C14-490

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).

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

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.

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.