Since its emergence in 2012, CRISPR technology has rapidly become the most advanced and efficient gene editing tool due to its superior performance, revolutionizing fields such as functional genomics, drug target screening, genetic disease therapy, cancer research, and crop breeding. In recognition of their groundbreaking contributions, scientists Emmanuelle Charpentier and Jennifer A. Doudna were awarded the Nobel Prize in Chemistry in 2020. This article provides an in-depth analysis of the core principles and mechanistic underpinnings of CRISPR technology.

Figure 1. Nobel Laureates in Chemistry 2020^[1]

1. Introduction to the CRISPR System

1.1 Composition of the CRISPR System

CRISPR is an acronym for "Clustered Regularly Interspaced Short Palindromic Repeats." The CRISPR system constitutes a natural defense mechanism in prokaryotes (including bacteria and archaea), evolved to protect against bacteriophage infections.

The CRISPR/Cas system consists of two primary components: the CRISPR genetic locus and the cas genes.

CRISPR Genetic Locus

The CRISPR locus is composed of three elements: the leader sequence, repeat sequences, and spacer sequences.

• Leader sequence: A several hundred base-pair-long AT-rich region located upstream of the CRISPR array, functioning as a promoter essential for transcription initiation.
• Spacer sequences: These are fragments of captured foreign DNA, serving as a "memory bank" for the CRISPR system. Upon re-invasion by homologous genetic material, the CRISPR/Cas system can precisely recognize and neutralize the threat.
• Repeat sequences: Typically 20–50 base pairs in length, containing 5–7 bp palindromic motifs that form hairpin structures upon transcription, thereby stabilizing the secondary structure of the RNA transcript. These repeats alternate with spacers and can number from a few to several hundred.

Cas Genes

Located adjacent to the CRISPR locus or dispersed throughout the genome, cas genes encode a variety of Cas proteins (e.g., Cas1–Cas10). These proteins play critical roles in the CRISPR/Cas system, working in concert with the CRISPR locus to form a robust defense mechanism against bacteriophage infection.

Figure 2. Schematic of the CRISPR Locus Structure^[2]

1.2 Classification of CRISPR Systems

CRISPR/Cas systems are categorized into two main classes—Class 1 and Class 2—based on differences in Cas protein composition and the nature of the effector complexes.

Class 1 Systems

These systems are characterized by a multi-protein effector module responsible for target genome recognition, inactivation of invading DNA, and processing of precursor CRISPR RNA (pre-crRNA).

Class 2 Systems

In contrast, Class 2 systems feature a single, multi-domain crRNA-binding protein, such as Cas9. This protein harbors all enzymatic activities required for interference, and in certain variants, also includes domains involved in pre-crRNA maturation.

Figure 3. Classification of CRISPR Systems^[3]

The cas genes in CRISPR/Cas systems can be functionally grouped into four overlapping modules: the adaptation module, expression and processing module, interference (or effector) module, and signaling or auxiliary module. These modules operate synergistically to enable genome editing capabilities.

• Adaptation module: Responsible for integrating new genetic information into the CRISPR array. It includes genes encoding Cas1 (essential for spacer integration) and Cas2 (a subunit of the adaptation complex), and may also involve Cas4 nuclease, Csn2 in type II-A systems, and reverse transcriptases in many type III systems.
• Expression and processing module: Governs pre-crRNA maturation. In most Class 1 systems, Cas6 directly mediates this process. In type II systems, bacterial RNase III (a non-Cas protein) is required. In many type V and all type VI systems, the large effector Cas protein contains an intrinsic catalytic domain for pre-crRNA processing.
• Interference or effector module: Mediates target recognition and nucleic acid cleavage. In Class 1 systems, this module comprises multiple Cas proteins (e.g., Cas3, Cas5–Cas8, Cas10, Cas11). In Class 2 systems, it is simplified to a single large protein, such as Cas9, Cas12, or Cas13.
• Signaling or auxiliary module: A broad collection of CRISPR-associated genes whose roles are largely predicted but not fully characterized.

Due to their simplicity and ease of use, research has primarily focused on Class 2 systems to identify novel tools for genome editing and molecular diagnostics. Class 2 CRISPR-Cas systems are further divided into three major types: type II (Cas9), type V (Cas12), and type VI (Cas13).

Type II Systems

The hallmark of type II systems is the CRISPR/Cas9 system. Cas1, Cas2, and Cas4 are involved in establishing the repeat-spacer array, while RNase III assists in crRNA maturation. The remaining functions are carried out by the Cas9 protein. Cas9 requires both tracrRNA and crRNA for activity: tracrRNA facilitates crRNA maturation and base-pairs with crRNA to form a tracrRNA:crRNA duplex that binds Cas9. The crRNA component directs sequence-specific binding to target DNA. Upon formation of the guide RNA–Cas9 complex, the two nuclease domains of Cas9—HNH and RuvC—become activated. The HNH domain cleaves the DNA strand complementary to the crRNA, while the RuvC domain cleaves the non-complementary strand, resulting in a double-strand break (DSB). This precise cleavage capability has established CRISPR/Cas9 as one of the most powerful and efficient tools in gene editing.

Type V Systems

Type V systems employ Cas12 as the single effector protein, exhibiting significant differences from type II systems. While type II systems utilize two nuclease domains (HNH and RuvC), type V systems possess only a single RuvC-like nuclease domain. In terms of guide RNA, Cas9 requires both tracrRNA and crRNA, whereas Cas12a functions with a single crRNA.

Notably, Cas9 generates blunt-ended DSBs, whereas Cas12a produces staggered (sticky) ends, a feature that expands its utility in gene editing and nucleic acid detection. Furthermore, systems such as Cas12a and Cas12b exhibit trans-cleavage activity. After sequence-specific recognition and cleavage of double-stranded DNA (dsDNA) guided by crRNA, Cas12a activates nonspecific single-stranded DNA (ssDNA) nuclease activity, enabling indiscriminate degradation of nearby ssDNA molecules. This property allows integration with technologies like LAMP, enhancing its application in diagnostic assays.

Figure 4. Major CRISPR/Cas Gene Editing Tools^[4]

Type VI Systems

Type VI systems utilize Cas13 as the single effector protein, capable of targeting and cleaving RNA. Cas13 contains two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, with the conserved RxxxxH motif serving as the catalytic site for its RNase activity. Cas13 can self-process pre-crRNA into mature crRNA using only a single crRNA guide. The crRNA-Cas13 complex then base-pairs with complementary target RNA, enabling cis-cleavage of the target transcript and thus achieving RNA knockdown. Additionally, Cas13 exhibits nonspecific trans-cleavage of surrounding RNA molecules, a feature that holds significant promise for RNA regulation and diagnostic applications.

2. Mechanism of Action of CRISPR/Cas Systems

The operation of CRISPR/Cas systems can be divided into three sequential phases:

• Acquisition of Variable Spacers

Upon invasion by foreign nucleic acids (e.g., phages or plasmids), CRISPR-containing bacteria and archaea integrate short fragments of exogenous DNA into their CRISPR array as new spacers.

• Expression of the CRISPR Locus

When homologous foreign nucleic acids re-enter the cell, the CRISPR array is transcribed into precursor crRNA (pre-crRNA). Concurrently, tracrRNA, which is complementary to pre-crRNA, is also transcribed. The tracrRNA first binds to Cas9 and then hybridizes with pre-crRNA via base pairing, forming a double-stranded RNA structure. This dsRNA associates with Cas9 to form a functional complex. In the presence of RNase III, pre-crRNA undergoes primary and secondary processing, during which redundant repeat and spacer sequences are removed, yielding mature crRNA capable of guiding sequence-specific targeting.

• Effector Phase (Target Interference)

Upon re-infection by homologous DNA, the CRISPR locus is transcribed, and after processing, a single-guide RNA (sgRNA) is generated. The sgRNA guides the Cas9 protein to precisely cleave the homologous DNA sequence, resulting in a double-strand break (DSB). The cell subsequently repairs the break via either non-homologous end joining (NHEJ) or homology-directed repair (HDR). This targeted interference mechanism effectively neutralizes invading genetic material.

Figure 5. CRISPR-Cas9-Mediated DNA Interference in Bacterial Adaptive Immunity^[5]

3. Principles of Genome Editing Using the CRISPR/Cas System (CRISPR/Cas9)

The CRISPR/Cas9 gene editing system relies on two core components: a guide RNA (gRNA) with targeting capability and the Cas9 protein. The underlying mechanism involves two fundamental processes: gRNA-guided Cas9-mediated DNA cleavage and subsequent DNA repair.

3.1 Cas9-Mediated Targeted DNA Cleavage

During targeted DNA cleavage, the CRISPR system identifies specific genomic loci through the combined action of crRNA (for sequence recognition) and tracrRNA (as a scaffold for Cas9 binding). When these are fused into a single-guide RNA (sgRNA), the sgRNA forms a complex with Cas9. This complex is guided to the target site via 20-nucleotide complementarity between the sgRNA and the genomic DNA. Cleavage occurs three nucleotides upstream of the protospacer adjacent motif (PAM), a short GC-rich sequence (e.g., 5′-NGG-3′) essential for target recognition. Cas9 contains two key nuclease domains: the HNH domain, which cleaves the crRNA-complementary DNA strand, and the RuvC-like domain, which cleaves the non-complementary strand. Cleavage takes place between the third and fourth nucleotides upstream of the PAM, generating a blunt-ended DSB. Once the Cas9/sgRNA complex induces a DSB, the cellular repair machinery is activated, enabling precise modulation of gene expression.

Figure 6. Mechanism of CRISPR-Cas9-Mediated Gene Editing^[6]

3.2 DNA Repair Pathways

1) Non-Homologous End Joining (NHEJ)

NHEJ is the predominant pathway for repairing DSBs. It involves end resection by nucleases and gap filling by DNA polymerases. However, this process is error-prone and often results in small insertions or deletions (indels) at the repair site. When such indels occur within protein-coding regions, they can disrupt the reading frame, leading to premature termination of translation and loss of protein function. This principle is widely exploited in gene knockout experiments. For instance, gRNAs are typically designed within exons near the translation start site to maximize the likelihood of frameshift mutations that abolish gene function. In contrast, indels in non-coding regions generally have minimal phenotypic consequences.

2）Homology-Directed Repair (HDR)

HDR is a high-fidelity repair mechanism that relies on homologous recombination between similar or identical DNA sequences. A key advantage of HDR is its precision—repair occurs without introducing unintended mutations. For example, to generate disease-relevant mutation models, researchers can introduce an exogenous DNA template containing the desired mutation. Following Cas9-induced cleavage, the cell can use this template via HDR to incorporate the mutation accurately into the genome.

CRISPR/Cas technology, as a representative of the next-generation gene editing platforms, holds immense potential across diverse scientific and therapeutic domains. In this article, we have elucidated the fundamental architecture and editing mechanisms of the CRISPR/Cas system. In subsequent articles, we will explore delivery strategies and applications of CRISPR/Cas technology in greater depth—stay tuned.

Ordering Information

Application	Product Type	Product Name	Catalog Number
General-purpose	SpCas9 with NLS	Cas9 Nuclease	14701ES
Compact size for delivery	Cas12a	ArCas12a Nuclease	14702ES
	Cas12b	AapCas12b Nuclease	14808ES
sgRNA preparation	sgRNA synthesis	Hifair® Precision sgRNA Synthesis Kit	11355ES
	sgRNA purification	Hieff NGS® RNA Cleaner	12602ES

References

[1] The Nobel Prize in Chemistry 2020, Retrieved October 7, 2020, from https://www.nobelprize.org/prizes/chemistry/

[2] Marraffini LA, Sontheimer EJ. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet. 2010;11(3):181-190.

[3] Makarova KS, Wolf YI, Iranzo J, et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020;18(2):67-83.

[4] Pickar-Oliver A, Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol. 2019;20(8):490-507.

[5] Jiang F, Doudna JA. CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys. 2017;46:505-529.

[6] Westermann L, Neubauer B, Köttgen M. Nobel Prize 2020 in Chemistry honors CRISPR: a tool for rewriting the code of life. Pflugers Arch. 2021 Jan;473(1):1-2.

Extended Reading

CRISPR Gene Editing Product Selection Guide

CRISPR/Cas Systems: Biological Formats and Delivery Strategies for Genome Editing