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Mouse Ctps2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ctps2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ctps2 gene (NCBI Reference Sequence: NM_001168568 ; Ensembl: ENSMUSG00000031360 ) is located on Mouse chromosome X. 19 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 18 (Transcript: ENSMUST00000033727). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ctps2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-31A20 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: Male chimeras hemizygous for a gene trapped allele appear normal at E9.5.

Exon 4 starts from about 19.23% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 3028 bp, and the size of intron 4 for 3'-loxP site insertion: 7890 bp. The size of effective cKO region: ~601 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 4 19 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ctps2 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(7101bp) | A(25.73% 1827) | C(21.19% 1505) | T(31.16% 2213) | G(21.91% 1556)

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% chrX + 162909959 162912958 3000 browser details YourSeq 371 266 1448 3000 90.8% chr10 - 127996125 128479769 483645 browser details YourSeq 203 799 1575 3000 81.2% chr10 + 82163632 82164190 559 browser details YourSeq 169 1269 1935 3000 81.8% chr13 - 111469687 111470058 372 browser details YourSeq 167 1229 1456 3000 86.4% chr2 + 29556857 29557078 222 browser details YourSeq 161 1237 1454 3000 86.2% chr7 - 24297119 24297326 208 browser details YourSeq 159 1245 1449 3000 87.3% chr17 + 88478318 88478516 199 browser details YourSeq 158 1198 1446 3000 92.6% chr6 - 22364370 22364676 307 browser details YourSeq 158 1184 1448 3000 84.3% chr11 - 75104848 75105061 214 browser details YourSeq 155 1238 1449 3000 87.2% chr15 - 80735943 80736149 207 browser details YourSeq 154 1274 1788 3000 81.0% chr10 + 63035361 63035694 334 browser details YourSeq 153 1026 1448 3000 90.1% chr6 - 51521646 51522215 570 browser details YourSeq 151 798 1575 3000 87.9% chr7 - 6952410 6953386 977 browser details YourSeq 151 1258 1448 3000 91.8% chr4 - 149671045 149671240 196 browser details YourSeq 149 1273 1450 3000 92.2% chrX - 75314922 75315108 187 browser details YourSeq 149 1258 1448 3000 88.9% chr9 - 88555355 88555540 186 browser details YourSeq 148 1269 1572 3000 84.3% chr8 + 86908027 86908310 284 browser details YourSeq 148 1257 1451 3000 92.1% chr11 + 78714418 78714612 195 browser details YourSeq 146 1274 1456 3000 91.1% chr8 + 69743936 69744120 185 browser details YourSeq 146 1274 1577 3000 89.4% chr13 + 119448716 119449360 645

Note: The 3000 bp section upstream of Exon 4 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% chrX + 162913560 162916559 3000 browser details YourSeq 285 1170 1874 3000 85.2% chr2 - 167204816 167205405 590 browser details YourSeq 273 1451 1874 3000 90.3% chr13 - 99324549 99324975 427 browser details YourSeq 265 1551 1875 3000 91.3% chr9 + 45214721 45215065 345 browser details YourSeq 263 1548 1861 3000 92.1% chr5 + 119888197 119912227 24031 browser details YourSeq 261 1551 1875 3000 90.2% chr6 - 83595511 83595835 325 browser details YourSeq 260 1536 1874 3000 88.9% chr2 - 76474717 76475056 340 browser details YourSeq 260 1546 1874 3000 89.7% chr1 - 106139614 106420311 280698 browser details YourSeq 259 1557 1870 3000 91.5% chr5 + 146889805 146890269 465 browser details YourSeq 259 1548 1874 3000 89.0% chr16 + 91988075 91988400 326 browser details YourSeq 257 1556 1874 3000 90.3% chr4 - 155351377 155351695 319 browser details YourSeq 257 1551 1872 3000 91.4% chr9 + 20574757 20575099 343 browser details YourSeq 257 1548 1875 3000 89.3% chr5 + 116666396 116666726 331 browser details YourSeq 257 1548 1872 3000 88.2% chr13 + 35958081 35958402 322 browser details YourSeq 256 1381 1872 3000 84.1% chr6 - 18480829 18481267 439 browser details YourSeq 256 1548 1875 3000 88.4% chr15 - 84361685 84362011 327 browser details YourSeq 256 1541 1875 3000 88.4% chr1 + 131028335 131028670 336 browser details YourSeq 255 1547 1874 3000 90.2% chr9 + 123522440 123522786 347 browser details YourSeq 255 1546 1870 3000 88.6% chr2 + 169201437 169201760 324 browser details YourSeq 255 1557 1875 3000 89.3% chr13 + 51776615 51776931 317

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ctps2 cytidine 5'-triphosphate synthase 2 [ Mus musculus (house mouse) ] Gene ID: 55936, updated on 12-Aug-2019

Gene summary

Official Symbol Ctps2 provided by MGI Official Full Name cytidine 5'-triphosphate synthase 2 provided by MGI Primary source MGI:MGI:1933185 See related Ensembl:ENSMUSG00000031360 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 Ctpsh; AI326475; A830031M15Rik Expression Ubiquitous expression in CNS E18 (RPKM 5.8), cerebellum adult (RPKM 5.0) and 26 other tissues See more Orthologs human all

Genomic context

Location: X; X F4 See Ctps2 in Genome Data Viewer

Exon count: 22

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (162901268..163034541)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (159339492..159472473)

Chromosome X - NC_000086.7

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Transcript information: This gene has 13 transcripts

Gene: Ctps2 ENSMUSG00000031360

Description cytidine 5'-triphosphate synthase 2 [Source:MGI Symbol;Acc:MGI:1933185] Gene Synonyms A830031M15Rik, CTPsH Location Chromosome X: 162,901,238-163,032,508 forward strand. GRCm38:CM001013.2 About this gene This gene has 13 transcripts (splice variants), 136 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ctps2-201 ENSMUST00000033727.13 3353 586aa ENSMUSP00000033727.7 Protein coding CCDS41202 P70303 TSL:1 GENCODE basic APPRIS P1

Ctps2-206 ENSMUST00000112303.7 3289 586aa ENSMUSP00000107922.1 Protein coding CCDS41202 P70303 TSL:1 GENCODE basic APPRIS P1

Ctps2-205 ENSMUST00000112302.7 3223 535aa ENSMUSP00000107921.1 Protein coding CCDS53240 P70303 TSL:1 GENCODE basic

Ctps2-204 ENSMUST00000112301.7 3099 586aa ENSMUSP00000107920.1 Protein coding CCDS41202 P70303 TSL:1 GENCODE basic APPRIS P1

Ctps2-202 ENSMUST00000101095.8 1665 521aa ENSMUSP00000098656.2 Protein coding - P70303 TSL:1 GENCODE basic

Ctps2-203 ENSMUST00000112300.1 741 151aa ENSMUSP00000107919.1 Protein coding - A2AEQ5 TSL:3 GENCODE basic

Ctps2-210 ENSMUST00000142927.1 903 No protein - lncRNA - - TSL:5

Ctps2-209 ENSMUST00000135094.7 592 No protein - lncRNA - - TSL:3

Ctps2-207 ENSMUST00000123089.7 522 No protein - lncRNA - - TSL:3

Ctps2-212 ENSMUST00000148949.7 432 No protein - lncRNA - - TSL:5

Ctps2-208 ENSMUST00000126956.1 373 No protein - lncRNA - - TSL:3

Ctps2-213 ENSMUST00000151424.1 368 No protein - lncRNA - - TSL:3

Ctps2-211 ENSMUST00000148199.1 199 No protein - lncRNA - - TSL:5

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151.27 kb Forward strand

162.90Mb 162.95Mb 163.00Mb Genes (Comprehensive set... Ctps2-202 >protein coding

Ctps2-212 >lncRNA Ctps2-208 >lncRNA Ctps2-203 >protein coding

Ctps2-207 >lncRNA Ctps2-209 >lncRNA

Ctps2-206 >protein coding

Ctps2-201 >protein coding

Ctps2-205 >protein coding

Ctps2-213 >lncRNA Ctps2-211 >lncRNA

Ctps2-204 >protein coding

Ctps2-210 >lncRNA

Contigs AL807822.7 > AL671975.12 > Genes < Gm15209-201processed pseudogene < S100g-201protein coding (Comprehensive set...

Regulatory Build

162.90Mb 162.95Mb 163.00Mb Reverse strand 151.27 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: ENSMUST00000033727

130.88 kb Forward strand

Ctps2-201 >protein coding

ENSMUSP00000033... Low complexity (Seg) TIGRFAM CTP synthase Superfamily P-loop containing nucleoside triphosphate hydrolase Class I glutamine amidotransferase-like

Pfam CTP synthase, N-terminal Glutamine amidotransferase

PROSITE profiles Glutamine amidotransferase PANTHER PTHR11550:SF2

CTP synthase Gene3D 3.40.50.300 Class I glutamine amidotransferase-like

CDD cd03113 CTP synthase GATase domain

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 586

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

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