Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse

Abstract

Graphical abstract

Tgfbr1 controls developmental plasticity between the hindlimb and external genitalia by remodeling their regulatory landscape

“…For the conditional Tgfbr1 inactivation approach we used three Tgfbr1 alleles, namely the Tgfbr1 null allele 37 and either Tgfbr1 flox , carrying LoxP sites flanking Exon 3 52 , or Tgfbr1 3 ex 3 -flox , containing a triplicated Exon 3 each surrounded by LoxP sites (Supplementary Fig. 1 ).…”

Tgfbr1 controls developmental plasticity between the hindlimb and external genitalia by remodeling their regulatory landscape

“…Generation of the Tgfbr1 Flox mouse lines was previously reported 48 . Tgfbr1 3ex3-flox resulted from the same experiments in which Tgfbr1 flox was generated, identified upon sequencing through the Tgfbr1 locus, which revealed that the exon 3 had been triplicated and the presence of LoxP sites in the introns between the triplicated exons and in those connecting them with exon 2 and exon 4.…”

“…For the conditional Tgfbr1 inactivation approach we used three Tgfbr1 alleles, namely the Tgfbr1 null allele 37 and either Tgfbr1 flox , carrying LoxP sites flanking Exon 3 48 , or Tgfbr1 3ex3-flox , containing a triplicated Exon 3 each surrounded by LoxP sites (supplementary Fig. 1).…”

“…We used a conditional approach to inactivate Tgfbr1 after the hindlimb and GT primordia were laid down relying on the Cdx2-Cre ERT transgenic driver 6 , a Tgfbr1 null allele 12 and either Tgfbr1 flox , carrying LoxP sites flanking Exon 3 13 , or Tgfbr1 3ex3-flox , containing a triplicated Exon 3 and associated LoxP sites that codes for a hypomorphic receptor (Supplementary Fig. 1A-D).…”

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE

“…This doesn't mean we can't use it for dsDNA. By targeting both dsDNA (DiNapoli et al, 2020[ 29 ]) and ssDNA (Bai et al, 2020[ 12 ]) templates in cells, experimental animals (Pineault et al, 2019[ 102 ]), plants (Mao et al, 2019[ 87 ]), model organisms (Bai et al, 2020[ 11 ]) and even humans (Ma et al, 2015[ 79 ]; Moon et al, 2019[ 91 ]), we can regulate insertions, deletions, replication, migration, transformation, copy number loss or gain (Anzalone et al, 2019[ 6 ]) with the CRISPR system. Different Cas enzymes are preferred according to the mechanisms by which they are effective in all these biological structures.…”