https://www.alphaknockout.com

Mouse Ctps2 Knockout Project (CRISPR/Cas9)

Objective: To create a Ctps2 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 2~3 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO 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 2 starts from the coding region. Exon 2~3 covers 19.17% of the coding region. The size of effective KO region: ~6746 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 19

Legends Exon of mouse Ctps2 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1686 bp section upstream of Exon 2 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 3 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(1686bp) | A(20.52% 346) | C(25.62% 432) | T(30.55% 515) | G(23.31% 393)

Note: The 1686 bp section upstream of Exon 2 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(24.85% 497) | C(21.1% 422) | T(32.95% 659) | G(21.1% 422)

Note: The 2000 bp section downstream of Exon 3 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 1686 1 1686 1686 100.0% chrX + 162901788 162903473 1686 browser details YourSeq 193 1135 1614 1686 94.5% chr19 + 45665137 45750425 85289 browser details YourSeq 76 1094 1300 1686 79.6% chr18 - 24345110 24345382 273 browser details YourSeq 66 1153 1369 1686 76.0% chr4 + 87074305 87074915 611 browser details YourSeq 56 1089 1197 1686 90.2% chr13 - 55016891 55017179 289 browser details YourSeq 54 1160 1386 1686 87.9% chr1 + 133184279 133184602 324 browser details YourSeq 52 1146 1264 1686 72.5% chr11 - 4556936 4557055 120 browser details YourSeq 51 1133 1213 1686 86.2% chr7 - 30889728 30889929 202 browser details YourSeq 48 1143 1219 1686 81.6% chr17 - 74939670 74939748 79 browser details YourSeq 48 1146 1233 1686 86.2% chr4 + 19687529 19687616 88 browser details YourSeq 47 1160 1241 1686 79.1% chr8 - 32810699 32810793 95 browser details YourSeq 47 1147 1213 1686 81.9% chr1 + 75536717 75536782 66 browser details YourSeq 45 1135 1215 1686 74.0% chr1 - 74272371 74272447 77 browser details YourSeq 43 1144 1210 1686 77.8% chr15 - 100186958 100187021 64 browser details YourSeq 41 1153 1241 1686 91.9% chr6 - 134634685 134634773 89 browser details YourSeq 41 1153 1209 1686 86.0% chr5 - 137011620 137011676 57 browser details YourSeq 40 1153 1212 1686 83.4% chr16 - 97194562 97194621 60 browser details YourSeq 40 1157 1240 1686 73.9% chr4 + 148406298 148406381 84 browser details YourSeq 40 1157 1208 1686 88.5% chr16 + 22510411 22510462 52 browser details YourSeq 39 1131 1180 1686 97.7% chr19 - 44065755 44128112 62358

Note: The 1686 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 2000 1 2000 2000 100.0% chrX + 162910181 162912180 2000 browser details YourSeq 371 44 1226 2000 90.8% chr10 - 127996125 128479769 483645 browser details YourSeq 203 577 1353 2000 81.2% chr10 + 82163632 82164190 559 browser details YourSeq 169 1047 1713 2000 81.8% chr13 - 111469687 111470058 372 browser details YourSeq 167 1007 1234 2000 86.4% chr2 + 29556857 29557078 222 browser details YourSeq 161 1015 1232 2000 86.2% chr7 - 24297119 24297326 208 browser details YourSeq 159 1023 1227 2000 87.3% chr17 + 88478318 88478516 199 browser details YourSeq 158 976 1224 2000 92.6% chr6 - 22364370 22364676 307 browser details YourSeq 158 962 1226 2000 84.3% chr11 - 75104848 75105061 214 browser details YourSeq 155 1016 1227 2000 87.2% chr15 - 80735943 80736149 207 browser details YourSeq 154 1052 1566 2000 81.0% chr10 + 63035361 63035694 334 browser details YourSeq 153 804 1226 2000 90.1% chr6 - 51521646 51522215 570 browser details YourSeq 151 576 1353 2000 87.9% chr7 - 6952410 6953386 977 browser details YourSeq 151 1036 1226 2000 91.8% chr4 - 149671045 149671240 196 browser details YourSeq 149 1051 1228 2000 92.2% chrX - 75314922 75315108 187 browser details YourSeq 149 1036 1226 2000 88.9% chr9 - 88555355 88555540 186 browser details YourSeq 148 1047 1350 2000 84.3% chr8 + 86908027 86908310 284 browser details YourSeq 148 1035 1229 2000 92.1% chr11 + 78714418 78714612 195 browser details YourSeq 146 1052 1234 2000 91.1% chr8 + 69743936 69744120 185 browser details YourSeq 146 1052 1355 2000 89.4% chr13 + 119448716 119449360 645

Note: The 2000 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: 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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