https://www.alphaknockout.com

Mouse Ifi27l2a Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ifi27l2a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ifi27l2a (NCBI Reference Sequence: NM_029803 ; Ensembl: ENSMUSG00000079017 ) is located on Mouse 12. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000055071). 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 Ifi27l2a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-62E20 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 normal susceptibility to infection with H1N1 or H7N7 influenza A virus, although sloughing of dead cells into bronchiolar lumen persists somewhat longer after infection than in infected controls.

Exon 3 is not frameshift exon, and covers 60.0% of the coding region. The size of intron 2 for 5'-loxP site insertion: 488 bp, and the size of intron 3 for 3'-loxP site insertion: 448 bp. The size of effective cKO region: ~630 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 3 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Ifi27l2a cKO region loxP site

Page 2 of 7 https://www.alphaknockout.com

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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7098bp) | A(24.96% 1772) | C(24.57% 1744) | T(26.04% 1848) | G(24.43% 1734)

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.

Page 3 of 7 https://www.alphaknockout.com

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 - 103443238 103446237 3000 browser details YourSeq 480 1245 1987 3000 93.2% chr7 - 30495431 30496227 797 browser details YourSeq 438 1239 1992 3000 90.4% chr7 - 30501973 30502529 557 browser details YourSeq 413 1245 1991 3000 89.2% chr14 - 118062419 118062935 517 browser details YourSeq 408 1245 1991 3000 89.0% chr8 - 65007757 65008288 532 browser details YourSeq 406 1245 1991 3000 90.5% chr19 + 47740533 47741041 509 browser details YourSeq 399 1245 1991 3000 89.7% chr14 + 65804368 65804897 530 browser details YourSeq 398 1245 1956 3000 90.0% chrX + 48735436 48735906 471 browser details YourSeq 397 1245 1991 3000 86.6% chr4 + 68113874 68114430 557 browser details YourSeq 396 1 3000 3000 86.0% chr12 - 103452227 103454665 2439 browser details YourSeq 393 1250 1970 3000 88.3% chr17 + 20398260 20398765 506 browser details YourSeq 388 1245 1960 3000 87.8% chr7 - 63366735 63367246 512 browser details YourSeq 387 1256 1956 3000 89.8% chr9 + 113477472 113477965 494 browser details YourSeq 385 1244 1960 3000 88.0% chr5 - 46554583 46555070 488 browser details YourSeq 376 1245 1953 3000 89.7% chr10 + 23685928 23686483 556 browser details YourSeq 375 957 1956 3000 88.2% chr1 + 90546362 90546881 520 browser details YourSeq 374 1245 1956 3000 88.4% chr12 + 77380529 77381008 480 browser details YourSeq 373 1245 1991 3000 86.6% chr1 - 33277002 33277506 505 browser details YourSeq 371 1245 1976 3000 88.5% chr6 - 139268836 139269332 497 browser details YourSeq 370 1253 1956 3000 89.0% chr6 + 57468124 57468599 476

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% chr12 - 103439608 103442607 3000 browser details YourSeq 902 99 1810 3000 89.1% chr12 - 103448771 103451479 2709 browser details YourSeq 92 1071 1742 3000 87.8% chr15 + 60641498 60642224 727 browser details YourSeq 84 99 244 3000 88.9% chr12 - 103455885 103456031 147 browser details YourSeq 28 1384 1421 3000 77.5% chr11 - 22097275 22097307 33 browser details YourSeq 24 2941 2989 3000 75.6% chr6 + 57639540 57639594 55 browser details YourSeq 22 1744 1768 3000 96.0% chr17 - 44482496 44482521 26 browser details YourSeq 22 1241 1262 3000 100.0% chr5 + 143405460 143405481 22 browser details YourSeq 22 1386 1407 3000 100.0% chr2 + 70389125 70389146 22 browser details YourSeq 22 1369 1390 3000 100.0% chr11 + 9888825 9888846 22

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

Page 4 of 7 https://www.alphaknockout.com

Gene and information: Ifi27l2a interferon, alpha-inducible protein 27 like 2A [ Mus musculus (house mouse) ] Gene ID: 76933, updated on 12-Aug-2019

Gene summary

Official Symbol Ifi27l2a provided by MGI Official Full Name interferon, alpha-inducible protein 27 like 2A provided by MGI Primary source MGI:MGI:1924183 See related Ensembl:ENSMUSG00000079017 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 Ifi27; Isg12; Isg12(b1); 2310061N23Rik Expression Biased expression in mammary gland adult (RPKM 553.4), subcutaneous fat pad adult (RPKM 344.9) and 14 other tissues Orthologs See more human all

Genomic context

Location: 12; 12 E See Ifi27l2a in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (103433873..103443680, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (104680377..104681890, complement)

Chromosome 12 - NC_000078.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 1 transcript

Gene: Ifi27l2a ENSMUSG00000079017

Description interferon, alpha-inducible protein 27 like 2A [Source:MGI Symbol;Acc:MGI:1924183] Gene Synonyms 2310061N23Rik, Ifi27 Location Chromosome 12: 103,442,167-103,443,680 reverse strand. GRCm38:CM001005.2 About this gene This gene has 1 transcript (splice variant), 39 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ifi27l2a-201 ENSMUST00000055071.8 463 90aa ENSMUSP00000054698.8 Protein coding CCDS49153 Q8R412 TSL:1 GENCODE basic APPRIS P1

21.51 kb Forward strand

103.435Mb 103.440Mb 103.445Mb 103.450Mb Ifi27-202 >protein coding (Comprehensive set...

Ifi27-203 >protein coding

Ifi27-204 >protein coding

Ifi27-201 >protein coding

Ifi27-206 >retained intron

Ifi27-205 >protein coding

Ifi27-208 >retained intron Ifi27-209 >retained intron

Ifi27-207 >protein coding

Contigs AC154347.2 > Genes < Ifi27l2a-201protein coding < Ifi27l2b-201protein coding (Comprehensive set...

Regulatory Build

103.435Mb 103.440Mb 103.445Mb 103.450Mb Reverse strand 21.51 kb

Regulation Legend Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000055071

< Ifi27l2a-201protein coding

Reverse strand 1.51 kb

ENSMUSP00000054... Transmembrane heli... Low complexity (Seg) Pfam Interferon alpha-inducible protein IFI6/IFI27-like PANTHER Interferon alpha-inducible protein IFI6/IFI27-like

PTHR16932:SF2 Gene3D IFI6/IFI27-like domain superfamily

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

Variant Legend missense variant

Scale bar 0 8 16 24 32 40 48 56 64 72 80 90

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

Page 7 of 7