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Mouse Mical1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mical1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mical1 gene (NCBI Reference Sequence: NM_138315 ; Ensembl: ENSMUSG00000019823 ) is located on Mouse chromosome 10. 24 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 24 (Transcript: ENSMUST00000019967). Exon 8~12 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mical1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-321I6 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:

Exon 8 starts from about 29.71% of the coding region. The knockout of Exon 8~12 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 748 bp, and the size of intron 12 for 3'-loxP site insertion: 692 bp. The size of effective cKO region: ~2115 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 24 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mical1 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(8615bp) | A(22.04% 1899) | C(27.08% 2333) | T(22.89% 1972) | G(27.99% 2411)

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% chr10 + 41477885 41480884 3000 browser details YourSeq 58 650 753 3000 77.9% chr6 - 121040380 121040483 104 browser details YourSeq 38 2439 2480 3000 95.3% chr7 + 112315010 112315051 42 browser details YourSeq 35 2517 2552 3000 100.0% chr15 + 4933999 4934036 38 browser details YourSeq 31 2847 2884 3000 97.1% chr4 + 125448908 125448945 38 browser details YourSeq 30 2517 2552 3000 80.7% chr4 - 153883198 153883228 31 browser details YourSeq 28 2518 2552 3000 91.5% chr14 - 76476429 76476466 38 browser details YourSeq 27 2517 2552 3000 96.6% chr14 - 96925266 96925308 43 browser details YourSeq 27 2517 2545 3000 89.3% chr7 + 125309999 125310026 28 browser details YourSeq 27 2517 2552 3000 96.6% chr14 + 74877680 74877724 45 browser details YourSeq 26 2519 2549 3000 93.6% chr16 + 65485555 65485586 32 browser details YourSeq 25 497 522 3000 100.0% chr5 - 115223992 115224018 27 browser details YourSeq 25 497 522 3000 100.0% chr15 - 11514999 11515026 28 browser details YourSeq 25 2518 2546 3000 93.2% chr3 + 152096306 152096334 29 browser details YourSeq 25 656 684 3000 96.3% chr2 + 65665471 65665501 31 browser details YourSeq 25 498 525 3000 84.7% chr14 + 39708506 39708531 26 browser details YourSeq 25 498 524 3000 96.3% chr13 + 39362476 39362502 27 browser details YourSeq 25 497 522 3000 100.0% chr11 + 117480210 117480237 28 browser details YourSeq 25 497 522 3000 100.0% chr1 + 93877326 93877353 28 browser details YourSeq 24 498 522 3000 100.0% chr3 - 122185005 122185031 27

Note: The 3000 bp section upstream of Exon 8 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% chr10 + 41483000 41485999 3000 browser details YourSeq 51 378 1163 3000 62.1% chr15 - 83716862 83717050 189 browser details YourSeq 38 2419 2460 3000 95.3% chr13 - 59799935 59799976 42 browser details YourSeq 37 2425 2463 3000 97.5% chr4 + 63486359 63486397 39 browser details YourSeq 36 2423 2463 3000 95.2% chr15 + 40650630 40650673 44 browser details YourSeq 35 2425 2463 3000 94.9% chr3 + 52442741 52442779 39 browser details YourSeq 35 64 113 3000 84.7% chr2 + 121544476 121544522 47 browser details YourSeq 35 2423 2459 3000 97.3% chr12 + 104815100 104815136 37 browser details YourSeq 34 2422 2460 3000 97.3% chr14 - 119017232 119017271 40 browser details YourSeq 34 2423 2459 3000 88.6% chr14 + 68657285 68657319 35 browser details YourSeq 34 64 110 3000 85.0% chr12 + 109080663 109080707 45 browser details YourSeq 33 2423 2457 3000 97.2% chr12 + 51816191 51816225 35 browser details YourSeq 33 2423 2457 3000 97.2% chr10 + 55551395 55551429 35 browser details YourSeq 32 64 113 3000 82.0% chr10 + 69267167 69267216 50 browser details YourSeq 31 64 110 3000 83.8% chr16 - 51265811 51265855 45 browser details YourSeq 29 2419 2454 3000 91.5% chr17 - 57279709 57279750 42 browser details YourSeq 27 82 110 3000 96.6% chr16 + 33590085 33590113 29 browser details YourSeq 26 1738 1768 3000 96.6% chr4 + 41943011 41943046 36 browser details YourSeq 26 2422 2449 3000 96.5% chr1 + 74469650 74469677 28 browser details YourSeq 25 82 110 3000 93.2% chr5 + 110884019 110884047 29

Note: The 3000 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Mical1 microtubule associated monooxygenase, calponin and LIM domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 171580, updated on 10-Oct-2019

Gene summary

Official Symbol Mical1 provided by MGI Official Full Name microtubule associated monooxygenase, calponin and LIM domain containing 1 provided by MGI Primary source MGI:MGI:2385847 See related Ensembl:ENSMUSG00000019823 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 MICAL; Nical; MICAL-1 Expression Broad expression in duodenum adult (RPKM 45.4), thymus adult (RPKM 34.0) and 19 other tissues See more Orthologs human all

Genomic context

Location: 10; 10 B2 See Mical1 in Genome Data Viewer

Exon count: 25

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (41475860..41487030)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (41196120..41206836)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 12 transcripts

Gene: Mical1 ENSMUSG00000019823

Description microtubule associated monooxygenase, calponin and LIM domain containing 1 [Source:MGI Symbol;Acc:MGI:2385847] Gene Synonyms Nical Location Chromosome 10: 41,476,314-41,487,032 forward strand. GRCm38:CM001003.2 About this gene This gene has 12 transcripts (splice variants), 190 orthologues, 36 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mical1- ENSMUST00000019967.15 3563 1048aa ENSMUSP00000019967.9 Protein coding CCDS35888 Q8VDP3 TSL:1 201 GENCODE basic APPRIS P2

Mical1- ENSMUST00000099934.10 3292 975aa ENSMUSP00000097519.4 Protein coding CCDS48547 Q8VDP3 TSL:1 202 GENCODE basic

Mical1- ENSMUST00000119962.7 3565 1048aa ENSMUSP00000113783.1 Protein coding - Q8VDP3 TSL:5 203 GENCODE basic APPRIS ALT2

Mical1- ENSMUST00000151486.1 418 43aa ENSMUSP00000116386.1 Protein coding - D3Z5P6 CDS 3' 212 incomplete TSL:2

Mical1- ENSMUST00000126436.1 3408 939aa ENSMUSP00000114969.1 Nonsense mediated - E9PUI4 TSL:5 207 decay

Mical1- ENSMUST00000134010.7 3645 No - Retained intron - - TSL:5 209 protein

Mical1- ENSMUST00000130838.1 726 No - Retained intron - - TSL:3 208 protein

Mical1- ENSMUST00000136681.1 696 No - Retained intron - - TSL:3 210 protein

Mical1- ENSMUST00000125099.1 677 No - Retained intron - - TSL:5 205 protein

Mical1- ENSMUST00000138657.1 537 No - Retained intron - - TSL:2 211 protein

Mical1- ENSMUST00000123798.1 488 No - Retained intron - - TSL:2 204 protein

Mical1- ENSMUST00000125730.1 423 No - Retained intron - - TSL:3 206 protein

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30.72 kb Forward strand

41.47Mb 41.48Mb 41.49Mb Genes (Comprehensive set... Mical1-201 >protein coding Ppil6-201 >protein coding

Mical1-203 >protein coding Ppil6-203 >retained intron

Mical1-209 >retained intron Mical1-204 >retained intron

Mical1-202 >protein coding

Mical1-207 >nonsense mediated decay

Mical1-212 >protein coding Mical1-210 >retained intronMical1-211 >retained intron

Mical1-208 >retained intron Mical1-205 >retained intron

Mical1-206 >retained intron

Contigs AC153360.6 > Genes < Smpd2-203retained intron (Comprehensive set...

< Smpd2-201protein coding

< Smpd2-206retained intron

< Smpd2-205retained intron

< Smpd2-202retained intron

< Smpd2-209nonsense mediated decay

< Smpd2-207retained intron

< Smpd2-204retained intron

< Smpd2-208retained intron

Regulatory Build

41.47Mb 41.48Mb 41.49Mb Reverse strand 30.72 kb

Regulation Legend

CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000019967

10.72 kb Forward strand

Mical1-201 >protein coding

ENSMUSP00000019... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily CH domain superfamily SSF57716

FAD/NAD(P)-binding domain superfamily SMART Calponin homology domain SM01203

Zinc finger, LIM-type Pfam FAD-binding domain Calponin homology domain bMERB domain

PROSITE profiles Zinc finger, LIM-type bMERB domain

Calponin homology domain PROSITE patterns Zinc finger, LIM-type

PANTHER PTHR24206

MICAL1 Gene3D 3.50.50.70 CH domain superfamily

2.10.110.10 CDD cd09358

Calponin homology domain

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

Variant Legend inframe insertion inframe deletion missense variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1048

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

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