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Mouse Spdya Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Spdya gene (NCBI Reference Sequence: NM_029254 ; Ensembl: ENSMUSG00000052525 ) is located on Mouse chromosome 17. 7 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000167641). 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 Spdya gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-456J14 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 inactivation of this gene results in impaired telomere attachment to the nuclear envelope during early meiosis, abnormal chromosome pairing and homologous synapsis, and meiotic prophase I arrest in male and female germ cells leading to infertility in both sexes.

Exon 2 starts from about 100% 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: 4098 bp, and the size of intron 2 for 3'-loxP site insertion: 2131 bp. The size of effective cKO region: ~732 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 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Spdya 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(7232bp) | A(29.85% 2159) | C(20.26% 1465) | T(32.65% 2361) | G(17.24% 1247)

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 + 71553009 71556008 3000 browser details YourSeq 209 995 1707 3000 94.2% chr18 + 66522605 66986681 464077 browser details YourSeq 139 954 1170 3000 91.8% chr15 + 97668756 97669250 495 browser details YourSeq 132 994 1617 3000 81.9% chr1 - 192118430 192118940 511 browser details YourSeq 132 1543 2064 3000 91.2% chr11 + 6375061 6375692 632 browser details YourSeq 130 999 1170 3000 94.2% chr11 - 112637650 112637844 195 browser details YourSeq 130 1010 1635 3000 77.4% chr1 - 87830437 87830670 234 browser details YourSeq 127 1024 1280 3000 90.6% chr10 - 94972023 94972346 324 browser details YourSeq 127 994 1170 3000 90.2% chr12 + 55929340 55929513 174 browser details YourSeq 125 1078 1711 3000 76.2% chr14 + 34410184 34410631 448 browser details YourSeq 123 1016 1617 3000 81.0% chr1 + 41451857 41452092 236 browser details YourSeq 122 994 1170 3000 87.3% chr1 + 122345739 122345892 154 browser details YourSeq 121 994 1241 3000 93.1% chr17 - 49651867 49652115 249 browser details YourSeq 119 1006 1170 3000 91.9% chr5 - 121110050 121110264 215 browser details YourSeq 119 997 1175 3000 83.7% chr16 - 87854677 87854844 168 browser details YourSeq 119 994 1151 3000 93.6% chr14 + 88702901 88703068 168 browser details YourSeq 118 1018 1374 3000 92.4% chr1 - 150987968 150988361 394 browser details YourSeq 117 1004 1167 3000 94.9% chr10 - 118029358 118029605 248 browser details YourSeq 117 996 1173 3000 95.6% chr13 + 48293443 48293687 245 browser details YourSeq 115 984 1133 3000 92.9% chr13 - 38787610 38787843 234

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 + 71556741 71559740 3000 browser details YourSeq 1790 645 2995 3000 92.9% chr1 - 18015134 18017752 2619 browser details YourSeq 87 1170 1302 3000 89.5% chr11 - 52897200 52897665 466 browser details YourSeq 83 1136 1300 3000 83.9% chr1 + 35940927 35941085 159 browser details YourSeq 82 1136 1302 3000 80.0% chr15 + 79579607 79579756 150 browser details YourSeq 80 1204 1307 3000 92.8% chr14 - 45428668 45428784 117 browser details YourSeq 80 1162 1295 3000 90.0% chr2 + 67610844 67610986 143 browser details YourSeq 72 1132 1266 3000 80.7% chr4 + 8925147 8925268 122 browser details YourSeq 72 1136 1280 3000 92.1% chr11 + 102186749 102187177 429 browser details YourSeq 71 1170 1302 3000 94.0% chr3 - 87863110 87863418 309 browser details YourSeq 69 1136 1278 3000 86.1% chr11 + 89956356 89956490 135 browser details YourSeq 68 1175 1294 3000 88.7% chr16 + 32650351 32650483 133 browser details YourSeq 66 1173 1278 3000 94.8% chr17 - 27988233 27988363 131 browser details YourSeq 65 1143 1278 3000 92.3% chr6 - 33894764 33894904 141 browser details YourSeq 65 1174 1293 3000 89.2% chr5 + 31301699 31301842 144 browser details YourSeq 63 1210 1299 3000 84.3% chr5 - 66061008 66061082 75 browser details YourSeq 63 2156 2258 3000 77.4% chr17 - 48079654 48079752 99 browser details YourSeq 63 1169 1288 3000 94.5% chr13 - 93528118 93528251 134 browser details YourSeq 63 1159 1301 3000 89.9% chr3 + 139524267 139524512 246 browser details YourSeq 63 1222 1302 3000 89.8% chr2 + 104823820 104823900 81

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: Spdya speedy/RINGO cell cycle regulator family, member A [ Mus musculus (house mouse) ] Gene ID: 70891, updated on 15-Oct-2019

Gene summary

Official Symbol Spdya provided by MGI Official Full Name speedy/RINGO cell cycle regulator family, member A provided by MGI Primary source MGI:MGI:1918141 See related Ensembl:ENSMUSG00000052525 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 GS4; Spdy1; MLZ-465; 4921517J08Rik; 4930548B21Rik Expression Biased expression in testis adult (RPKM 7.5), CNS E18 (RPKM 0.8) and 10 other tissues See more Orthologs human all

Genomic context

Location: 17; 17 E1.3 See Spdya in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (71526895..71589533)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (71901401..71938873)

Chromosome 17 - NC_000083.6

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Transcript information: This gene has 4 transcripts

Gene: Spdya ENSMUSG00000052525

Description speedy/RINGO cell cycle regulator family, member A [Source:MGI Symbol;Acc:MGI:1918141] Gene Synonyms 4921517J08Rik, 4930548B21Rik, Spdy1, speedy A1, speedy A2, speedy/ringo Location Chromosome 17: 71,552,061-71,589,533 forward strand. GRCm38:CM001010.2 About this gene This gene has 4 transcripts (splice variants), 186 orthologues, is a member of 1 Ensembl protein family and is associated with 17 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Spdya-204 ENSMUST00000167641.7 1891 310aa ENSMUSP00000125912.1 Protein coding CCDS50178 Q5IBH7 TSL:5 GENCODE basic APPRIS P1

Spdya-202 ENSMUST00000124001.7 1247 283aa ENSMUSP00000118426.1 Protein coding CCDS50179 B2RWC8 Q5IBH7 TSL:1 GENCODE basic

Spdya-203 ENSMUST00000144142.1 933 310aa ENSMUSP00000118994.1 Protein coding CCDS50178 Q5IBH7 TSL:1 GENCODE basic APPRIS P1

Spdya-201 ENSMUST00000064420.11 1877 309aa ENSMUSP00000063214.5 Protein coding - B8JJB9 TSL:1 GENCODE basic

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57.47 kb Forward strand 71.55Mb 71.56Mb 71.57Mb 71.58Mb 71.59Mb Genes Mettl4-ps1-202 >lncRNA (Comprehensive set...

Mettl4-ps1-201 >transcribed unprocessed pseudogene

Spdya-204 >protein coding

Spdya-202 >protein coding

Spdya-201 >protein coding

Spdya-203 >protein coding

Contigs CT010429.11 >

Genes < Trmt61b-201lncRNA (Comprehensive set...

< Trmt61b-202lncRNA

< Trmt61b-203lncRNA

Regulatory Build

71.55Mb 71.56Mb 71.57Mb 71.58Mb 71.59Mb Reverse strand 57.47 kb

Regulation Legend

CTCF 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

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Transcript: ENSMUST00000167641

37.47 kb Forward strand

Spdya-204 >protein coding

ENSMUSP00000125... MobiDB lite Low complexity (Seg) Pfam Cell cycle regulatory protein Speedy PANTHER PTHR31545

PTHR31545:SF4

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 310

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

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