Gene editing modify specific sites within the genome through gene deletions, insertions or conversions to study functionally unknown genes or conduct gene therapy. It is also used to change the biological traits of organisms to establish new varieties. Gene editing techniques include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9) (CRISPR/Cas9).
Gene Editing
Gene editing technology is a set of advanced molecular biology tools that directly modify the genome of an organism, such as the CRISPR-Cas9 system. Through gene editing, genetic information of organisms can be selectively inserted, deleted, or modified to achieve precise control of gene expression. Gene editing technology also raises ethical and safety issues, so scientists and policymakers need to carefully weigh and regulate its use in practical applications.
Related Experimental Schemes
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing gene transcription or activating RNA degradation. This mechanism was discovered in plants in 1998 by Andrew Fire and Craig Mello. Today, this phenomenon can be observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals.
CRISPR-Cas9 knockout in cultured mammalian cells uses an sgRNA to direct Cas9 to a complementary genomic sequence adjacent to a compatible PAM; Cas9 creates a targeted DNA double-strand break, and repair by non-homologous end joining can introduce insertions or deletions that disrupt the coding sequence or functional genomic element. The readout of knockout is detection of edited alleles and loss of gene product or phenotype, commonly by PCR/Sanger-sequence trace decomposition, targeted sequencing, immunoblotting, immunostaining, or flow cytometry when the target protein is detectable at the cell surface.
Base editing introduces targeted single-base substitutions without intentionally generating double-stranded DNA breaks or requiring donor DNA templates; cytosine base editors mainly mediate C•G-to-T•A changes, while adenine base editors mediate A•T-to-G•C changes. The readout is the fraction of sequencing reads carrying the intended base conversion at the target site, together with bystander edits, indels, and off-target edits when measured.
Prime editing is a CRISPR-derived genome editing method that uses a Cas9 H840A nickase fused to an engineered M-MLV reverse transcriptase and a prime-editing guide RNA (pegRNA), where the pegRNA specifies the genomic target through its spacer and encodes the desired edit through a primer-binding site and reverse-transcription template. The editor nicks one DNA strand, the exposed 3' DNA end hybridizes to the pegRNA primer-binding site, reverse transcription copies the edited sequence into a 3' flap, and cellular DNA repair resolves the intermediate to generate substitutions, small insertions, or small deletions without requiring a donor DNA template or a programmed double-strand break. The standard mammalian workflow designs candidate pegRNAs and, when using PE3 or PE3b, an additional nicking sgRNA; delivers prime-editor and guide components into cells; allows editing to occur; extracts genomic DNA; amplifies the target locus; and quantifies precise edits, indels, and byproducts by amp
CRISPRi and CRISPRa use catalytically inactive Cas9, typically SpCas9 D10A/H840A, as an RNA-guided DNA-binding platform that targets genomic loci through sgRNA complementarity and an adjacent PAM without generating Cas9 nuclease-mediated DNA cleavage. CRISPRi represses transcription by recruiting dCas9 alone or dCas9 fused to repressor domains such as KRAB to promoters or transcription start site regions, while CRISPRa activates transcription by recruiting activation domains such as VP64, VPR, or SAM components to promoter-proximal regions. The primary readout is target-gene expression change, commonly measured by RT-qPCR, RNA-seq, reporter fluorescence, or protein-level assays, and the readout reflects transcriptional repression or activation at the targeted endogenous locus.
CRISPR-Cas9 mouse zygote editing introduces Cas9 nuclease and guide RNA into one-cell embryos so that Cas9 creates a guide-directed double-strand break at the target locus; repair by non-homologous end joining can generate indels, while repair with an added donor template can generate defined knock-in or point-mutation alleles. The readout is embryo, pup, or founder genotype, usually assessed by PCR, restriction-fragment analysis, Sanger sequencing, TIDE/sequence-trace analysis, or targeted sequencing; successful editing is interpreted as the presence of indels, intended HDR alleles, or both at the target locus.
CRISPR-Cas9 editing of human pluripotent stem cells uses a guide RNA to direct Cas9 to a genomic target, where Cas9 creates a double-strand break that is repaired mainly by non-homologous end joining for knockout mutations or by homology-directed repair when a donor template is supplied for precise knock-in or sequence correction. The readout is generated by genotyping edited bulk populations or single-cell-derived clones, using PCR, sequencing, restriction-based assays, reporter fluorescence, or allele-specific analysis to distinguish unedited alleles, indels, precise donor-mediated edits, biallelic deletions, and unwanted on-target lesions.
CRISPR-Cas9 RNP editing uses preassembled Cas9 protein and guide RNA to direct sequence-specific DNA cleavage at a genomic target, after which cellular DNA repair generates insertions/deletions for knockout or uses an added donor template for knock-in. In primary immune cells, published protocols deliver Cas9 RNPs mainly by electroporation or nucleofection because these methods can introduce protein-RNA complexes into difficult-to-transfect T cells, B cells, NK cells, monocytes, myeloid cells, and innate lymphoid cells without viral Cas9 expression.
CRISPR-Cas9 HDR knock-in uses a guide RNA to direct Cas9 to a genomic target adjacent to a PAM, where Cas9 creates a double-strand break; if a donor DNA template with homology to the cut region is present, cellular HDR can copy the donor sequence into the genome, producing a precise substitution, tag, reporter, or insertion rather than an indel. The readout is the fraction of alleles or cells carrying the intended donor-derived edit, measured by junction PCR, restriction-fragment analysis, Sanger sequencing, amplicon deep sequencing, flow cytometry for reporter knock-in, or clone genotyping; NHEJ indels and partial or non-HDR insertions are measured in parallel because they compete with or confound precise HDR outcomes.
CRISPR-Cas9 zebrafish embryo editing introduces targeted double-strand breaks in genomic DNA by delivering Cas9 nuclease with a guide RNA into one-cell-stage embryos; repair by endogenous DNA-repair pathways produces indels or donor-mediated insertions that can be detected by phenotype, PCR-based genotyping, heteroduplex assays, Sanger sequencing, or amplicon sequencing. The readout reflects the frequency and spectrum of edited alleles in mosaic F0 embryos or transmitted F1 animals; because injected embryos can carry multiple alleles, founder screening and sequence confirmation are required before establishing stable mutant lines.
