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Mouse Cdk5rap2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cdk5rap2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cdk5rap2 gene (NCBI Reference Sequence: NM_145990 ; Ensembl: ENSMUSG00000039298 ) is located on Mouse chromosome 4. 37 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 37 (Transcript: ENSMUST00000144099). 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 Cdk5rap2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-98J17 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 phenotype varies by strain background. Severely affected mutants exhibit small size, severe anemia, and neonatal death. Mildly affected mutants are viable with mild macrocytic anemia, reduced fertility and radiation senstitivity.

Exon 3 starts from about 2.29% 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: 2161 bp, and the size of intron 3 for 3'-loxP site insertion: 20943 bp. The size of effective cKO region: ~568 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 37 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Cdk5rap2 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(7068bp) | A(25.34% 1791) | C(22.82% 1613) | T(29.36% 2075) | G(22.48% 1589)

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% chr4 - 70401563 70404562 3000 browser details YourSeq 252 1 298 3000 92.6% chr12 - 21793435 21908421 114987 browser details YourSeq 241 1 279 3000 94.6% chr12 + 18604398 18723919 119522 browser details YourSeq 229 1 311 3000 92.3% chr7 - 133745575 133745978 404 browser details YourSeq 228 1 298 3000 92.0% chr12 + 19481119 19690392 209274 browser details YourSeq 218 2 274 3000 92.7% chr16 + 21713933 21714461 529 browser details YourSeq 197 1 249 3000 93.4% chr12 + 75654902 75655259 358 browser details YourSeq 191 2 274 3000 94.1% chr18 + 77018161 77018722 562 browser details YourSeq 190 41 308 3000 91.7% chr17 - 46902125 46902468 344 browser details YourSeq 183 1 313 3000 93.8% chr4 + 149962561 149962957 397 browser details YourSeq 175 2 290 3000 87.6% chrX + 111523700 111523989 290 browser details YourSeq 174 28 311 3000 87.9% chr11 - 85328291 85328791 501 browser details YourSeq 173 1 354 3000 88.8% chr16 + 23554104 23554421 318 browser details YourSeq 169 1 183 3000 96.2% chr4 + 116795670 116795852 183 browser details YourSeq 169 1 202 3000 94.4% chr10 + 41877827 41878088 262 browser details YourSeq 167 1 313 3000 87.1% chr8 + 72776659 72776844 186 browser details YourSeq 166 1 308 3000 86.7% chr11 - 75974524 75974724 201 browser details YourSeq 166 2 527 3000 84.2% chr4 + 4939730 4939946 217 browser details YourSeq 165 2 183 3000 95.6% chr2 - 168908226 168908408 183 browser details YourSeq 165 1 183 3000 95.7% chr13 + 30060180 30060377 198

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% chr4 - 70397995 70400994 3000 browser details YourSeq 432 2233 2818 3000 88.6% chr1 + 65406436 65407028 593 browser details YourSeq 391 2235 2834 3000 88.3% chr1 + 150220648 150221264 617 browser details YourSeq 383 2236 2834 3000 86.1% chr16 + 20791478 20792093 616 browser details YourSeq 382 2213 2798 3000 85.1% chr4 - 5874255 5874864 610 browser details YourSeq 381 2242 2847 3000 88.0% chr8 - 24355246 24355862 617 browser details YourSeq 375 2203 2768 3000 86.3% chr6 + 115685015 115685593 579 browser details YourSeq 374 2236 2788 3000 86.6% chr16 - 35013656 35014220 565 browser details YourSeq 372 2239 2788 3000 86.5% chr2 - 83637337 83637871 535 browser details YourSeq 371 2236 2826 3000 84.4% chr10 + 57349677 57350270 594 browser details YourSeq 370 2188 2796 3000 85.4% chr15 + 64489844 64490477 634 browser details YourSeq 368 2190 2834 3000 86.2% chr4 - 18667566 18668428 863 browser details YourSeq 366 2239 2788 3000 84.9% chrX + 139058640 139059202 563 browser details YourSeq 360 2217 2834 3000 85.4% chr2 - 61238699 61239329 631 browser details YourSeq 353 2236 2834 3000 85.4% chr17 - 51677547 51678120 574 browser details YourSeq 350 2185 2834 3000 85.9% chr2 + 76399635 76400363 729 browser details YourSeq 349 2185 2834 3000 83.0% chr13 + 97355072 97355707 636 browser details YourSeq 348 2186 2758 3000 84.8% chr15 - 94089579 94090161 583 browser details YourSeq 346 2257 2817 3000 83.3% chr8 + 41459636 41460199 564 browser details YourSeq 346 2297 2793 3000 88.2% chr12 + 74321651 74322157 507

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: Cdk5rap2 CDK5 regulatory subunit associated protein 2 [ Mus musculus (house mouse) ] Gene ID: 214444, updated on 12-Aug-2019

Gene summary

Official Symbol Cdk5rap2 provided by MGI Official Full Name CDK5 regulatory subunit associated protein 2 provided by MGI Primary source MGI:MGI:2384875 See related Ensembl:ENSMUSG00000039298 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 an; mKIAA1633; 2900018K03Rik Expression Broad expression in testis adult (RPKM 11.0), CNS E11.5 (RPKM 5.7) and 23 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 C2 See Cdk5rap2 in Genome Data Viewer

Exon count: 37

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (70216855..70410435, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (69884058..70071401, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 6 transcripts

Gene: Cdk5rap2 ENSMUSG00000039298

Description CDK5 regulatory subunit associated protein 2 [Source:MGI Symbol;Acc:MGI:2384875] Gene Synonyms 2900018K03Rik, an Location Chromosome 4: 70,216,856-70,410,443 reverse strand. GRCm38:CM000997.2 About this gene This gene has 6 transcripts (splice variants), 193 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 45 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cdk5rap2- ENSMUST00000144099.7 11847 1822aa ENSMUSP00000119891.1 Protein coding CCDS38779 Q8K389 TSL:1 206 GENCODE basic APPRIS P1

Cdk5rap2- ENSMUST00000138561.7 1171 390aa ENSMUSP00000116928.1 Protein coding - Q5SP75 CDS 5' and 3' 204 incomplete TSL:5

Cdk5rap2- ENSMUST00000140108.1 711 92aa ENSMUSP00000119151.1 Protein coding - F6R2K5 CDS 5' 205 incomplete TSL:3

Cdk5rap2- ENSMUST00000076541.12 5248 76aa ENSMUSP00000075856.6 Nonsense mediated - H7BX43 TSL:1 201 decay

Cdk5rap2- ENSMUST00000124251.1 582 No - lncRNA - - TSL:5 202 protein

Cdk5rap2- ENSMUST00000126416.1 517 No - lncRNA - - TSL:3 203 protein

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213.59 kb Forward strand 70.25Mb 70.30Mb 70.35Mb 70.40Mb Contigs AL929409.5 > AL845502.5 > Genes (Comprehensive set... < Cdk5rap2-206protein coding

< Cdk5rap2-201nonsense mediated decay

< Cdk5rap2-204protein coding < Cdk5rap2-203lncRNA

< Cdk5rap2-202lncRNA < Gm26320-201misc RNA

< Ywhaq-ps3-201transcribed processed pseudogene

< Cdk5rap2-205protein coding

Regulatory Build

70.25Mb 70.30Mb 70.35Mb 70.40Mb Reverse strand 213.59 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene pseudogene processed transcript

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Transcript: ENSMUST00000144099

< Cdk5rap2-206protein coding

Reverse strand 193.59 kb

ENSMUSP00000119... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Centrosomin, N-terminal motif 1 PANTHER PTHR46930

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1822

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

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