Point mutations are a type of genetic mutation involving a change to a single nucleotide in the DNA or RNA sequence—typically a substitution of one nucleotide for another. The term can also broadly refer to the insertion or deletion of a single nucleotide. In a broader sense, point mutations include base substitutions, single-base insertions, or deletions; in the narrow sense, it refers specifically to single base substitutions. These substitutions are classified as either transitions or transversions.

Point mutations play a crucial role across various research fields. In basic scientific research, they are used to explore gene or protein functional domains by introducing targeted mutations. When studying disease mechanism studies and model development, point mutations help replicate driver mutations to investigate tumorigenesis and drug resistance. In drug discovery and screening, they are essential for testing drug sensitivity and identifying candidate compounds targeting specific mutations.

There are three widely used approaches for constructing point mutation line:

Base editing systems are generally categorized into two main types, depending on the fused deaminase enzyme: cytosine base editors (CBE) and adenine base editors (ABE).

CBEs employ a cytosine deaminase fused to a Cas9 nickase to convert cytosine (C) within a target region into uracil (U) without causing double-strand breaks (DSBs). During subsequent DNA repair or replication, this conversion ultimately results in a C→T (or occasionally C→G) substitution.

Similarly, ABEs use a fusion of adenine deaminase with Cas9 nickase, which, guided by sgRNA, binds to the target genomic DNA. The adenine deaminase then deaminates adenine (A) in single-stranded DNA, converting it to inosine (I), which is read as guanine (G) during replication—thereby achieving an A•T to G•C base pair conversion.

Advantages and Limitations of Base Editors:
Base editors do not rely on DSBs, leading to relatively high mutation efficiencies. However, due to the non-specific activity of deaminases, unintended mutations may occur near the PAM site (typically within 4–7 nucleotides). Additionally, base editors currently offer a limited range of possible nucleotide substitutions and cannot support arbitrary base changes.

1. RNP Method

In this approach, a ribonucleoprotein (RNP) complex is formed in vitro by combining gRNA and Cas9 protein. The RNP complex is then co-transfected into cells along with a single-stranded oligonucleotide (ssODN) donor template. The RNP precisely recognizes and cleaves the target genomic site, after which homology-directed repair (HDR) occurs using the ssODN template to introduce the desired point mutation. This method is broadly applicable, offers high editing efficiency, a streamlined workflow, and a short project turnaround time.

2. Plasmid with Selection Marker

This method involves co-transfecting cells with a plasmid that co-expresses gRNA and Cas9, along with a donor plasmid. The donor plasmid carries two homology arms flanking a selectable marker expression cassette flanked by loxP sites, with the desired point mutation encoded within the homology arms. This strategy is less restricted by the location of the target mutation and enables enrichment of positive clones through selection markers, thus improving the success rate.

3. AAV-Donor Method

In this approach, the donor template is delivered via an adeno-associated virus (AAV) vector to mediate HDR. The AAV genome is single-stranded DNA, which remains episomal and persists in the cell for an extended period, greatly enhancing HDR efficiency. This method is particularly well-suited for hard-to-transfect cell lines, such as suspension cells.

Prime Editing enables precise introduction of point mutations, as well as small insertions or deletions, without relying on double-strand breaks (DSBs) or exogenous donor DNA templates.

Basic Principle of Prime Editing:
First, the spacer region of the prime editing guide RNA (pegRNA) binds to the target DNA strand, positioning the pegRNA in complex with Cas9 nickase fused to reverse transcriptase (nCas9-RT) at the genomic locus. The nCas9 enzyme introduces a nick in the non-target strand. The primer binding site (PBS) on the pegRNA then anneals to the nicked DNA, and reverse transcriptase uses the reverse transcription template (RTT) within the pegRNA to synthesize a DNA strand containing the desired edit.

At this stage, two competing DNA sequences coexist at the nick site—one carrying the edited sequence and one retaining the original sequence. These sequences form a 3' or 5' flap. Through this competition, if the edited strand is incorporated into the target DNA strand, a mismatch is created, which is then resolved by the cellular mismatch repair system, ultimately resulting in either the desired edited sequence or the original sequence being retained.

Compared to base editors (BEs), Prime Editing offers a broader range of editing capabilities. While BEs are limited to four types of base transitions (such as C→T or A→G). Prime Editing can introduce all 12 possible base substitutions, as well as small insertions and deletions, with higher precision. Moreover, Prime Editing provides a clear advantage for applications requiring multi-base edits or targeting sites beyond the editable window of BEs.

Although traditional homology-directed repair (HDR) theoretically allows for any desired edit, it relies on double-strand breaks (DSBs), which often trigger non-homologous end joining (NHEJ), resulting in a high frequency of unintended insertions or deletions (indels). In contrast, Prime Editing operates without the need for DSBs, yielding cleaner editing outcomes with fewer byproducts—making it particularly well-suited for applications requiring highly precise genome editing.

Point mutations, as a crucial mutation type in genetic research, play an irreplaceable role in elucidating disease mechanisms and developing therapeutic strategies. This article has detailed the fundamental principles and respective advantages and disadvantages of three common methods for constructing point mutations, providing valuable references for related research.