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Mouse Scamp2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Scamp2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Scamp2 gene (NCBI Reference Sequence: NM_022813.3 ; Ensembl: ENSMUSG00000040188 ) is located on Mouse chromosome 9. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000045791). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Scamp2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-364G16 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 2 starts from about 5.88% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 16082 bp, and the size of intron 4 for 3'-loxP site insertion: 1233 bp. The size of effective cKO region: ~2858 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 2 3 4 5 6 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Scamp2 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(9358bp) | A(23.81% 2228) | C(24.82% 2323) | T(26.38% 2469) | G(24.98% 2338)

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% chr9 + 57573944 57576943 3000 browser details YourSeq 348 1206 1612 3000 93.2% chrX - 140635868 140636282 415 browser details YourSeq 344 1206 1604 3000 96.3% chr11 + 97311921 98034683 722763 browser details YourSeq 339 1215 1612 3000 93.0% chr6 - 38373461 38373855 395 browser details YourSeq 336 1205 1612 3000 93.2% chr4 - 133949922 133950302 381 browser details YourSeq 332 1206 1604 3000 92.7% chr19 - 37058018 37058403 386 browser details YourSeq 330 1207 1604 3000 91.6% chr11 - 19999702 20000098 397 browser details YourSeq 327 1206 1600 3000 92.5% chr16 + 11262726 11263108 383 browser details YourSeq 326 1206 1604 3000 92.6% chr8 + 111476443 111476839 397 browser details YourSeq 325 1209 1604 3000 92.3% chr12 + 81278640 81279008 369 browser details YourSeq 321 1207 1604 3000 93.3% chr11 + 79271752 79272313 562 browser details YourSeq 317 1207 1604 3000 94.0% chr16 - 18295516 18439443 143928 browser details YourSeq 314 1206 1604 3000 96.5% chr8 - 11654565 11655098 534 browser details YourSeq 310 1206 1604 3000 94.3% chr3 - 116606434 116606943 510 browser details YourSeq 292 1168 1604 3000 87.6% chr1 - 138857628 138858032 405 browser details YourSeq 287 1242 1600 3000 95.3% chr18 + 80202127 80202690 564 browser details YourSeq 285 1219 1604 3000 89.5% chr4 + 127001383 127001743 361 browser details YourSeq 284 1216 1604 3000 93.3% chr9 - 78468313 78510382 42070 browser details YourSeq 278 1206 1603 3000 90.2% chr11 + 77672154 77672503 350 browser details YourSeq 263 1191 1559 3000 93.7% chr11 - 88062569 88063169 601

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% chr9 + 57579802 57582801 3000 browser details YourSeq 175 623 2963 3000 90.0% chr11 - 76403033 76663730 260698 browser details YourSeq 127 654 2811 3000 84.6% chr12 - 80199851 80315332 115482 browser details YourSeq 104 656 2810 3000 92.0% chr11 - 23187684 23253857 66174 browser details YourSeq 90 2698 2896 3000 89.7% chr16 + 84728601 84728928 328 browser details YourSeq 82 2703 2966 3000 86.8% chr1 + 191118194 191118504 311 browser details YourSeq 78 629 2750 3000 91.5% chr16 - 38328736 38456541 127806 browser details YourSeq 78 2468 2846 3000 92.4% chr11 - 98189861 98190242 382 browser details YourSeq 77 2658 2786 3000 81.4% chr5 - 143150808 143150964 157 browser details YourSeq 76 2531 2753 3000 92.4% chr9 + 87085714 87086023 310 browser details YourSeq 71 226 530 3000 87.4% chr17 + 83494131 83494483 353 browser details YourSeq 67 627 717 3000 92.5% chr8 - 124766791 124767168 378 browser details YourSeq 67 620 717 3000 88.6% chr1 - 127398089 127398193 105 browser details YourSeq 66 624 1123 3000 67.5% chr17 - 14215673 14215759 87 browser details YourSeq 66 623 737 3000 89.3% chr11 + 94190910 94191150 241 browser details YourSeq 65 2710 2845 3000 83.0% chr4 + 48158259 48158391 133 browser details YourSeq 65 627 717 3000 90.0% chr11 + 85940485 85940580 96 browser details YourSeq 64 2692 2846 3000 89.2% chr8 - 123524453 123524619 167 browser details YourSeq 63 2710 2846 3000 78.5% chr14 - 79358787 79358919 133 browser details YourSeq 61 623 697 3000 90.7% chr4 + 43869267 43869341 75

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Scamp2 secretory carrier membrane protein 2 [ Mus musculus (house mouse) ] Gene ID: 24044, updated on 26-Jun-2020

Gene summary

Official Symbol Scamp2 provided by MGI Official Full Name secretory carrier membrane protein 2 provided by MGI Primary source MGI:MGI:1346518 See related Ensembl:ENSMUSG00000040188 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 Sc2; AI875466 Expression Ubiquitous expression in large intestine adult (RPKM 92.7), testis adult (RPKM 90.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 B See Scamp2 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (57560944..57588798)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (57408751..57436605)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 6 transcripts

Gene: Scamp2 ENSMUSG00000040188

Description secretory carrier membrane protein 2 [Source:MGI Symbol;Acc:MGI:1346518] Gene Synonyms Sc2 Location Chromosome 9: 57,560,943-57,588,795 forward strand. GRCm38:CM001002.2 About this gene This gene has 6 transcripts (splice variants), 360 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Scamp2- ENSMUST00000045791.10 2340 329aa ENSMUSP00000038350.9 Protein coding CCDS23224 Q3TAL2 TSL:1 201 Q9ERN0 GENCODE basic APPRIS P1

Scamp2- ENSMUST00000216986.1 697 200aa ENSMUSP00000150920.1 Protein coding - A0A1L1SUU4 CDS 3' 205 incomplete TSL:3

Scamp2- ENSMUST00000215942.1 193 47aa ENSMUSP00000148996.1 Protein coding - A0A1L1SQD1 CDS 3' 203 incomplete TSL:5

Scamp2- ENSMUST00000217200.1 331 No - Processed - - TSL:2 206 protein transcript

Scamp2- ENSMUST00000216667.1 794 No - Retained intron - - TSL:2 204 protein

Scamp2- ENSMUST00000213662.1 560 No - Retained intron - - TSL:2 202 protein

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47.85 kb Forward strand 57.56Mb 57.57Mb 57.58Mb 57.59Mb Genes (Comprehensive set... Scamp2-201 >protein coding Ulk3-201 >protein coding

Scamp2-205 >protein coding Scamp2-206 >processed transcript Ulk3-203 >retained intron

Scamp2-202 >retained intron Scamp2-204 >retained intron

Scamp2-203 >protein coding Ulk3-205 >nonsense mediated decay

Ulk3-202 >retained intron

Ulk3-206 >retained intron

Ulk3-204 >retained intron

Ulk3-207 >retained intron

Contigs < AC164548.3 < AC122528.3 Genes < Mpi-201protein coding (Comprehensive set...

< Mpi-203retained intron

< Mpi-205nonsense mediated decay

< Mpi-202retained intron

Regulatory Build

57.56Mb 57.57Mb 57.58Mb 57.59Mb Reverse strand 47.85 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000045791

27.85 kb Forward strand

Scamp2-201 >protein coding

ENSMUSP00000038... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam SCAMP PANTHER PTHR10687:SF7

SCAMP

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 329

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

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