Gene Tar getting

Gene Tar getting

What is the correct sequence for generating knockout mice?

Generation of knockout mice by ES cell method follows ordered protocol. Homology arms plus positive-negative selection markers are cloned into targeting vector containing floxed or null allele. Vector electroporated into cultured embryonic stem cells where homologous recombination occurs. G418 and ganciclovir selection enriches correctly targeted clones confirmed by Southern blot analysis. Targeted ES cells microinjected into blastocysts implanted into pseudopregnant foster mothers produce chimeric offspring. Chimeras bred for germline transmission yield heterozygous knockout founders, later intercrossed to homozygous knockout lines.

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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Which system has single base-pair specificity through protein-DNA interaction?

Zinc finger nucleases link three to six fingers each contacting three base pairs, giving triplet recognition with context dependent cross-talk limiting true single base resolution. CRISPR relies on RNA-DNA pairing with mismatch tolerance. TALENs use central domain of 33-35 amino acid repeats where residues 12 and 13 called repeat variable di-residues dictate single nucleotide specificity: NI binds A, HD binds C, NG binds T, NN binds G. Each repeat recognizes exactly one base pair independently, providing genuine single base pair resolution through protein-DNA interaction ideal for precise targeting without context dependence.

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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What is essential for inversion to happen in Cre-lox system?

In Cre-loxP system Cre binds 13 base pair inverted repeats surrounding 8 base pair asymmetric core of loxP. For inversion versus deletion decision, relative direction of core determines outcome. When core sequences point towards each other on same chromosome, meaning loxP sites in opposite orientation, Cre catalyzed recombination reverses intervening segment without loss. Direct repeats cause deletion. Thus requirement for inversion is antiparallel loxP orientation ensuring DNA strand exchanged leads to reversal rather than circular excision, employed in FLEX switches for reversible gene control.

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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In Cre-Off (DO) vectors, the gene is active before recombination and:

Cre-Off or double floxed inverted open reading frame systems express gene constitutively before recombination. Coding sequence flanked by incompatible lox sites in forward orientation remains active under promoter. Cre mediated inversion using opposite orientation loxP or lox2272 flips gene into antisense orientation, often followed by excision locking orientation, extinguishing transcription or producing non-functional transcript. Therefore activity before Cre allows labeling or function, after Cre signal terminates. This inversion dependent off switch enables Cre mediated silencing in specific neurons for circuit mapping.

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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What defines class 2 CRISPR systems?

CRISPR systems are classified into Class 1 with multi-protein effector complexes and Class 2 with single large effector protein mediating interference. Class 1 includes Types I, III, IV requiring Cascade complex for targeting. Class 2 includes Types II, V and VI containing Cas9, Cas12 and Cas13 respectively. Single effector architecture simplifies repurposing for genome editing since one protein plus guide RNA suffices. This distinction based on effector complexity explains why Cas9 dominates biotechnology despite natural diversity of CRISPR mechanisms across bacteria and archaea.

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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Which mutation type is created by NHEJ repair after Cas9 cut?

Cas9 induced double strand break activates cellular repair pathways. Homology directed repair uses donor template for precise correction but operates mainly in S phase. Predominant pathway in mammalian somatic cells is non-homologous end joining which directly ligates broken ends without template, often introducing small errors. Ku proteins and DNA ligase IV mediated NHEJ results in insertion or deletion of few nucleotides at cut site, causing frameshift or premature stop codon. This error-prone repair underlies knockout generation by CRISPR through disruptive indels.

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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The Cas9-based CRISPR mechanism in type II system uses:

Type II CRISPR system of Streptococcus pyogenes minimal interference module requires trans-activating crRNA tracrRNA. Pre-crRNA containing repeats is annealed to tracrRNA forming duplex recognized by RNase III for processing into mature guide RNAs. TracrRNA also hybridizes to crRNA to form dual guide structure that activates Cas9 conformational change enabling PAM scanning and DNA cleavage. Unlike type I requiring Cas3 helicase nuclease or type V using Cas12 alone, type II canonical system depends on tracrRNA for biogenesis and Cas9 loading, later fused as single guide RNA.

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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Which platform has lowest off-target mutation rate?

Off-target mutations determine safety for therapeutic genome editing. CRISPR tolerates mismatches distal to PAM generating relatively higher off-target cleavage. ZFNs and TALENs show lower tolerance due to longer protein-DNA interface and requirement for dimerization, yet still recognize near cognate sites. Homing endonucleases or meganucleases like I-SceI recognize 18-24 base pair asymmetric sequences, often exceeding 20 base pairs of specificity, resulting in rarity of cognate site in complex genome and extremely low off-target rate. Their long recognition site accounts for highest specificity among editing nucleases.

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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Which feature is common between TALENs and ZFNs?

Zinc finger nucleases and transcription activator-like effector nucleases are chimeric nucleases sharing cleavage module. Both fuse sequence specific DNA binding domain to non-specific FokI endonuclease catalytic domain from Flavobacterium okeanokoites. FokI must dimerize to cut DNA, so two ZFN or TALEN monomers bind opposite strands with spacer. DNA binding domains differ: zinc fingers versus TALE repeats, but nuclease domain identical. CRISPR instead uses Cas9 nuclease. Conservation of FokI domain explains similar requirements for FokI dimerization and spacing rules in both platforms.

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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Which genome editing platform uses protein-DNA interactions to determine specificity?

Genome editing specificity can derive from protein-DNA contacts versus RNA-DNA base pairing. CRISPR Cas9 relies on guide RNA pairing and PAM. RNA interference uses small RNA-mRNA complementarity. Zinc finger nucleases use engineered arrays of Cys2His2 zinc finger domains, each recognizing three nucleotides via alpha helix contacting major groove. Tandem fingers create specific protein-DNA interface fused to FokI nuclease. Thus ZFN specificity is entirely determined by protein side chain interactions with DNA bases, requiring modular assembly and selection to achieve desired target without RNA intermediate.

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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Cre-FLEX system allows for:

Engineered Cre dependent vectors utilize double floxed inverted orientation or FLEX switch. Wild type and mutant loxP pairs are arranged antiparallel flanking gene in inverted orientation. In absence of Cre, transcription proceeds in wrong orientation or cassette blocked. Upon Cre expression, sequential recombination events invert coding sequence into correct orientation then excise two lox sites locking it permanently active or inactive. This enables irreversible flipping. Such reversible flipping before locking allows tight Cre dependent control for optogenetics, chemogenetics and cell type specific gene expression manipulation.

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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Which recombinase is used for inversion and excision of DNA fragments in eukaryotes?

Several recombinases perform site-specific DNA rearrangements. RecA mediates homologous recombination, not site specific excision. Flp recognizes FRT sites in yeast system analogous to Cre. Cas9 is RNA guided nuclease, not recombinase. Cre from bacteriophage P1 is widely used in eukaryotes for both deletion when loxP sites are directly repeated and inversion when oppositely oriented. Its ability to function without cofactors, high efficiency in mammalian nuclei, and reversibility control via mutant lox variants makes Cre preferred for conditional knockouts, lineage tracing and chromosome engineering.

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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