https://www.alphaknockout.com

Mouse Gjd2 Knockout Project (CRISPR/Cas9)

Objective: To create a Gjd2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Gjd2 gene (NCBI Reference Sequence: NM_010290 ; Ensembl: ENSMUSG00000068615 ) is located on Mouse chromosome 2. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000090275). Exon 1~2 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Nullizygous mutations can cause loss of electrical synapses, impaired synchronous activity of inhibitory networks, altered spike synchrony in OB glomeruli, absent coupling of alpha-ganglion cells in retina, and abnormal cued conditioning, nerve fiber andsingle cell responses, and insulin secretion.

Exon 1 starts from about 0.1% of the coding region. Exon 1~2 covers 100.0% of the coding region. The size of effective KO region: ~2101 bp. The KO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2

Legends Exon of mouse Gjd2 Knockout region

Page 2 of 8 https://www.alphaknockout.com

Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of start codon is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Overview of the Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of stop codon is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Page 3 of 8 https://www.alphaknockout.com

Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(20.85% 417) | C(29.5% 590) | T(23.2% 464) | G(26.45% 529)

Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(26.2% 524) | C(18.85% 377) | T(33.25% 665) | G(21.7% 434)

Note: The 2000 bp section downstream of stop codon is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

Page 4 of 8 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr2 - 114013135 114015134 2000 browser details YourSeq 27 1856 1883 2000 100.0% chr10 + 76360431 76360521 91 browser details YourSeq 22 164 185 2000 100.0% chr15 + 25936825 25936846 22

Note: The 2000 bp section upstream of start codon is BLAT searched against the genome. No significant similarity is found.

BLAT Search Results (down)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr2 - 114009032 114011031 2000 browser details YourSeq 34 770 867 2000 92.7% chr3 - 126068135 126068259 125 browser details YourSeq 32 842 877 2000 88.6% chr14 - 112351512 112351546 35 browser details YourSeq 30 838 870 2000 96.9% chr2 + 113748801 113748833 33 browser details YourSeq 28 834 866 2000 93.8% chr10 + 3635891 3635923 33 browser details YourSeq 27 834 866 2000 84.4% chr3 + 29454701 29454732 32 browser details YourSeq 26 854 879 2000 100.0% chr3 - 145573041 145573066 26 browser details YourSeq 26 1073 1103 2000 93.4% chr13 - 20502379 20502729 351 browser details YourSeq 25 835 866 2000 90.7% chr3 + 16548876 16548908 33 browser details YourSeq 24 446 474 2000 96.2% chr7 + 29523166 29523196 31

Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found.

Page 5 of 8 https://www.alphaknockout.com

Gene and protein information: Gjd2 gap junction protein, delta 2 [ Mus musculus (house mouse) ] Gene ID: 14617, updated on 1-Oct-2019

Gene summary

Official Symbol Gjd2 provided by MGI Official Full Name gap junction protein, delta 2 provided by MGI Primary source MGI:MGI:1334209 See related Ensembl:ENSMUSG00000068615 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Gja9; cx36; connexin36 Expression Biased expression in CNS E18 (RPKM 8.1), whole brain E14.5 (RPKM 6.7) and 6 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 E4 See Gjd2 in Genome Data Viewer Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (114009601..114014378, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (113835337..113839355, complement)

Chromosome 2 - NC_000068.7

Page 6 of 8 https://www.alphaknockout.com

Transcript information: This gene has 1 transcript

Gene: Gjd2 ENSMUSG00000068615

Description gap junction protein, delta 2 [Source:MGI Symbol;Acc:MGI:1334209] Gene Synonyms Cx36, Gja9, connexin36 Location Chromosome 2: 114,009,601-114,013,619 reverse strand. GRCm38:CM000995.2 About this gene This gene has 1 transcript (splice variant), 230 orthologues, 19 paralogues, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gjd2-201 ENSMUST00000090275.4 2879 321aa ENSMUSP00000087742.4 Protein coding CCDS16563 O54851 TSL:1 GENCODE basic APPRIS P1

24.02 kb Forward strand

114.00Mb 114.01Mb 114.02Mb Genes A530058N18Rik-208 >lncRNA (Comprehensive set...

A530058N18Rik-209 >lncRNA

A530058N18Rik-204 >lncRNA

A530058N18Rik-203 >lncRNA

A530058N18Rik-201 >lncRNA

A530058N18Rik-207 >lncRNA

A530058N18Rik-206 >retained intron

A530058N18Rik-205 >lncRNA

A530058N18Rik-202 >lncRNA

Contigs AL935137.6 > AL844569.4 >

Genes (Comprehensive set... < Gjd2-201protein coding

Regulatory Build

114.00Mb 114.01Mb 114.02Mb Reverse strand 24.02 kb

Regulation Legend

CTCF Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000090275

< Gjd2-201protein coding

Reverse strand 4.02 kb

ENSMUSP00000087... Transmembrane heli... Low complexity (Seg) SMART Connexin, N-terminal Gap junction protein, cysteine-rich domain

Prints Gap junction delta-2 protein (Cx36)

Connexin Pfam Connexin, N-terminal PROSITE patterns Connexin, conserved site Connexin, conserved site

PANTHER PTHR11984:SF32

Connexin Gene3D Connexin, N-terminal domain superfamily

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 321

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

Page 8 of 8