https://www.alphaknockout.com

Mouse Pank1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Pank1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Pank1 gene (NCBI Reference Sequence: NM_001114339 ; Ensembl: ENSMUSG00000033610 ) is located on Mouse chromosome 19. 7 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 7 (Transcript: ENSMUST00000036584). 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 Pank1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-70P9 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 mutant has increased body weight, polyphagia, decreased serum triglyceride and glucose levels after fasting.

Exon 3 starts from about 55.17% 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: 13383 bp, and the size of intron 3 for 3'-loxP site insertion: 5230 bp. The size of effective cKO region: ~754 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Pank1 Homology arm cKO region loxP site

Page 2 of 7 https://www.alphaknockout.com

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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7254bp) | A(24.79% 1798) | C(22.24% 1613) | T(30.78% 2233) | G(22.19% 1610)

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.

Page 3 of 7 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr19 - 34827624 34830623 3000 browser details YourSeq 81 1791 1927 3000 96.7% chr15 + 37471779 37730621 258843 browser details YourSeq 78 1791 1874 3000 95.0% chr12 - 10449688 10449769 82 browser details YourSeq 76 1791 1893 3000 95.3% chr14 - 35930780 35930884 105 browser details YourSeq 72 1819 1934 3000 83.2% chr16 + 8221634 8221733 100 browser details YourSeq 71 1808 1905 3000 83.4% chr1 - 168499323 168499400 78 browser details YourSeq 70 1791 1875 3000 88.0% chr3 - 49685235 49685317 83 browser details YourSeq 70 1791 1870 3000 92.4% chr13 - 83870526 83870604 79 browser details YourSeq 69 1791 1875 3000 95.0% chr12 - 98950223 98950319 97 browser details YourSeq 69 1791 1863 3000 98.7% chr1 + 143871582 143871828 247 browser details YourSeq 68 1791 1878 3000 94.9% chr17 - 47509677 47509908 232 browser details YourSeq 68 1796 1875 3000 92.5% chr11 - 79791750 79791829 80 browser details YourSeq 68 1791 1874 3000 94.8% chr1 - 155232258 155232349 92 browser details YourSeq 68 1786 1863 3000 94.9% chr15 + 86626584 86626667 84 browser details YourSeq 67 1809 1903 3000 96.0% chr14 - 21621579 21621675 97 browser details YourSeq 67 1791 1863 3000 97.2% chr10 + 116788885 116788986 102 browser details YourSeq 66 1791 1864 3000 97.2% chr1 - 176945658 176945783 126 browser details YourSeq 63 1819 1931 3000 94.4% chr16 + 39487209 39487337 129 browser details YourSeq 63 1809 1937 3000 94.4% chr10 + 94128914 94129052 139 browser details YourSeq 62 1798 1870 3000 95.8% chr11 + 114637076 114637184 109

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% chr19 - 34823870 34826869 3000 browser details YourSeq 49 1844 1912 3000 80.7% chr11 - 8114725 8114789 65 browser details YourSeq 45 1834 1890 3000 89.5% chr11 + 35969033 35969089 57 browser details YourSeq 44 1844 1898 3000 90.8% chr6 - 91812628 91812683 56 browser details YourSeq 44 1827 1890 3000 92.4% chr11 - 35558831 35558900 70 browser details YourSeq 44 1841 1894 3000 86.8% chr7 + 64345416 64345468 53 browser details YourSeq 44 1293 1369 3000 79.5% chr10 + 61469661 61469736 76 browser details YourSeq 43 1357 1889 3000 54.0% chr15 - 80814394 80814443 50 browser details YourSeq 42 2203 2276 3000 76.5% chr14 - 69773161 69773226 66 browser details YourSeq 42 1346 1404 3000 92.0% chr13 - 95212865 95212928 64 browser details YourSeq 42 1853 1910 3000 80.4% chr1 + 105254797 105254849 53 browser details YourSeq 39 2325 2400 3000 95.4% chr12 - 108021895 108022281 387 browser details YourSeq 39 1357 1887 3000 50.0% chr12 - 12837451 12837498 48 browser details YourSeq 39 1841 1891 3000 88.3% chr1 - 37700931 37700981 51 browser details YourSeq 39 1844 1890 3000 91.5% chr16 + 55842305 55842351 47 browser details YourSeq 39 2325 2368 3000 88.1% chr12 + 70449183 70449224 42 browser details YourSeq 39 1843 1891 3000 89.8% chr11 + 23165925 23165973 49 browser details YourSeq 39 1841 1889 3000 89.8% chr1 + 112451495 112451543 49 browser details YourSeq 38 1844 1891 3000 89.6% chr2 - 45986126 45986173 48 browser details YourSeq 38 1841 1890 3000 88.7% chr10 + 78404003 78404051 49

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 https://www.alphaknockout.com

Gene and protein information: Pank1 pantothenate kinase 1 [ Mus musculus (house mouse) ] Gene ID: 75735, updated on 10-Oct-2019

Gene summary

Official Symbol Pank1 provided by MGI Official Full Name pantothenate kinase 1 provided by MGI Primary source MGI:MGI:1922985 See related Ensembl:ENSMUSG00000033610 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 Pank; Pank1b Expression Ubiquitous expression in ovary adult (RPKM 4.1), liver adult (RPKM 3.2) and 28 other tissues See more Orthologs human all

Genomic context

Location: 19 C1; 19 See Pank1 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (34806935..34883423, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (34885384..34953945, complement)

Chromosome 19 - NC_000085.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 4 transcripts

Gene: Pank1 ENSMUSG00000033610

Description pantothenate kinase 1 [Source:MGI Symbol;Acc:MGI:1922985] Gene Synonyms 4632412I06Rik, 5430426F23Rik, Pank1, Pank1a, Pank1b Location Chromosome 19: 34,806,940-34,879,455 reverse strand. GRCm38:CM001012.2 About this gene This gene has 4 transcripts (splice variants), 210 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 14 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Pank1-201 ENSMUST00000036584.12 7414 548aa ENSMUSP00000043562.5 Protein coding CCDS50424 Q8K4K6 TSL:1 GENCODE basic

Pank1-202 ENSMUST00000112460.2 2760 373aa ENSMUSP00000108079.1 Protein coding CCDS37966 Q543J7 Q8K4K6 TSL:1 GENCODE basic APPRIS P1

Pank1-204 ENSMUST00000238065.1 2342 251aa ENSMUSP00000157555.1 Protein coding - A0A494B9A0 GENCODE basic

Pank1-203 ENSMUST00000237577.1 1138 No protein - Retained intron - - -

92.52 kb Forward strand

34.80Mb 34.82Mb 34.84Mb 34.86Mb 34.88Mb Genes Gm50116-201 >processed pseudogene (Comprehensive set...

Contigs < AC125259.8 AC113244.19 > Genes (Comprehensive set... < Pank1-201protein coding

< Pank1-202protein coding

< Pank1-204protein coding

< Mir107-201miRNA < Pank1-203retained intron

Regulatory Build

34.80Mb 34.82Mb 34.84Mb 34.86Mb 34.88Mb Reverse strand 92.52 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

pseudogene processed transcript RNA gene

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000036584

< Pank1-201protein coding

Reverse strand 72.52 kb

ENSMUSP00000043... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) TIGRFAM Type II pantothenate kinase Superfamily SSF53067 Pfam Type II pantothenate kinase PANTHER PTHR12280

PTHR12280:SF23 Gene3D 3.30.420.40

1.10.8.780

3.30.420.510

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

Variant Legend missense variant stop retained variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 548

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

Page 7 of 7