Q.53 Which of the following molecular approaches can be used to generate complete
knock–out of a target gene in plants?
(A) Homologous recombination
(B) CRISPR–Cas9
(C) Antisense RNA technique
(D) Activation tagging

CRISPR-Cas9 is the correct answer (B) for generating complete knock-out of a target gene in plants. This technique creates precise double-strand breaks repaired by error-prone mechanisms, leading to frameshift mutations that fully disrupt gene function. Homologous recombination can also achieve this but is far less efficient in plants compared to CRISPR-Cas9.​

Option Analysis

Homologous Recombination (A): This method replaces or disrupts a target gene through precise DNA integration using homologous sequences. While demonstrated in plants like tobacco, its low efficiency (e.g., 7/51 transgenics in early studies) makes it impractical for routine use.​

CRISPR-Cas9 (B): Guide RNA directs Cas9 nuclease to create targeted double-strand breaks, repaired via non-homologous end joining (NHEJ) to introduce insertions/deletions that cause complete loss-of-function. Widely applied in crops like tomato, melon, and Arabidopsis with high efficiency (up to 45% mutation rate).​

Antisense RNA Technique (C): Expresses complementary RNA to hybridize with target mRNA, blocking translation or triggering degradation via RNA interference. This achieves knockdown (partial reduction) rather than genomic knockout, as DNA remains intact.​

Activation Tagging (D): T-DNA with strong enhancers randomly inserts near genes to overexpress them, producing gain-of-function phenotypes. It does not knock out genes but activates them.​

CRISPR-Cas9 revolutionized complete knock-out of target gene in plants by enabling precise genomic edits for functional studies and crop improvement. This SEO-optimized guide covers molecular approaches for complete knock-out target gene plants, tailored for CSIR NET life sciences preparation.

Why Complete Knock-Out Matters

Complete gene knock-out permanently disrupts target gene function at the DNA level, unlike transient knockdown methods. Essential for studying gene roles in plant metabolism, stress response, and development.​

CRISPR-Cas9: Gold Standard Method

CRISPR-Cas9 uses sgRNA to guide Cas9 for targeted cuts, triggering NHEJ repairs that yield frameshifts. Achieves 39-71% efficiency in tomato and melon protoplasts/plants, producing stable null mutants.​

Homologous Recombination Limitations

Relies on homology-directed repair for gene replacement/disruption but faces low HR rates in plants (e.g., rare successes in Arabidopsis). Supplanted by CRISPR for practicality.​

Non-Knockout Alternatives

• Antisense RNA: Post-transcriptional silencing via mRNA degradation; incomplete knockout.​

• Activation tagging: Enhances gene expression; opposite of knock-out.​

CRISPR-Cas9 dominates for complete knock-out target gene plants in modern biotechnology.