Mouse transgenesis: Redefinition with CRISPR - Tutorial 5 by Yap Wei Hsum . https://padlet.com/weihsumyap1/buo6xtblb5m8 BIO61504 Animal Biotechnology Topic 4 discussion paper (tutorial) - Compare conventional site directed homologous recombination, Easi-CRISPR, and i-GONAD in terms of their methodology in creating transgenic mouse en-us 2016-06-09 13:44:11 UTC 2025-12-20 06:10:25 UTC hello@padlet.com Group 1 https://padlet.com/weihsumyap1/buo6xtblb5m8/wish/2379701862 Homologous recombinant: A cloning procedure is undertaken to insert the construct into a plasmid vector as a template to replace the endogenous locus. This template could be a drug-selection cassette only (knockout) or an exon flanked with two loxP sites, or a more complex feature (knock in). These vectors contain a positive and negative selection cassette. The plasmid is then electroporated into the ES cells and then drug selected in vitro. After verification that the sequence is correctly inserted, the cells are microinjected into a blastocyst, before being surgically transferred into pseudopregnant females. The chimeric progenies will be genotyped to ensure the expected construct is correctly inserted into the genome by homologous recombination.

Easi-CRISPR: Involves targeting by two sgRNAs which flank the endogenous exon and are complexed with Cas9 to form a ribonucleoprotein complex for cellular delivery. The exon is replaced after the DSB of the DNA and repaired with a long-stranded oligonucleotide template containing two loxP sites and spanning the entire exon.

i-GONAD: in situ delivery of the CRISPR/Cas9 reagents to the mouse oviduct by electroporation to allow Cas9 protein access to the zygote DNA to edit the genome. Generation of complex alleles using improved-genome editing via oviductal nucleic acid delivery (i-GONAD) technology. One or two single guide RNAs (sgRNA) are designed to either disrupt a critical exon (knockout) or remove an entire exon for replacement with a repair template (knock in). The sgRNAs
are synthesized, or in vitro transcribed, and then complexed with the tracrRNA and then Cas9 protein to form a ribonucleoprotein (RNP) complex. The RNPs are in situ electroporated with a long single-stranded oligonucleotide repair template (ssODN) into the oviduct of a pregnant female. The progenies are genotyped to ascertain successful editing of the gene of interest.]]> 2022-11-11 04:21:33 UTC https://padlet.com/weihsumyap1/buo6xtblb5m8/wish/2379701862 Group 2 amirahfaudzi02 https://padlet.com/weihsumyap1/buo6xtblb5m8/wish/2379705576 conventional site directed homologous recombination:
Their method necessitates the culture and genetic modification of mouse embryonic stem cells by homologous recombination, with selection cassettes replacing a critical exon for a knockout allele, or two loxP sites flanking a critical exon in addition to the selection cassettes for a knockin allele, being a routine strategy for allelic replacement. The genetically modified embryonic stem cells are then drug selected and microinjected into mouse blastocysts. The microinjected
blastocysts are finally implanted into pseudopregnant females by surgical transfer.

Easi-CRISPR:
- improve the generation of conditional alleles in mice using programmable nucleases.
- repair DNA after DSB is higher for the homology-directed repair pathway than homologous recombination,
- the delivery of a longer repair template would result in a higher efficiency for generating mutant alleles.
- involves targeting by two sgRNAs which flank the endogenous exon and are complexed with Cas9 to form a ribonucleoprotein complex for cellular delivery.
- The exon is replaced after the DSB of the DNA and repaired with a long-stranded oligonucleotide template containing two loxP sites and spanning the entire exon.

i-GONAD:
Generation of complex alleles using improved-genome editing via oviductal nucleic acid delivery (i-GONAD) technology. One or two single guide RNAs (sgRNA) are designed to either disrupt a critical exon (knockout) or remove an entire exon for replacement with a repair template (knockin). The sgRNAs are synthesized, or in vitro transcribed, and then complexed with the tracrRNA and then Cas9 protein to form a ribonucleoprotein (RNP) complex. The RNPs are in situ electroporated with a long single-stranded oligonucleotide repair template (ssODN) into the oviduct of a pregnant female. The progenies are genotyped to ascertain successful editing of the gene of interest]]> 2022-11-11 04:26:23 UTC https://padlet.com/weihsumyap1/buo6xtblb5m8/wish/2379705576 G3 https://padlet.com/weihsumyap1/buo6xtblb5m8/wish/2379712513
Conventional site directed homologous recombinant:


  • rapid production of knockout
  • conditional alleles or mice carrying single point mutations
  • the generation of more complex alleles is more challenging and requires at least two sgRNAs and a repair template in the form of two short single-stranded DNA (ssDNA) repair templates each containing a loxP site
  • the process of generating these conditional alleles using programmable nucleases remains lengthy and relatively inefficient (no recombination and mutation)


 


Easi-CRISPR:


  • Involves targeting by two sgRNAs which flank the endogenous exon and are complexed with Cas9 to form a ribonucleoprotein complex for cellular delivery
  •  High efficiency in editing and allelic replacement, averaging a 50% success rate, and up to 100% editing for certain alleles
  • Require highly trained staff and the use of an expensive microinjection apparatus



i-GONAD:


  • Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female


  • delivers genome editing nucleic acids and CRISPR components into embryos in situ. 
  • CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. 
  •  generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. 
     it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills.

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