-3', where N can be any nucleotide.

Upon confirming the PAM sequence, Cas9 unravels the target DNA double helix to assess the complementarity between sgRNA and target DNA. Ideally, when no mismatches are found, Cas9 undergoes conformational changes to stabilize the DNA-RNA hybrid. Its nuclease domains become activated, ready for DNA cleavage.

DNA cleavage: creation of double-stranded breaks

The Cas9 NUC lobe domains cleave both strands of the DNA, with HNH cutting the strand complementary to the sgRNA and RuvC cutting the non-complementary strand. The result is a double-stranded break that triggers the host cell's innate DNA repair mechanisms.

Repair pathways: Non-homologous end joining (NHEJ) and homology-directed repair (HDR)

The subsequent DNA repair mechanism is what essentially brings about genome editing. Repair can occur in two ways:

1. Non-homologous end joining (NHEJ) rejoins DNA ends without a template, resulting in random insertions and deletions that disable gene function. This method is often leveraged for gene knockout applications.
2. Homology-directed repair (HDR) is a high-fidelity repair mechanism using a donor DNA template that informs precise gene editing. HDR is commonly used for gene knock-ins or corrections, particularly in treating genetic disorders.

Cas9 variants and precision engineering

Traditional Cas9-based CRISPR systems may face bottlenecks that impact site specificity and cutting efficiency, potentially resulting in off-target edits. Furthermore, the requirement of the NGG PAM site restricts the scope of target sites that CRISPR can aim at. Several Cas9 nuclease variants have been developed to overcome these challenges.

Hi-Fi Cas9: enhanced specificity and reduced off-targets

While wild-type Cas9 may tolerate mismatches between sgRNA and the target DNA, high-fidelity Cas9 variants contain synthetic mutations in their REC or NUC lobes, which make the nuclease less tolerant to mismatches. Hi-Fi Cas9 variants, such as SpCas9-HF1, eSpCas9(1.1) and HypaCas9, minimize off-target editing by preventing non-specific editing at sequences that show similarities to the target site.⁴

dCas9: catalytically inactive variant for gene regulation and epigenetic control

Dead Cas9 (dCas9) is a Cas9 nuclease variant containing HNH and RuvC mutations. While it can still bind to target DNA sequences, the mutations deactivate its cleavage capability. It is mainly used in CRISPR interference (CRISPRi) applications, where the dCas9 binds to a target sequence to repress its transcription through steric hindrance.⁵ Furthermore, it can modulate epigenetic enzyme activity to regulate gene expression.⁶

Nickase Cas9: single-strand cleavage for refined editing

Cas9 nickases (nCas9) have either RuvC or HNH (but not both) mutated, so the enzyme only cuts one DNA strand. Researchers utilize this technology using a nCas9 with a pair of gRNAs targeting opposite DNA strands, creating a staggered DSB rather than a blunt-ended one. Thus, nCas9 significantly improves specificity and reduces off-target editing.⁷

Design and use of high-fidelity variants

High-fidelity Cas9 variants are designed through structure-guided mutagenesis and evaluated by in vitro and in vivo screening methods to validate their specificity. Furthermore, they are fine-tuned in a context-dependent manner based on the genome type, the target site structure and the host cell type.

Enhancing genome editing efficiency and specificity

Successful applications of the CRISPR-Cas9 technology depend on meticulous design strategies that balance precision, efficiency and safety. Below are key considerations:

Importance of PAM site selection

PAM site selection is integral to the optimal positioning of Cas9. Therefore, an appropriate PAM site near the target sequence must be identified to ensure the target is accessible by the CRISPR-Cas9 system.

Guide RNA design considerations

Efficient and targeted gene editing requires effective sgRNA design. Key criteria include the following:

1. The sequence must not be complementary to regions similar to other genomic sites, especially near the PAM region⁸
2. It must be complementary to a sequence confirmed to have functional significance for the cell
3. The guanine-cytidine (GC) content must be optimal (~40-60%) to stabilize sgRNA-DNA binding⁹
4. When necessary, the sgRNA must be chemically modified to prevent secondary structures, such as hairpins, that may interfere with binding¹⁰

Techniques to reduce off-target activity

1. High-fidelity Cas9 variants
2. Truncated sgRNAs with 17-18 nucleotides instead of 20¹¹
3. Using paired Cas9 nickases to target opposite strands of the DNA7
4. Rational-design approaches to enhance stability and reduce rigidity¹²
5. Effective delivery methods to maintain the Cas9 presence in the cell optimal, preventing its excessive cleavage activity

Factors affecting editing efficiency

Other factors that affect editing efficiency include cell types and chromatin accessibility. Dividing cells yield better efficiency than quiescent or terminally differentiated cells, such as neurons.¹³ Furthermore, epigenetic modifications may alter the chromatin structure at the target site and prevent its accessibility.¹⁴

Cas9 delivery formats and lab protocols

The efficiency and safety of CRISPR-Cas9 systems heavily rely on seamless delivery systems.

Delivery options

Cas9 can be delivered into cells in the following formats:

1. Ribonucleoprotein (RNP) complex comprising the Cas9 protein and the sgRNA for transient expression
2. Cas9 mRNA co-delivery with sgRNA for transient expression
3. Plasma DNA encoding both Cas9 and sgRNA for prolonged expression
4. Lentiviral vectors for stable expression

Transfection methods

The following methods can be used for introducing CRISPR components directly into cells:

1. Electroporation: Uses electric pulses to permeabilize the cell membrane to allow uptake of sgRNA and Cas9
2. Lipofection: Delivery of Cas9 and sgRNA encapsulated in lipid nanoparticles
3. Microinjection: Direct injection into the cytoplasm or the nucleus RNP complex formation and delivery

Using an RNP complex ensures rapid genome editing action with no risk of genomic integration and reduced risks of off-target editing. It is prepared by pre-assembling and incubating the recombinant Cas9 protein with sgRNA.

Protocols for using recombinant Cas9 in laboratory settings

An RNP-based CRISPR-Cas9 workflow is conducted as follows:

1. sgRNA design
2. Preparation of the Cas9-sgRNA RNP complex
3. Delivery of RNP to cells
4. Incubation for 24–72 hours to allow editing and recovery
5. Evaluating editing efficiency using PCR, next-generation sequencing and quantitative cellular assays

Applications and Advances

Therapeutic gene editing and disease modelling

CRISPR-Cas9-based gene editing is diligently studied to develop treatments for various cancers and genetic disorders, including sickle cell disease, muscular dystrophy and β-thalassemia.¹⁵ Simultaneously, CRISPR knockout and knock-in studies aid drug discovery by disclosing the genetic perturbations underlying complex diseases.¹⁶

Innovations in Cas9 engineering

Cas9 engineering could address specificity and safety issues to broaden the therapeutic utility of CRISPR-Cas9. Proper sgRNA selection could be streamlined using CRISPR libraries and machine learning algorithms that predict on-target and off-target scores.¹⁷ Rational-design approaches and chemical modifications can augment target specificity, while Cas9 variants can be implemented to refine cutting precision.

Ethical and safety considerations in genome editing

Despite the possibilities CRISPR-Cas9 editing can bring in therapeutic settings, its power sparks ethical and safety concerns. The risks associated with off-target editing and resulting genomic instability require strict regulatory oversight. Furthermore, germline editing is highly debatable due to its implications for the genome in future generations.¹⁸ Perhaps the most important aspect of the discussion is to ensure equal access to CRISPR-Cas9 gene editing, especially among underprivileged populations with limited resources.

References

1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007;315(5819):1709-1712.
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. Allemailem KS, Alsahli MA, Almatroudi A, Alrumaihi F, Alkhaleefah FK, Rahmani AH, et al. Current updates of CRISPR/Cas9‐mediated genome editing and targeting within tumor cells: an innovative strategy of cancer management. Cancer Commun 2022;42(12):1257-1287.
4. Kovalev MA, Davletshin AI, Karpov DS. Engineering Cas9: Next generation of genomic editors. Appl Microbiol Biotechnol 2024;108(1):209.
5. Pandey H, Yadav B, Shah K, Kaur R, Choudhary D, Sharma N, et al. A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications. Protein Expr Purif 2024;220:106500.
6. Cai R, Lv R, Shi Xe, Yang G, Jin J. CRISPR/dCas9 tools: epigenetic mechanism and application in gene transcriptional regulation. Int J Mol Sci 2023;24(19):14865.
7. Asmamaw Mengstie M, Teshome Azezew M, Asmamaw Dejenie T, Teshome AA, Tadele Admasu F, Behaile Teklemariam A, et al. Recent advancements in reducing the off-target effect of CRISPR-Cas9 genome editing. Biol Targets Ther 2024:21-28.
8. Manghwar H, Li B, Ding X, Hussain A, Lindsey K, Zhang X, et al. CRISPR/Cas systems in genome editing: methodologies and tools for sgRNA design, off‐target evaluation, and strategies to mitigate off‐target effects. Adv Sci 2020;7(6):1902312.
9. 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.
10. Riesenberg S, Helmbrecht N, Kanis P, Maricic T, Pääbo S. Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage. Nat Commun 2022;13(1):489.
11. Chey YC, Gierus L, Lushington C, Arudkumar JC, Geiger A, Staker LG, et al. Enhancing gRNA Transcript levels by Reducing the Scaffold Poly-T Tract for Optimal SpCas9-and SaCas9-mediated Gene Editing. BMC Genomics 2025;26:138.
12. Liu X, Xiong W, Qi Q, Zhang Y, Ji H, Cui S, et al. Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing. Nucleic Acids Res 2022;50(8):4769-4783.
13. Javaid N, Choi S. CRISPR/Cas system and factors affecting its precision and efficiency. Front Cell Dev Biol 2021;9:761709.
14. Chen E, Lin-Shiao E, Trinidad M, Saffari Doost M, Colognori D, Doudna JA. Decorating chromatin for enhanced genome editing using CRISPR-Cas9. PNAS 2022;119(49):e2204259119.
15. Sharma G, Sharma AR, Bhattacharya M, Lee S-S, Chakraborty C. CRISPR-Cas9: a preclinical and clinical perspective for the treatment of human diseases. Mol Ther 2021;29(2):571-586.
16. Khurana A, Sayed N, Singh V, Khurana I, Allawadhi P, Rawat PS, et al. A comprehensive overview of CRISPR/Cas 9 technology and application thereof in drug discovery. J Cell Biochem 2022;123(10):1674-1698.
17. Ding S, Zheng J, Jia C. DeepMEns: an ensemble model for predicting sgRNA on-target activity based on multiple features. Brief Funct Genomics 2025;24:elae043.
18. 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.

recent-articles