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Mouse Cdk5r1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Cdk5r1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Cdk5r1 (NCBI Reference Sequence: NM_009871 ; Ensembl: ENSMUSG00000048895 ) is located on Mouse 11. 1 exon is identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000053413). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cdk5r1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-91N11 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 the gene results in structural abnormalities of the brain such as a small corpus callosum and delaminated cerebral cortex. Mice show hyperactivity and decreased locomotion in response to stimulants.

Exon 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~954 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region gRNA region

Wildtype allele A T G T G 5' A 3'

1

Targeting vector A T G T G A

Targeted allele A T G T G A

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Cdk5r1 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(6921bp) | A(20.69% 1432) | C(25.56% 1769) | T(24.78% 1715) | G(28.97% 2005)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr11 + 80474510 80477509 3000 browser details YourSeq 77 213 473 3000 74.6% chr1 + 194831006 194831240 235 browser details YourSeq 66 239 475 3000 79.1% chr11 + 76535276 76535480 205 browser details YourSeq 54 185 267 3000 82.4% chr1 - 167812415 167812492 78 browser details YourSeq 50 204 267 3000 91.7% chrY + 17558050 17558120 71 browser details YourSeq 50 204 267 3000 91.7% chrY + 12844829 12844899 71 browser details YourSeq 49 406 475 3000 81.0% chr19 + 60591624 60591689 66 browser details YourSeq 48 217 272 3000 92.9% chr9 + 20732820 20732875 56 browser details YourSeq 46 215 272 3000 89.7% chr14 - 17163160 17163217 58 browser details YourSeq 42 214 267 3000 88.9% chr11 + 60118779 60118832 54 browser details YourSeq 39 413 474 3000 76.4% chr12 - 51124603 51124660 58 browser details YourSeq 39 215 267 3000 86.8% chr19 + 46410581 46410633 53 browser details YourSeq 39 2139 2189 3000 78.6% chr19 + 27584013 27584055 43 browser details YourSeq 38 1151 1195 3000 95.3% chr11 + 43672298 43672344 47 browser details YourSeq 37 216 266 3000 86.3% chrX + 89617061 89617111 51 browser details YourSeq 36 228 267 3000 95.0% chr5 - 101763185 101763224 40 browser details YourSeq 36 413 475 3000 85.4% chr3 - 28977339 28977399 61 browser details YourSeq 36 216 267 3000 84.7% chr1 - 141175671 141175722 52 browser details YourSeq 31 429 471 3000 86.1% chr13 - 80822051 80822093 43 browser details YourSeq 30 234 267 3000 94.2% chr14 + 46488634 46488667 34

Note: The 3000 bp section upstream of Exon 1 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% chr11 + 80478431 80481430 3000 browser details YourSeq 36 1326 1364 3000 100.0% chr1 - 185163498 185163550 53 browser details YourSeq 26 1339 1364 3000 100.0% chr4 + 148251171 148251196 26 browser details YourSeq 25 1339 1364 3000 100.0% chr11 + 116577390 116577416 27

Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Cdk5r1 cyclin-dependent kinase 5, regulatory subunit 1 (p35) [ Mus musculus (house mouse) ] Gene ID: 12569, updated on 10-Oct-2019

Gene summary

Official Symbol Cdk5r1 provided by MGI Official Full Name cyclin-dependent kinase 5, regulatory subunit 1 (p35) provided by MGI Primary source MGI:MGI:101764 See related Ensembl:ENSMUSG00000048895 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 p25; p35; Cdk5r; D11Bwg0379e Orthologs human all

Genomic context

Location: 11 B5; 11 47.94 cM See Cdk5r1 in Genome Data Viewer Exon count: 1

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (80477023..80481184)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (80290548..80294681)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 2 transcripts

Gene: Cdk5r1 ENSMUSG00000048895

Description cyclin-dependent kinase 5, regulatory subunit 1 (p35) [Source:MGI Symbol;Acc:MGI:101764] Gene Synonyms D11Bwg0379e, p25, p35 Location Chromosome 11: 80,477,023-80,481,184 forward strand. GRCm38:CM001004.2 About this gene This gene has 2 transcripts (splice variants), 242 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 41 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Cdk5r1-201 ENSMUST00000053413.11 4162 307aa ENSMUSP00000099514.4 Protein coding CCDS25134 P61809 Q542T9 TSL:NA GENCODE basic APPRIS P1

Cdk5r1-202 ENSMUST00000147694.1 659 138aa ENSMUSP00000120964.1 Protein coding - B1AQH0 CDS 3' incomplete TSL:1

24.16 kb Forward strand 80.47Mb 80.48Mb 80.49Mb (Comprehensive set... Psmd11-214 >protein coding Cdk5r1-201 >protein coding

Psmd11-201 >protein coding Cdk5r1-202 >protein coding

Psmd11-204 >retained intron

Psmd11-209 >nonsense mediated decay

Psmd11-215 >nonsense mediated decay

Psmd11-205 >protein coding

Psmd11-203 >protein coding

Psmd11-213 >protein coding

Psmd11-202 >nonsense mediated decay

Contigs AL591146.21 > Genes < Myo1d-201protein coding (Comprehensive set...

Regulatory Build

80.47Mb 80.48Mb 80.49Mb Reverse strand 24.16 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000053413

4.16 kb Forward strand

Cdk5r1-201 >protein coding

ENSMUSP00000099... MobiDB lite Low complexity (Seg) Superfamily Cyclin-like superfamily

Pfam Cyclin-dependent kinase 5 activator

PIRSF Cyclin-dependent kinase 5 activator

PANTHER PTHR23401

PTHR23401:SF2 Gene3D 1.10.472.10

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 307

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

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