SF2312

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).

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.

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.

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.

Constitutive germline knockout models are generated by producing a heritable loss-of-function allele in the mouse genome, typically through complete gene disruption in embryonic stem (ES) cells followed by germline transmission or through CRISPR/Cas-mediated editing of zygotes, resulting in offspring that carry a stable null allele in all tissues. Classical approaches rely on homologous recombination in ES cells to introduce targeted gene disruptions, which are then transmitted through chimeric mice to the germline. More recent genome editing strategies use CRISPR/Cas systems to induce double-strand breaks and non-homologous end joining (NHEJ), frequently generating frameshift mutations that abolish gene function, enabling faster generation of knockout alleles directly in embryos. Germline transmission or direct germline editing ensures that the mutation is present in all cells of the resulting animal, allowing systemic functional analysis of gene loss.

Gram staining of tissue sections is a histochemical technique used to differentiate Gram-positive and Gram-negative bacteria within histological specimens based on differences in bacterial cell wall structure and dye retention, adapted from classical bacteriological Gram staining into tissue-compatible “histological Gram stain” variants. In tissue applications, modifications of the Brown-Hopps and Brown-Brenn methods are commonly used to improve differentiation of microorganisms embedded within host connective tissue and to reduce overstaining or loss of Gram-negative signal, which are known limitations of earlier approaches. The principle relies on crystal violet-iodine complex retention in Gram-positive organisms and subsequent decolorization and counterstaining steps that allow contrast visualization of Gram-negative organisms against tissue background.

CRISPR/Cas9 knockout animal modeling uses guide RNA to direct Cas9 to a genomic target, where Cas9 creates a DNA double-strand break; repair by error-prone non-homologous end joining generates insertions or deletions that can disrupt coding sequence and produce knockout alleles. Classic animal-model workflows deliver Cas9 mRNA or Cas9 protein with sgRNA into fertilized zygotes by microinjection or electroporation, then transfer edited embryos into pseudopregnant recipients and genotype founders for target-site mutations.