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Mouse Ica1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ica1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ica1 gene (NCBI Reference Sequence: NM_010492 ; Ensembl: ENSMUSG00000062995 ) is located on Mouse chromosome 6. 14 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000038403). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ica1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-82N21 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygous mutation of this gene results in diabetes and spontaneous lethality at 4-5 months of age on a NOD background, however mice on a 129/Sv background are normal. Onset of diabetes starts 4 weeks later than wild-typeNOD mice and mutants are resistant to cyclophospamide-accelerated diabetes.

Exon 3 starts from about 1.26% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 3614 bp, and the size of intron 3 for 3'-loxP site insertion: 4821 bp. The size of effective cKO region: ~663 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ica1 Homology arm cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region 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 GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7163bp) | A(28.14% 2016) | C(21.33% 1528) | T(31.31% 2243) | G(19.21% 1376)

Note: The sequence of homologous arms and cKO region 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr6 - 8755002 8758001 3000 browser details YourSeq 284 2268 2840 3000 86.3% chr8 + 79438511 79439093 583 browser details YourSeq 276 2386 2871 3000 87.0% chr3 - 66040004 66040493 490 browser details YourSeq 271 2386 2857 3000 84.8% chr15 + 73560248 73560729 482 browser details YourSeq 243 2386 2895 3000 86.5% chr7 + 81139610 81140123 514 browser details YourSeq 242 2433 2898 3000 84.6% chr14 - 10766337 10766804 468 browser details YourSeq 241 2404 2855 3000 84.4% chr12 - 109816099 109816537 439 browser details YourSeq 241 2407 2842 3000 84.9% chr12 - 69660768 69661195 428 browser details YourSeq 238 2386 2839 3000 88.8% chr10 + 78062021 78062597 577 browser details YourSeq 235 2386 2895 3000 86.6% chr11 - 35714935 35715762 828 browser details YourSeq 233 2386 2874 3000 82.9% chr18 - 41901167 41901667 501 browser details YourSeq 232 2311 2844 3000 81.0% chr19 + 27051678 27052227 550 browser details YourSeq 229 2522 2890 3000 85.0% chr7 + 66234357 66234730 374 browser details YourSeq 228 2406 2888 3000 82.8% chr11 + 103448666 103449142 477 browser details YourSeq 228 2298 2899 3000 86.7% chr11 + 87645103 87645721 619 browser details YourSeq 220 2404 2871 3000 87.5% chr10 + 126022370 126022842 473 browser details YourSeq 217 2386 2888 3000 85.8% chr2 - 68556731 68557248 518 browser details YourSeq 216 2386 3000 3000 84.7% chr11 - 34832150 34833028 879 browser details YourSeq 216 2406 2856 3000 84.4% chr13 + 110830461 110830922 462 browser details YourSeq 212 2409 2871 3000 84.3% chr10 - 3618078 3618533 456

Note: The 3000 bp section upstream of Exon 3 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 3000 1 3000 3000 100.0% chr6 - 8751339 8754338 3000 browser details YourSeq 71 754 838 3000 95.0% chr11 + 109935484 109935603 120 browser details YourSeq 70 753 846 3000 94.0% chr1 + 161554618 161554714 97 browser details YourSeq 69 2107 2217 3000 97.3% chr10 + 125944305 125944483 179 browser details YourSeq 62 742 816 3000 95.6% chr9 + 45474680 45474798 119 browser details YourSeq 61 2129 2218 3000 85.3% chr1 - 128780229 128780308 80 browser details YourSeq 60 756 837 3000 94.2% chr5 - 98017717 98017837 121 browser details YourSeq 60 756 843 3000 91.7% chr10 - 97052675 97052763 89 browser details YourSeq 60 740 854 3000 79.8% chr11 + 29701588 29701683 96 browser details YourSeq 56 760 852 3000 77.8% chr12 - 19572136 19572201 66 browser details YourSeq 56 760 852 3000 77.8% chr12 + 24004208 24004273 66 browser details YourSeq 56 760 852 3000 77.8% chr12 + 22658351 22658416 66 browser details YourSeq 53 765 836 3000 95.0% chr5 - 132299252 132299336 85 browser details YourSeq 52 753 806 3000 98.2% chr4 - 66166377 66166430 54 browser details YourSeq 52 753 808 3000 96.5% chr10 - 43055237 43055292 56 browser details YourSeq 52 2084 2194 3000 95.0% chr16 + 32013701 32013954 254 browser details YourSeq 51 2171 2230 3000 96.6% chr5 + 92213825 92213954 130 browser details YourSeq 50 753 808 3000 96.3% chr17 - 63582516 63582571 56 browser details YourSeq 50 740 805 3000 92.9% chr1 + 182905594 182905666 73 browser details YourSeq 49 756 828 3000 94.5% chr6 - 60617636 60617708 73

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ica1 islet cell autoantigen 1 [ Mus musculus (house mouse) ] Gene ID: 15893, updated on 12-Aug-2019

Gene summary

Official Symbol Ica1 provided by MGI Official Full Name islet cell autoantigen 1 provided by MGI Primary source MGI:MGI:96391 See related Ensembl:ENSMUSG00000062995 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as 69kDa; ICA69 Expression Broad expression in testis adult (RPKM 6.7), cortex adult (RPKM 5.0) and 19 other tissues See more Orthologs human all

Genomic context

Location: 6 A1; 6 4.38 cM See Ica1 in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (8630527..8778540, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (8580527..8728484, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 12 transcripts

Gene: Ica1 ENSMUSG00000062995

Description islet cell autoantigen 1 [Source:MGI Symbol;Acc:MGI:96391] Gene Synonyms 69kDa, ICA69 Location Chromosome 6: 8,630,527-8,778,488 reverse strand. GRCm38:CM000999.2 About this gene This gene has 12 transcripts (splice variants), 206 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 26 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ica1- ENSMUST00000038403.11 2075 478aa ENSMUSP00000040062.5 Protein coding CCDS19911 P97411 TSL:1 201 GENCODE basic APPRIS P2

Ica1- ENSMUST00000115520.7 1876 478aa ENSMUSP00000111182.1 Protein coding CCDS19911 P97411 TSL:5 204 GENCODE basic APPRIS P2

Ica1- ENSMUST00000115519.7 1706 465aa ENSMUSP00000111181.1 Protein coding - D3Z118 TSL:5 203 GENCODE basic APPRIS ALT2

Ica1- ENSMUST00000115518.7 1256 310aa ENSMUSP00000111180.1 Protein coding - D3Z119 TSL:1 202 GENCODE basic

Ica1- ENSMUST00000153390.7 1138 277aa ENSMUSP00000117734.1 Protein coding - D3Z376 CDS 3' 211 incomplete TSL:5

Ica1- ENSMUST00000126430.1 565 141aa ENSMUSP00000116861.1 Protein coding - F7BG11 CDS 5' 206 incomplete TSL:3

Ica1- ENSMUST00000151758.1 441 45aa ENSMUSP00000117112.1 Protein coding - D3Z699 CDS 3' 210 incomplete TSL:3

Ica1- ENSMUST00000126039.7 404 73aa ENSMUSP00000118010.1 Protein coding - D3Z020 CDS 3' 205 incomplete TSL:3

Ica1- ENSMUST00000127398.7 226 66aa ENSMUSP00000118194.1 Protein coding - F6UY19 CDS 5' 207 incomplete TSL:3

Ica1- ENSMUST00000156695.7 1493 302aa ENSMUSP00000138459.1 Nonsense mediated - S4R217 TSL:5 212 decay

Ica1- ENSMUST00000135113.1 960 No - lncRNA - - TSL:1 208 protein

Ica1- ENSMUST00000145870.1 470 No - lncRNA - - TSL:2 209 protein

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167.96 kb Forward strand 8.64Mb 8.66Mb 8.68Mb 8.70Mb 8.72Mb 8.74Mb 8.76Mb 8.78Mb Contigs AC158670.5 > Genes (Comprehensive set... < Ica1-201protein coding

< Ica1-204protein coding

< Ica1-203protein coding

< Ica1-212nonsense mediated decay

< Ica1-209lncRNA < Ica1-211protein coding

< Ica1-207protein coding < Ica1-208lncRNA

< Ica1-202protein coding

< Ica1-206protein coding < Ica1-205protein coding

< Ica1-210protein coding

Regulatory Build

8.64Mb 8.66Mb 8.68Mb 8.70Mb 8.72Mb 8.74Mb 8.76Mb 8.78Mb Reverse strand 167.96 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000038403

< Ica1-201protein coding

Reverse strand 147.96 kb

ENSMUSP00000040... MobiDB lite Superfamily AH/BAR domain superfamily

SMART Arfaptin homology (AH) domain Islet cell autoantigen Ica1, C-terminal

Pfam Arfaptin homology (AH) domain Islet cell autoantigen Ica1, C-terminal PROSITE profiles Arfaptin homology (AH) domain PANTHER Islet cell autoantigen 1/Ica1-like

Islet cell autoantigen 1 Gene3D AH/BAR domain superfamily CDD cd07661

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

Variant Legend

stop gained missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 478

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

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