SEAN CARRINGTON

ARTICLE REVIEW: GENERATION OF MEGABASE-SCALE DELETIONS, INVERSIONS,

AND DUPLICATIONS INVOLVING THE CONTACTIN-6 GENE IN MICE BY

CRISPER/CAS9 TECHNOLOGY

The background of this study involves Copy Number Variation (CNV) of the human

CNTN6 gene which encodes for the contactin-6 protein was found to be the cause of severe

neurodevelopmental impairments which is also commonly associated with facial dysmorphias

due to deletions or duplications. However, the deleterious point mutations for CNTN6 did not

show any clinical phenotypes. Thus, the study wanted to focus an experimental model to study

CNV of the CNTN6 locus by generating mice that carry large deletions, duplications, and

inversion mutations in association with the CNTN6 gene.

Three genes were considered candidates for causing intellectual impairment based on the

analysis of deletions and duplications involved in the peritelomeric region pter-p25.3: CHL1

(encodes neural cell adhesion molecule), CNTN4, and CNTN6 (CNT genes encode for

contactin-4/6 proteins). Copy number variations that were subject to microdeletions or

microduplications involving one or more of the genes were associated with severe

neurodevelopmental disorders such as intellectual disability, developmental delay, and other

disorders. However, CNTN6 was considered to be the main subject due to how patients carrying

the deletion were seen to have CNTN6 act alone or in association with CHL1 and CNTN4 genes.

The CNTN6 gene is within the immunoglobin superfamily of neural cell-adhesion molecules that

promote neurite outgrowth and synaptogenesis most prominently in the sensory-motor pathways

which is imperative for correct orientation of dendrite growth in addition to synapse formation.

Knockout mice that were chosen for the experiment were homologous for the CNTN6

gene and were viable and fertile. The mice brain regarding the formation and organization of
 SEAN CARRINGTON

nuclei and layers appeared normal at the beginning of the experiment, but CNTN6 deficiency

subjection showed defection in motor coordination. In the megbase-scale chromosome

modification of CNTN6, CRISPR/Cas9 was used for generating deletions, duplications, and

inversions by using gRNAs for target sites, flanking the DNA fragment containing CNTN6 gene

located on mouse chromosome 6 at position 103,510,581–106,700,141 (GRCm38/mm10).

gRNAs were injected with Cas9 mRNA and ssODN into the cytoplasm of 599 zygotes and

transplanted 256 viable 1-cell embryos into 11 recipient CD-1 females. 41 live offspring were

birthed which were then subjected to genotyping of the F0 offspring by PCR analysis in which

identification of 11 offspring carried deletions, duplications, and inversions of the CNTN6 gene.

11 of the genetically modified F0 appeared to be viable and healthy; six F0 female and four F0

male were crossed with C57BL for the birth of F1 offspring implying fertility of both the

genetically modified male and female. All f1 offspring derived so too appeared to be healthy

without any visible anomalies.

Thus in the result and conclusion of the study, all genetically modified F0 offspring were

viable and present with deletions, duplications, and inversions of the CNTN6 gene which served

as new mouse lines in studying the effects of CNV of the CNTN6 gene. The method used within

this study permits for “rapid in vivo modeling of large chromosomal rearrangements in mice,”

(Korablev, et al., 2017). They had created mice carrying megabase-scale deletions, duplications,

and inversions involving the full-sized CNTN6 gene by using the gene editing tool

CRISPER/Cas9. The creation of such mice allow for the study of severe neurodevelopment

impairments caused by such mutations in humans using these mice that potentially model disease

due to CNV rather than CNTN6 knockout mice.