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#gene editing

9 public questions tagged with this topic.

Transgenic Plant -2

Zinc finger nucleases (ZFNs) consist of:

Zinc finger nucleases represent first generation programmable nucleases fusing custom DNA recognition to cleavage effector. Each ZFN monomer architecture includes tandem array of Cys2-His2 zinc finger motifs originally derived from transcription factor Zif268 and Sp1, each finger ~30 amino acids folded into beta-beta-alpha structure where Zn2+ tetrahedrally coordinated by two cysteines in beta hairpin and two histidines in alpha helix stabilizing domain. Recognition helix positions -1 to 6 contact major groove bases via hydrogen bonds and van der Waals contacts. Array of three to six fingers recognizes 9 to 18 base pairs contiguously. C-terminal FokI endonuclease domain from Flavobacterium okeanokoites comprises nonspecific cleavage domain 196 amino acids requiring dimerization for double strand break formation leaving 4 bp 5' overhangs. Flexible linker GSGGS between finger array and FokI permits optimal spacing. Pair of ZFNs binding opposite strands tail-to-tail spaced 5-7 bp allows FokI dimer formation cutting intervening sequence, creating DSB stimulating homologous recombination or mutagenic NHEJ for targeted knockout or knockin applications in crops and human therapeutics for CCR5 editing and hemophilia correction.

Ref: Kim et al. PNAS 1996 ZFN; Carroll Annu Rev Biochem 2014 ZFN; Chandrasegaran 2016.

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Transgenic Plant -2

Gene subtraction strategy in plants mainly uses:

Genetic subtraction seeks to downregulate undesirable endogenous functions rather than introduce novel enzymatic activity. Molecular tools include antisense RNA where transgene produces reverse complement transcript hybridizing to target mRNA, and RNA interference where hairpin construct generates double-stranded RNA processed by Dicer into small interfering RNAs that guide Argonaute mediated cleavage of complementary mRNA. These approaches produce knockdown phenotype resembling loss-of-function mutant, allowing functional genomics and trait improvement. Compared to gene addition that confers insect or herbicide resistance via foreign protein production, subtraction modifies quality traits by blocking enzymes causing softening, browning, or allergen accumulation. Construct design requires partial gene fragment in antisense orientation under strong promoter, no new coding capacity needed. Efficiency depends on target mRNA abundance, siRNA accessibility, and avoidance of off-target effects. Therefore antisense RNA and RNAi constitute central technologies enabling gene subtraction strategy in transgenic plants for postharvest quality and metabolic engineering. Applications include non-browning potatoes reducing acrylamide formation during frying by silencing vacuolar invertase and asparagine synthetase. Systemic spread of silencing signal mediated by small RNAs enhances efficiency but may cause off-target effects in related gene families requiring careful construct design and bioinformatic analysis to ensure specificity of gene subtraction approach.

Ref: Watson Molecular Biology Gene 7th ed antisense RNAi; Fire Nature 1998 RNAi; NCBI NBK21471 silencing subtraction; Hannon Nature 2002 mechanism.

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Gene Therapy

CRISPR, ZFN and TALEN are used in gene therapy for:

CRISPR-Cas9, ZFN, TALEN constitute programmable site-specific endonucleases enabling precise genome manipulation. CRISPR uses single-guide RNA 20 nt spacer complementary to genomic target adjacent NGG PAM recognized Streptococcus pyogenes Cas9 possessing HNH domain cleaving complementary strand RuvC noncomplementary generating blunt double-strand break 3 bp upstream PAM. Zinc-finger arrays modules each recognizing 3 bp triplets linked FokI nuclease dimerize cutting within spacer, while TALEN repeats repeat-variable di-residues HD recognizing cytosine NI adenine NG thymine NN guanine provide single-base flexibility. Cellular repair via error-prone nonhomologous end-joining introduces indels knocking out CCR5 for HIV resistance, while homology-directed repair donor template 400 bp homology arms flanking break precisely corrects sickle HBB glutamate 6 valine mutation or inserts therapeutic transgene into safe harbor AAVS1 chromosome 19 constitutive expression under endogenous promoter. Permanent genomic alteration distinguishes editing from transient augmentation offering curative potential single intervention approved exagamglogene autotemcel for sickle cell. This mechanistic insight guides vector optimization, dosing strategies, and clinical safety monitoring essential for translational development and regulatory evaluation.

Ref: NIH Genome Editing CRISPR ZFN TALEN Overview; Nature Biotech Comparison 2020; NCBI Gene Editing Therapy https://www.ncbi.nlm.nih.gov/books/NBK542207/.

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DNA-Protein Interaction analysis

Site-directed mutagenesis helps in:

Site-directed mutagenesis provides powerful reverse genetics approach to determine functionally critical residues within protein. By changing specific codon to encode alternate amino acid, mutant protein can be tested for loss of enzymatic catalysis, DNA binding, protein-protein interaction or stability. Comparing activity of wild-type versus mutant variants through biochemical assays or complementation studies identifies residues essential for active site geometry, allostery or post-translational modification. This targeted approach reveals structure-function relationships, validates computational predictions and guides drug design, unlike random fragmentation, exon mapping or simple sequencing applications.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Tar getting

CRISPR interference phase involves:

Bacterial CRISPR adaptive immunity comprises three functional phases. Adaptation involves spacer acquisition by Cas1-Cas2. Expression involves transcription of CRISPR array into pre-crRNA processed into mature crRNAs with tracrRNA assistance. Interference phase represents actual defense where effector complex loaded with crRNA surveys invading DNA for complementary protospacer adjacent to PAM and mediates degradation. In type II systems Cas9 itself cleaves target DNA three base pairs upstream of PAM, generating double strand breaks, thereby neutralizing bacteriophage replication during interference phase efficiently and rapidly.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Tar getting

Which condition ensures correct homologous recombination?

Positive-negative selection strategy identifies homologous recombination. Neomycin resistance inside homology arms provides positive selection; only vector integrated cells survive G418. HSV-tk outside arms provides negative selection; random integrants retain tk and convert ganciclovir to toxic triphosphate. True homologous recombinants lose tk through double crossover. Ideal phenotype is G418 resistance due to neo inclusion and inability to grow under ganciclovir pressure indicating tk presence elimination, often reported as G418 resistant and ganciclovir sensitive in screening nomenclature, allowing enrichment of targeted embryonic stem cells.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Tar getting

What would result from placing loxP sites flanking exon 2 of a gene in same orientation?

Placing loxP sites in same orientation flanking exon 2 and intronic regions preserves gene function until Cre acts. Cre recombinase synapses two directly repeated loxP sites, forming a loop and catalyzing strand exchange. Recombination excises the looped DNA as circular molecule containing exon 2 and one loxP site, leaving single loxP scar in chromosome. Loss of coding exon causes frameshift mediated nonsense or deletion of essential domain, achieving conditional knockout after tissue specific Cre activation without affecting other tissues developmentally.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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