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Mouse Pcsk6 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pcsk6 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pcsk6 gene (NCBI Reference Sequence: NM_011048 ; Ensembl: ENSMUSG00000030513 ) is located on Mouse chromosome 7. 22 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 22 (Transcript: ENSMUST00000055576). Exon 7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pcsk6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-187G21 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: Homozygous mutation of this gene results in partial lethality by E15.5. Embryos develop situs ambiguus with left pulmonary isomerism or craniofacial malformations including cyclopia, or both.

Exon 7 starts from about 27.6% of the coding region. The knockout of Exon 7 will result in frameshift of the gene. The size of intron 6 for 5'-loxP site insertion: 21153 bp, and the size of intron 7 for 3'-loxP site insertion: 6005 bp. The size of effective cKO region: ~673 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 7 22 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pcsk6 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(7173bp) | A(26.43% 1896) | C(23.34% 1674) | T(26.4% 1894) | G(23.83% 1709)

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% chr7 + 65949670 65952669 3000 browser details YourSeq 248 1406 1749 3000 92.8% chr16 + 32450395 32450776 382 browser details YourSeq 241 1424 1750 3000 92.7% chr10 + 105605501 105606152 652 browser details YourSeq 236 1415 1719 3000 93.4% chr1 - 180466933 180467697 765 browser details YourSeq 235 1423 1733 3000 93.7% chr5 - 113725020 113725487 468 browser details YourSeq 231 1420 1721 3000 91.7% chr13 - 35722601 35722976 376 browser details YourSeq 230 1435 1718 3000 92.0% chr2 - 91417560 91417905 346 browser details YourSeq 230 1420 1718 3000 93.0% chr16 - 11162986 11163315 330 browser details YourSeq 228 1453 1715 3000 94.6% chrX - 94040121 94040430 310 browser details YourSeq 228 1429 1719 3000 90.8% chr4 + 41847817 41848173 357 browser details YourSeq 227 1420 1718 3000 91.7% chr3 - 135668447 135668761 315 browser details YourSeq 227 1420 1715 3000 92.9% chr8 + 3326575 3326873 299 browser details YourSeq 227 1420 1725 3000 93.8% chr18 + 36623364 36623713 350 browser details YourSeq 226 1435 1718 3000 91.6% chr11 + 95933607 95933947 341 browser details YourSeq 224 1422 1720 3000 93.5% chr3 - 88589432 88590082 651 browser details YourSeq 224 1420 1718 3000 93.8% chr15 - 67511605 67511936 332 browser details YourSeq 223 1422 1718 3000 92.8% chr5 - 128978153 128978795 643 browser details YourSeq 223 1420 1719 3000 92.8% chr12 - 40924398 40924718 321 browser details YourSeq 223 1448 1719 3000 94.1% chr2 + 128725465 128725787 323 browser details YourSeq 222 1423 1724 3000 92.1% chr9 + 41625587 41625948 362

Note: The 3000 bp section upstream of Exon 7 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% chr7 + 65953343 65956342 3000 browser details YourSeq 94 1028 1163 3000 85.2% chr9 + 108420060 108589997 169938 browser details YourSeq 88 651 1084 3000 83.1% chr13 + 30160614 30161041 428 browser details YourSeq 87 922 1127 3000 72.7% chr9 + 107335140 107335323 184 browser details YourSeq 84 970 1135 3000 89.7% chr2 + 6255250 6255427 178 browser details YourSeq 83 1005 1131 3000 85.6% chr1 + 89910967 89911097 131 browser details YourSeq 82 1002 1127 3000 82.6% chr18 + 20577123 20577248 126 browser details YourSeq 81 1013 1135 3000 82.4% chr4 - 140553799 140553920 122 browser details YourSeq 81 1013 1135 3000 83.0% chr11 + 58096624 58096746 123 browser details YourSeq 80 1001 1127 3000 81.6% chr10 - 93617690 93617816 127 browser details YourSeq 78 1013 1134 3000 82.0% chr15 + 74648561 74648682 122 browser details YourSeq 77 1016 1131 3000 86.0% chr6 + 124948020 124948137 118 browser details YourSeq 76 1041 1252 3000 82.4% chr10 - 8894045 8894269 225 browser details YourSeq 75 1045 1318 3000 70.3% chr6 - 124876993 124877120 128 browser details YourSeq 73 1060 1157 3000 83.9% chr11 - 119932800 119932893 94 browser details YourSeq 73 912 1098 3000 89.2% chr11 - 101359257 101359451 195 browser details YourSeq 72 1028 1128 3000 86.2% chr15 - 100897126 101187667 290542 browser details YourSeq 72 971 1166 3000 82.4% chr15 - 83242966 83243159 194 browser details YourSeq 72 971 1138 3000 77.4% chrX + 65250547 65250696 150 browser details YourSeq 71 1055 1247 3000 90.7% chr16 + 33924771 33925030 260

Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Pcsk6 proprotein convertase subtilisin/kexin type 6 [ Mus musculus (house mouse) ] Gene ID: 18553, updated on 10-Sep-2019

Gene summary

Official Symbol Pcsk6 provided by MGI Official Full Name proprotein convertase subtilisin/kexin type 6 provided by MGI Primary source MGI:MGI:102897 See related Ensembl:ENSMUSG00000030513 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 SPC4; Pace4; C86343; b2b2830Clo Expression Ubiquitous expression in liver adult (RPKM 17.9), cerebellum adult (RPKM 13.1) and 26 other tissuesS ee more Orthologs human all

Genomic context

Location: 7 C; 7 35.36 cM See Pcsk6 in Genome Data Viewer

Exon count: 22

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (65862136..66050386)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (73007022..73195272)

Chromosome 7 - NC_000073.6

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Transcript information: This gene has 6 transcripts

Gene: Pcsk6 ENSMUSG00000030513

Description proprotein convertase subtilisin/kexin type 6 [Source:MGI Symbol;Acc:MGI:102897] Gene Synonyms PACE4, SPC4, b2b2830Clo Location Chromosome 7: 65,861,734-66,050,386 forward strand. GRCm38:CM001000.2 About this gene This gene has 6 transcripts (splice variants), 189 orthologues, 9 paralogues, is a member of 1 Ensembl protein family and is associated with 27 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pcsk6-201 ENSMUST00000055576.11 4405 959aa ENSMUSP00000053742.5 Protein coding CCDS52267 F6XJP7 TSL:5 GENCODE basic APPRIS P3

Pcsk6-202 ENSMUST00000098391.10 4207 946aa ENSMUSP00000095992.4 Protein coding CCDS71978 E9Q4D0 TSL:1 GENCODE basic APPRIS ALT2

Pcsk6-204 ENSMUST00000176209.1 3734 798aa ENSMUSP00000135033.1 Protein coding - H3BJL4 CDS 5' incomplete TSL:5

Pcsk6-203 ENSMUST00000176199.7 619 164aa ENSMUSP00000135851.1 Protein coding - H3BLN2 CDS 3' incomplete TSL:3

Pcsk6-205 ENSMUST00000177272.1 681 No protein - Retained intron - - TSL:5

Pcsk6-206 ENSMUST00000177369.1 583 No protein - Retained intron - - TSL:5

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208.65 kb Forward strand 65.90Mb 65.95Mb 66.00Mb 66.05Mb Genes (Comprehensive set... Pcsk6-203 >protein coding Pcsk6-206 >retained intron Pcsk6-205 >retained intron

Pcsk6-201 >protein coding

Pcsk6-202 >protein coding

Pcsk6-204 >protein coding

Snrpa1-206 >protein coding

Gm45213-201 >protein coding

Snrpa1-207 >lncRNA

Snrpa1-202 >lncRNA

Contigs < AC165968.3 < AC132595.3 AC148973.4 > Genes < Gm20684-201lncRNA (Comprehensive set...

Regulatory Build

65.90Mb 65.95Mb 66.00Mb 66.05Mb Reverse strand 208.65 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000055576

188.36 kb Forward strand

Pcsk6-201 >protein coding

ENSMUSP00000053... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily SSF54897 Galactose-binding-like domain superfamily

Peptidase S8/S53 domain superfamily Growth factor receptor cysteine-rich domain superfamily SMART EGF-like domain

Furin-like repeat Prints Peptidase S8, subtilisin-related Pfam Peptidase S8, pro-domain P domain Growth factor receptor domain 4 PLAC

Peptidase S8/S53 domain PROSITE profiles P domain PLAC

PROSITE patterns Peptidase S8, subtilisin, His-active site

Peptidase S8, subtilisin, Asp-active site

Peptidase S8, subtilisin, Ser-active site PANTHER PTHR42884:SF8

PTHR42884 Gene3D Peptidase S8, pro-domain superfamily 2.10.220.10

Peptidase S8/S53 domain superfamily Galactose-binding-like domain superfamily CDD Kexin/furin catalytic domain Furin-like repeat

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 959

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

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