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Mouse Morc3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Morc3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Morc3 gene (NCBI Reference Sequence: NM_001045529 ; Ensembl: ENSMUSG00000039456 ) is located on Mouse chromosome 16. 17 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 17 (Transcript: ENSMUST00000202261). 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 Morc3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-221E11 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 null allele die at or within a day of birth.

Exon 3 starts from about 4.0% 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: 2750 bp, and the size of intron 3 for 3'-loxP site insertion: 813 bp. The size of effective cKO region: ~633 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 4 17 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Morc3 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(7133bp) | A(26.15% 1865) | C(19.37% 1382) | T(33.3% 2375) | G(21.18% 1511)

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% chr16 + 93840945 93843944 3000 browser details YourSeq 251 1056 1499 3000 90.9% chr9 + 22407384 22408011 628 browser details YourSeq 240 1144 1533 3000 85.6% chr17 + 27044576 27044917 342 browser details YourSeq 229 1158 1499 3000 94.3% chr4 + 132903361 132903763 403 browser details YourSeq 225 980 1499 3000 93.8% chr9 - 56902613 56903172 560 browser details YourSeq 225 1132 1499 3000 93.5% chr15 - 6618807 6619165 359 browser details YourSeq 222 980 1499 3000 87.8% chr13 + 55390227 55390579 353 browser details YourSeq 219 1180 1839 3000 95.1% chr5 + 114186580 114187331 752 browser details YourSeq 206 1132 1499 3000 90.4% chr5 + 24396242 24396574 333 browser details YourSeq 205 1180 1499 3000 95.2% chr2 + 181558942 181559566 625 browser details YourSeq 204 1189 1499 3000 91.3% chr9 - 110030248 110030507 260 browser details YourSeq 204 1027 1504 3000 94.1% chr13 - 43664039 43664512 474 browser details YourSeq 204 1320 1874 3000 88.8% chr7 + 30109550 30110041 492 browser details YourSeq 203 1180 1499 3000 91.3% chr16 + 17184087 17184362 276 browser details YourSeq 202 958 1867 3000 85.0% chr1 - 182074878 182075199 322 browser details YourSeq 201 1029 1499 3000 97.3% chr7 - 44963326 44963827 502 browser details YourSeq 201 1320 1662 3000 97.7% chr19 - 59106527 59107038 512 browser details YourSeq 201 1021 1499 3000 95.9% chr6 + 33855036 33855564 529 browser details YourSeq 198 1320 1855 3000 91.5% chr6 - 49192089 49192563 475 browser details YourSeq 197 1320 1769 3000 88.7% chr13 - 119787899 119788173 275

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% chr16 + 93844578 93847577 3000 browser details YourSeq 111 1141 1401 3000 83.0% chr16 + 93837503 93837682 180 browser details YourSeq 103 1143 1545 3000 93.2% chr15 - 102872140 103166666 294527 browser details YourSeq 81 1142 1512 3000 93.5% chr15 + 12380343 12474388 94046 browser details YourSeq 77 1405 1550 3000 92.3% chr6 - 148523541 148943084 419544 browser details YourSeq 77 1401 1563 3000 93.4% chr13 - 43659698 43659911 214 browser details YourSeq 72 1401 1550 3000 91.8% chr11 - 77891385 77891673 289 browser details YourSeq 71 1476 1617 3000 77.3% chr4 + 151959151 151959258 108 browser details YourSeq 66 1480 1855 3000 77.7% chr8 - 85661386 85661699 314 browser details YourSeq 64 1302 1463 3000 89.2% chrX - 122001370 122001789 420 browser details YourSeq 61 1519 1867 3000 92.0% chr5 - 124553527 124553959 433 browser details YourSeq 60 1146 1542 3000 88.7% chr11 - 76068661 76069229 569 browser details YourSeq 60 1420 1550 3000 91.6% chr4 + 109007207 109007476 270 browser details YourSeq 57 1480 1568 3000 94.0% chr11 - 117719292 117719382 91 browser details YourSeq 57 1489 1617 3000 95.3% chr3 + 149634255 149634417 163 browser details YourSeq 56 1479 1563 3000 93.8% chr2 + 74493337 74493424 88 browser details YourSeq 55 1481 1568 3000 92.2% chr12 + 87346418 87346507 90 browser details YourSeq 55 1421 1497 3000 89.9% chr10 + 108755098 108755395 298 browser details YourSeq 54 2300 2495 3000 77.1% chr12 - 25337324 25337495 172 browser details YourSeq 53 1491 1761 3000 93.4% chr2 + 23031485 23032008 524

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: Morc3 microrchidia 3 [ Mus musculus (house mouse) ] Gene ID: 338467, updated on 12-Aug-2019

Gene summary

Official Symbol Morc3 provided by MGI Official Full Name microrchidia 3 provided by MGI Primary source MGI:MGI:2136841 See related Ensembl:ENSMUSG00000039456 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 NXP2; Zcwcc3; AI452146; BF318192; D16Jhu32e; 1110051N18Rik Expression Ubiquitous expression in CNS E18 (RPKM 8.1), CNS E11.5 (RPKM 7.9) and 27 other tissues See more Orthologs human all

Genomic context

Location: 16; 16 C4 See Morc3 in Genome Data Viewer

Exon count: 18

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (93832121..93876077)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (93832366..93876318)

Chromosome 16 - NC_000082.6

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Transcript information: This gene has 10 transcripts

Gene: Morc3 ENSMUSG00000039456

Description microrchidia 3 [Source:MGI Symbol;Acc:MGI:2136841] Gene Synonyms 1110051N18Rik, D16Jhu32e, Zcwcc3 Location Chromosome 16: 93,832,121-93,876,073 forward strand. GRCm38:CM001009.2 About this gene This gene has 10 transcripts (splice variants), 137 orthologues, 1 paralogue, 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

Morc3- ENSMUST00000202261.4 4179 942aa ENSMUSP00000144369.1 Protein coding CCDS79490 F7BJB9 TSL:1 206 GENCODE basic APPRIS P1

Morc3- ENSMUST00000044068.9 4178 942aa ENSMUSP00000040152.6 Protein coding - F7BJB9 TSL:5 201 GENCODE basic APPRIS P1

Morc3- ENSMUST00000201097.2 1386 397aa ENSMUSP00000144058.2 Protein coding - A0A0J9YU83 CDS 3' 202 incomplete TSL:5

Morc3- ENSMUST00000201754.3 1298 92aa ENSMUSP00000144357.1 Nonsense mediated - A0A0J9YUV3 TSL:1 205 decay

Morc3- ENSMUST00000232639.1 726 58aa ENSMUSP00000156167.1 Nonsense mediated - A0A338P7B1 CDS 5' 210 decay incomplete

Morc3- ENSMUST00000232425.1 2114 No - Retained intron - - - 209 protein

Morc3- ENSMUST00000202663.1 882 No - Retained intron - - TSL:3 207 protein

Morc3- ENSMUST00000201497.1 735 No - Retained intron - - TSL:2 203 protein

Morc3- ENSMUST00000201706.1 634 No - Retained intron - - TSL:3 204 protein

Morc3- ENSMUST00000231891.1 246 No - Retained intron - - - 208 protein

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63.95 kb Forward strand 93.83Mb 93.84Mb 93.85Mb 93.86Mb 93.87Mb 93.88Mb Genes (Comprehensive set... Morc3-201 >protein coding Chaf1b-204 >retained intron

Morc3-206 >protein coding Chaf1b-201 >protein coding

Morc3-203 >retained intron Morc3-208 >retained intron Morc3-210 >nonsense mediated decay Chaf1b-202 >protein coding

Morc3-202 >protein coding Chaf1b-206 >protein coding

Morc3-205 >nonsense mediated decay Chaf1b-203 >protein coding

Morc3-209 >retained intron

Morc3-207 >retained intronMorc3-204 >retained intron

Contigs < AC168220.1 Genes < Gm30881-201lncRNA (Comprehensive set...

Regulatory Build

93.83Mb 93.84Mb 93.85Mb 93.86Mb 93.87Mb 93.88Mb Reverse strand 63.95 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000202261

43.95 kb Forward strand

Morc3-206 >protein coding

ENSMUSP00000144... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Histidine kinase/HSP90-like ATPase superfamily Pfam PF13589 Morc, S5 domain 2-like

Zinc finger, CW-type PROSITE profiles Zinc finger, CW-type PANTHER PTHR23336

PTHR23336:SF17 Gene3D Histidine kinase/HSP90-like ATPase superfamily 3.30.40.100

CDD cd16931

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

Variant Legend frameshift variant missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 942

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

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