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Mouse Ifi27 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ifi27 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ifi27 gene (NCBI Reference Sequence: NM_026790 ; Ensembl: ENSMUSG00000064215 ) is located on Mouse chromosome 12. 7 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000085065). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ifi27 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-308L21 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: Mice homozygous for a knock-out allele exhibit protection from induced restenosis.

Exon 2 starts from about 100% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1071 bp, and the size of intron 2 for 3'-loxP site insertion: 989 bp. The size of effective cKO region: ~618 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 2 3 4 5 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Ifi27 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(7118bp) | A(27.23% 1938) | C(22.32% 1589) | T(26.95% 1918) | G(23.5% 1673)

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% chr12 + 103432207 103435206 3000 browser details YourSeq 147 2490 2832 3000 86.5% chr8 - 94470187 94856854 386668 browser details YourSeq 139 2471 2834 3000 89.7% chr1 + 184429520 184429886 367 browser details YourSeq 135 2461 2834 3000 84.7% chr7 - 112199971 112200343 373 browser details YourSeq 129 2492 2832 3000 82.6% chr14 - 47420529 47420850 322 browser details YourSeq 124 2491 2805 3000 86.9% chr18 - 19712096 19712598 503 browser details YourSeq 123 2490 2834 3000 90.2% chr4 + 57427201 57427629 429 browser details YourSeq 123 2478 2760 3000 86.5% chr17 + 63041628 63041912 285 browser details YourSeq 121 2483 2813 3000 81.6% chr2 - 71935111 71935378 268 browser details YourSeq 120 2490 2760 3000 88.9% chr9 - 62870821 62871092 272 browser details YourSeq 115 2665 2834 3000 83.3% chrX - 95674824 95674972 149 browser details YourSeq 114 2489 2833 3000 90.7% chr10 - 89708910 89709574 665 browser details YourSeq 113 2461 2817 3000 94.4% chr5 - 126105730 126106107 378 browser details YourSeq 112 2490 2834 3000 83.8% chr10 - 3331990 3332310 321 browser details YourSeq 109 2490 2816 3000 83.6% chr8 - 77551112 77551419 308 browser details YourSeq 109 2665 2834 3000 83.1% chr12 - 11307746 11307897 152 browser details YourSeq 109 2665 2834 3000 82.5% chr4 + 131074054 131074201 148 browser details YourSeq 109 2665 2839 3000 82.0% chr1 + 171585941 171586097 157 browser details YourSeq 108 2490 2834 3000 91.6% chr2 - 157850251 157850662 412 browser details YourSeq 108 2663 2833 3000 82.1% chr8 + 66699803 66699953 151

Note: The 3000 bp section upstream of Exon 2 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% chr12 + 103435825 103438824 3000 browser details YourSeq 293 2402 2951 3000 83.3% chrX + 37119930 37120508 579 browser details YourSeq 279 2281 2956 3000 83.9% chr6 + 96564646 96565342 697 browser details YourSeq 227 2328 2956 3000 85.0% chr11 + 30769590 30770403 814 browser details YourSeq 224 2261 2878 3000 78.5% chr10 + 128328508 128329132 625 browser details YourSeq 218 2419 2958 3000 83.7% chrX - 9152382 9152957 576 browser details YourSeq 218 2404 2958 3000 79.5% chr7 - 26353110 26353768 659 browser details YourSeq 218 2482 2956 3000 84.7% chr16 - 35835341 35835780 440 browser details YourSeq 214 2328 2956 3000 83.5% chr18 + 38534252 38534854 603 browser details YourSeq 214 2281 2856 3000 82.5% chr1 + 164581679 165126857 545179 browser details YourSeq 211 2484 2956 3000 85.0% chr7 + 100239651 100240166 516 browser details YourSeq 211 2419 2958 3000 80.3% chr6 + 48803171 48803753 583 browser details YourSeq 210 2489 2954 3000 75.7% chr8 + 79494399 79494884 486 browser details YourSeq 208 2281 2936 3000 82.7% chr4 - 86566262 86566899 638 browser details YourSeq 200 2281 3000 3000 80.6% chr11 - 105367578 105368258 681 browser details YourSeq 197 2330 2818 3000 74.5% chr3 - 100279280 100279766 487 browser details YourSeq 197 2403 2956 3000 81.7% chr12 - 117919188 117919745 558 browser details YourSeq 195 2411 2878 3000 82.0% chr10 - 42439837 42440301 465 browser details YourSeq 192 2328 2818 3000 79.0% chr16 + 8774389 8774846 458 browser details YourSeq 191 2328 2819 3000 77.6% chr15 + 38982898 39075414 92517

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ifi27 interferon, alpha-inducible protein 27 [ Mus musculus (house mouse) ] Gene ID: 52668, updated on 24-Oct-2019

Gene summary

Official Symbol Ifi27 provided by MGI Official Full Name interferon, alpha-inducible protein 27 provided by MGI Primary source MGI:MGI:1277180 See related Ensembl:ENSMUSG00000064215 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 ISG12a; Ifi27l1; D12Ertd647e; 1110013J02Rik; 2900026P10Rik Expression Broad expression in adrenal adult (RPKM 234.6), mammary gland adult (RPKM 205.0) and 16 other tissues See more

Genomic context

Location: 12 E; 12 52.93 cM See Ifi27 in Genome Data Viewer Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (103434189..103440245)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (104672399..104678455)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 9 transcripts

Gene: Ifi27 ENSMUSG00000064215

Description interferon, alpha-inducible protein 27 [Source:MGI Symbol;Acc:MGI:1277180] Gene Synonyms 1110013J02Rik, 2900026P10Rik, D12Ertd647e, Ifi27l1 Location Chromosome 12: 103,434,211-103,440,239 forward strand. GRCm38:CM001005.2 About this gene This gene has 9 transcripts (splice variants), 115 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ifi27-205 ENSMUST00000085065.11 977 260aa ENSMUSP00000082142.5 Protein coding CCDS36529 Q8C6G0 TSL:1 GENCODE basic APPRIS ALT2

Ifi27-204 ENSMUST00000079294.11 963 250aa ENSMUSP00000078275.5 Protein coding CCDS36530 E9Q1C5 TSL:1 GENCODE basic APPRIS ALT2

Ifi27-201 ENSMUST00000066701.12 889 215aa ENSMUSP00000068729.6 Protein coding CCDS26130 Q9D1E1 TSL:1 GENCODE basic APPRIS P4

Ifi27-202 ENSMUST00000076702.11 828 205aa ENSMUSP00000075994.5 Protein coding CCDS26131 Q70LN0 TSL:1 GENCODE basic APPRIS ALT2

Ifi27-203 ENSMUST00000076788.11 708 165aa ENSMUSP00000076068.5 Protein coding CCDS26132 Q70LM9 TSL:1 GENCODE basic APPRIS ALT2

Ifi27-207 ENSMUST00000140838.1 410 105aa ENSMUSP00000117403.1 Protein coding - D3Z4L8 CDS 3' incomplete TSL:1

Ifi27-208 ENSMUST00000148824.1 629 No protein - Retained intron - - TSL:2

Ifi27-206 ENSMUST00000122905.7 625 No protein - Retained intron - - TSL:2

Ifi27-209 ENSMUST00000153670.1 527 No protein - Retained intron - - TSL:2

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26.03 kb Forward strand 103.43Mb 103.44Mb 103.45Mb Genes (Comprehensive set... Ifi27-202 >protein coding

Ifi27-203 >protein coding

Ifi27-204 >protein coding

Ifi27-201 >protein coding

Ifi27-206 >retained intron

Ifi27-205 >protein coding

Ifi27-208 >retained intron

Ifi27-207 >protein coding

Ifi27-209 >retained intron

Contigs AC154347.2 > Genes < Ddx24-201protein coding < Ifi27l2a-201protein coding (Comprehensive set...

< Ddx24-202protein coding

< Ddx24-206protein coding

< Ddx24-204retained intron

< Ddx24-205non stop decay

< Ddx24-209protein coding

Regulatory Build

103.43Mb 103.44Mb 103.45Mb Reverse strand 26.03 kb

Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000085065

5.98 kb Forward strand

Ifi27-205 >protein coding

ENSMUSP00000082... Low complexity (Seg) Pfam Interferon alpha-inducible protein IFI6/IFI27-like PANTHER PTHR16932:SF15

Interferon alpha-inducible protein IFI6/IFI27-like Gene3D IFI6/IFI27-like domain superfamily

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

Variant Legend frameshift variant missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 260

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

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