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Mouse Cdc5l Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cdc5l conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cdc5l gene (NCBI Reference Sequence: NM_152810 ; Ensembl: ENSMUSG00000023932 ) is located on Mouse chromosome 17. 16 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000024727). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cdc5l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-88C12 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 1.91% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 5348 bp, and the size of intron 2 for 3'-loxP site insertion: 1257 bp. The size of effective cKO region: ~604 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 2 3 4 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cdc5l 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(7104bp) | A(25.39% 1804) | C(19.82% 1408) | T(32.25% 2291) | G(22.54% 1601)

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% chr17 - 45428293 45431292 3000 browser details YourSeq 1563 871 2865 3000 93.3% chr14 - 14503573 14883193 379621 browser details YourSeq 1500 881 2865 3000 92.0% chr13 + 64653646 64655464 1819 browser details YourSeq 1478 701 2506 3000 93.3% chr6 + 73683724 73858749 175026 browser details YourSeq 1443 943 2865 3000 91.0% chr10 - 48799921 48801687 1767 browser details YourSeq 1430 320 2505 3000 90.3% chr17 - 93955031 93956982 1952 browser details YourSeq 1412 1050 2865 3000 93.7% chr3 - 9043619 9045476 1858 browser details YourSeq 1408 340 2505 3000 89.6% chr9 + 10145940 10147861 1922 browser details YourSeq 1393 319 2504 3000 89.6% chr4 - 75673880 75675820 1941 browser details YourSeq 1371 701 2501 3000 92.7% chr1 + 166656302 166657938 1637 browser details YourSeq 1347 701 2485 3000 92.0% chr6 + 36822150 36823770 1621 browser details YourSeq 1346 701 2504 3000 91.5% chr2 - 150066414 150068044 1631 browser details YourSeq 1325 706 2398 3000 90.5% chr12 - 42487164 42488960 1797 browser details YourSeq 1321 709 2505 3000 90.3% chr11 + 38266848 38268477 1630 browser details YourSeq 1312 730 2504 3000 91.2% chr13 + 10276131 10277743 1613 browser details YourSeq 1294 707 2486 3000 90.8% chr5 - 40056685 40058302 1618 browser details YourSeq 1292 727 2501 3000 90.3% chr13 - 75565476 75567085 1610 browser details YourSeq 1289 706 2507 3000 90.3% chr9 - 12942810 12944478 1669 browser details YourSeq 1282 705 2505 3000 90.0% chr12 + 101305399 101307016 1618 browser details YourSeq 1278 701 2433 3000 91.9% chr2 - 60655243 60656810 1568

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% chr17 - 45424689 45427688 3000 browser details YourSeq 294 680 1837 3000 96.3% chr10 + 118039828 118253697 213870 browser details YourSeq 272 1007 1837 3000 90.1% chr10 - 118245771 118246061 291 browser details YourSeq 270 1007 1837 3000 89.7% chr10 - 118348241 118348531 291 browser details YourSeq 270 1007 1837 3000 89.7% chr10 - 118342817 118343107 291 browser details YourSeq 270 1007 1837 3000 89.7% chr10 - 118331970 118332260 291 browser details YourSeq 270 1007 1837 3000 89.7% chr10 - 118326531 118326821 291 browser details YourSeq 269 1007 1837 3000 90.0% chr10 - 118240342 118240632 291 browser details YourSeq 269 1007 1837 3000 90.0% chr10 + 118258836 118259126 291 browser details YourSeq 268 1007 1837 3000 89.4% chr10 - 118337393 118337683 291 browser details YourSeq 268 1007 1837 3000 89.4% chr10 - 118353667 118353957 291 browser details YourSeq 129 452 714 3000 83.3% chr14 - 70228513 70228698 186 browser details YourSeq 123 487 737 3000 89.0% chr11 + 115721217 115721584 368 browser details YourSeq 122 487 671 3000 91.3% chr5 + 114896121 114896682 562 browser details YourSeq 118 466 699 3000 88.4% chr1 + 155512791 155513320 530 browser details YourSeq 111 484 711 3000 83.1% chr8 + 105208895 105209040 146 browser details YourSeq 110 487 666 3000 92.4% chr18 + 77798192 77798512 321 browser details YourSeq 110 484 756 3000 81.2% chr17 + 84780889 84781030 142 browser details YourSeq 106 484 699 3000 83.9% chr11 - 4935974 4936101 128 browser details YourSeq 105 484 697 3000 80.0% chr14 + 76184051 76184192 142

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cdc5l cell division cycle 5-like (S. pombe) [ Mus musculus (house mouse) ] Gene ID: 71702, updated on 12-Aug-2019

Gene summary

Official Symbol Cdc5l provided by MGI Official Full Name cell division cycle 5-like (S. pombe) provided by MGI Primary source MGI:MGI:1918952 See related Ensembl:ENSMUSG00000023932 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as PCDC5RP; AA408004; 1200002I02Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 14.3), CNS E14 (RPKM 10.2) and 28 other tissues See more Orthologs human all

Genomic context

Location: 17; 17 B3 See Cdc5l in Genome Data Viewer

Exon count: 17

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (45391887..45433707, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (45528836..45570656, complement)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 3 transcripts

Gene: Cdc5l ENSMUSG00000023932

Description cell division cycle 5-like (S. pombe) [Source:MGI Symbol;Acc:MGI:1918952] Gene Synonyms 1200002I02Rik, PCDC5RP Location Chromosome 17: 45,391,884-45,433,737 reverse strand. GRCm38:CM001010.2 About this gene This gene has 3 transcripts (splice variants), 207 orthologues, 15 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cdc5l-201 ENSMUST00000024727.9 3027 802aa ENSMUSP00000024727.8 Protein coding CCDS37627 Q3UCF2 Q6A068 TSL:1 GENCODE basic APPRIS P1

Cdc5l-203 ENSMUST00000232910.1 418 No protein - Retained intron - - -

Cdc5l-202 ENSMUST00000232900.1 764 No protein - lncRNA - - -

61.85 kb Forward strand 45.39Mb 45.40Mb 45.41Mb 45.42Mb 45.43Mb 45.44Mb Genes B230354K17Rik-202 >lncRNA (Comprehensive set...

B230354K17Rik-204 >lncRNA

B230354K17Rik-201 >lncRNA

B230354K17Rik-203 >lncRNA

Contigs AC169676.2 > < AC163677.2 Genes (Comprehensive set... < Cdc5l-201protein coding < Spats1-202lncRNA

< Cdc5l-202lncRNA < Cdc5l-203retained intron

Regulatory Build

45.39Mb 45.40Mb 45.41Mb 45.42Mb 45.43Mb 45.44Mb Reverse strand 61.85 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000024727

< Cdc5l-201protein coding

Reverse strand 41.85 kb

ENSMUSP00000024... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Homeobox-like domain superfamily SMART SANT/Myb domain Pfam PF13921 Pre-mRNA splicing factor component Cdc5p/Cef1

PROSITE profiles Myb domain PANTHER PTHR45885

Gene3D 1.10.10.60 CDD cd11659

SANT/Myb domain

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

Variant Legend stop gained missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 802

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

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