https://www.alphaknockout.com

Mouse Mfap5 Knockout Project (CRISPR/Cas9)

Objective: To create a Mfap5 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mfap5 gene (NCBI Reference Sequence: NM_015776 ; Ensembl: ENSMUSG00000030116 ) is located on Mouse chromosome 6. 10 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000148517). Exon 6~8 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: Mice homozygous for a knock-out allele exhibit decreased circulating neutrophils.

Exon 6 starts from about 32.72% of the coding region. Exon 6~8 covers 30.08% of the coding region. The size of effective KO region: ~4146 bp. The KO region does not have any other known gene.

Page 1 of 9 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 6 7 8 10

Legends Exon of mouse Mfap5 Knockout region

Page 2 of 9 https://www.alphaknockout.com

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

Forward Reverse Complement

Sequence 12

Note: The 964 bp section upstream of Exon 6 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 748 bp section downstream of Exon 8 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 9 https://www.alphaknockout.com

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

Sequence 12

Summary: Full Length(964bp) | A(28.84% 278) | C(22.61% 218) | T(26.45% 255) | G(22.1% 213)

Note: The 964 bp section upstream of Exon 6 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.

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

Sequence 12

Summary: Full Length(748bp) | A(30.75% 230) | C(20.99% 157) | T(24.73% 185) | G(23.53% 176)

Note: The 748 bp section downstream of Exon 8 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 9 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 964 1 964 964 100.0% chr6 + 122520930 122521893 964 browser details YourSeq 176 427 776 964 90.4% chr4 + 136166599 136280577 113979 browser details YourSeq 171 423 772 964 93.5% chr6 - 34847674 35096013 248340 browser details YourSeq 166 427 758 964 92.4% chr18 + 32287545 32288099 555 browser details YourSeq 155 449 776 964 92.4% chr10 - 128162169 128542481 380313 browser details YourSeq 148 427 773 964 90.8% chr11 - 119272466 119272815 350 browser details YourSeq 134 468 774 964 92.5% chr4 + 58945211 58945778 568 browser details YourSeq 126 629 775 964 90.1% chr16 - 9694395 9694535 141 browser details YourSeq 125 621 778 964 87.5% chr1 + 131165985 131166129 145 browser details YourSeq 124 642 788 964 92.5% chr6 - 30066431 30066578 148 browser details YourSeq 123 642 776 964 95.6% chr4 + 43462574 43462708 135 browser details YourSeq 123 642 776 964 95.6% chr15 + 81953439 81953573 135 browser details YourSeq 122 642 783 964 93.0% chr5 - 118341588 118341729 142 browser details YourSeq 122 642 800 964 92.4% chr17 + 88830119 88830648 530 browser details YourSeq 121 642 776 964 94.9% chr18 - 9998804 9998938 135 browser details YourSeq 121 642 776 964 94.9% chr1 - 136638674 136638808 135 browser details YourSeq 121 642 782 964 93.0% chr10 + 42497033 42497173 141 browser details YourSeq 120 642 773 964 95.5% chr4 - 33234508 33234639 132 browser details YourSeq 120 642 776 964 94.9% chr11 - 74753494 74754023 530 browser details YourSeq 120 438 769 964 91.7% chr10 - 116957291 117182171 224881

Note: The 964 bp section upstream of Exon 6 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 748 1 748 748 100.0% chr6 + 122526040 122526787 748 browser details YourSeq 155 435 629 748 88.7% chr7 + 27236973 27237166 194 browser details YourSeq 152 430 635 748 87.9% chr11 + 78429171 78429366 196 browser details YourSeq 151 436 617 748 92.3% chr4 + 135887873 135888055 183 browser details YourSeq 146 417 631 748 92.9% chr4 - 137630905 137631123 219 browser details YourSeq 133 452 634 748 90.4% chr13 - 107536996 107537181 186 browser details YourSeq 132 452 634 748 84.6% chr15 + 101201311 101201481 171 browser details YourSeq 131 452 628 748 91.2% chr4 - 107640748 107640931 184 browser details YourSeq 131 434 587 748 89.9% chr3 - 79608657 79608804 148 browser details YourSeq 131 458 627 748 86.3% chr4 + 116642265 116642426 162 browser details YourSeq 131 442 597 748 89.5% chr16 + 57178803 57178954 152 browser details YourSeq 130 452 637 748 86.8% chr7 - 126917619 126917798 180 browser details YourSeq 130 442 634 748 89.5% chr8 + 107409489 107409667 179 browser details YourSeq 128 452 635 748 93.9% chr7 - 19679309 19679503 195 browser details YourSeq 128 433 625 748 92.3% chr13 + 99465863 99466060 198 browser details YourSeq 127 433 629 748 87.1% chr5 - 129010927 129011117 191 browser details YourSeq 127 464 632 748 85.0% chr12 - 112565991 112566150 160 browser details YourSeq 126 433 583 748 92.1% chr6 + 31303230 31303386 157 browser details YourSeq 124 452 634 748 86.7% chr1 + 23337892 23338066 175 browser details YourSeq 122 435 586 748 90.2% chr5 + 120757611 120757762 152

Note: The 748 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 https://www.alphaknockout.com

Gene and protein information: Mfap5 microfibrillar associated protein 5 [ Mus musculus (house mouse) ] Gene ID: 50530, updated on 12-Aug-2019

Gene summary

Official Symbol Mfap5 provided by MGI Official Full Name microfibrillar associated protein 5 provided by MGI Primary source MGI:MGI:1354387 See related Ensembl:ENSMUSG00000030116 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 MAGP-2; MFAP-5 Expression Biased expression in subcutaneous fat pad adult (RPKM 54.4), mammary gland adult (RPKM 48.6) and 10 other tissues Orthologs See more human all

Genomic context

Location: 6; 6 F1 See Mfap5 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (122513584..122529290)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (122463694..122479308)

Chromosome 6 - NC_000072.6

Page 6 of 9 https://www.alphaknockout.com

Transcript information: This gene has 7 transcripts

Gene: Mfap5 ENSMUSG00000030116

Description microfibrillar associated protein 5 [Source:MGI Symbol;Acc:MGI:1354387] Gene Synonyms MAGP-2 Location Chromosome 6: 122,505,845-122,529,290 forward strand. GRCm38:CM000999.2 About this gene This gene has 7 transcripts (splice variants), 179 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mfap5-207 ENSMUST00000148517.7 1506 164aa ENSMUSP00000122863.1 Protein coding CCDS20496 Q9QZJ6 TSL:1 GENCODE basic APPRIS P2

Mfap5-201 ENSMUST00000032210.13 946 152aa ENSMUSP00000032210.8 Protein coding CCDS85155 F8WJ99 TSL:3 GENCODE basic

Mfap5-202 ENSMUST00000118626.7 748 156aa ENSMUSP00000113742.1 Protein coding - D3Z4D7 TSL:3 GENCODE basic APPRIS ALT2

Mfap5-203 ENSMUST00000121656.1 579 69aa ENSMUSP00000112596.1 Protein coding - D3YW93 TSL:3 GENCODE basic

Mfap5-206 ENSMUST00000142896.7 425 122aa ENSMUSP00000116769.1 Protein coding - D3Z3L7 CDS 3' incomplete TSL:5

Mfap5-204 ENSMUST00000125728.7 328 28aa ENSMUSP00000118521.1 Protein coding - D3Z0U1 CDS 3' incomplete TSL:5

Mfap5-205 ENSMUST00000126357.1 2491 No protein - Retained intron - - TSL:1

Page 7 of 9 https://www.alphaknockout.com

43.45 kb Forward strand 122.50Mb 122.51Mb 122.52Mb 122.53Mb Genes (Comprehensive set... Mfap5-204 >protein coding Mfap5-205 >retained intron

Mfap5-207 >protein coding

Mfap5-201 >protein coding

Mfap5-202 >protein coding

Mfap5-203 >protein coding

Mfap5-206 >protein coding

Contigs AC155946.7 >

Genes < Rimklb-205protein coding (Comprehensive set...

Regulatory Build

122.50Mb 122.51Mb 122.52Mb 122.53Mb Reverse strand 43.45 kb

Regulation Legend

CTCF 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 8 of 9 https://www.alphaknockout.com

Transcript: ENSMUST00000148517

15.71 kb Forward strand

Mfap5-207 >protein coding

ENSMUSP00000122... Cleavage site (Sign... Pfam Microfibril-associated glycoprotein PANTHER PTHR16485:SF6

Microfibril-associated glycoprotein

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 164

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

Page 9 of 9