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Mouse Ints6 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ints6 conditional knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ints6 (NCBI Reference Sequence: NM_008715 ; Ensembl: ENSMUSG00000035161 ) is located on Mouse 14. 19 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 18 (Transcript: ENSMUST00000053959). Exon 5~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ints6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-182O10 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: Mice homozygous for a transgenic gene disruption exhibit embryonic lethality at E7.

Exon 5 starts from about 16.23% of the coding region. The knockout of Exon 5~6 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 27426 bp, and the size of intron 6 for 3'-loxP site insertion: 453 bp. The size of effective cKO region: ~2665 bp. The cKO 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 5 6 7 19 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ints6 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(9142bp) | A(29.86% 2730) | C(16.67% 1524) | G(17.86% 1633) | T(35.6% 3255)

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% chr14 - 62716733 62719732 3000 browser details YourSeq 151 265 455 3000 92.9% chr9 + 70684328 70684916 589 browser details YourSeq 149 265 443 3000 94.2% chr4 - 123359106 123359498 393 browser details YourSeq 143 267 440 3000 94.0% chr10 + 128680529 128937435 256907 browser details YourSeq 142 267 443 3000 93.0% chr4 - 45129275 45129475 201 browser details YourSeq 141 271 442 3000 92.9% chr7 + 127062586 127062773 188 browser details YourSeq 140 273 442 3000 92.3% chr13 - 51828327 51828513 187 browser details YourSeq 139 264 442 3000 92.8% chr17 - 56025560 56025752 193 browser details YourSeq 139 261 442 3000 92.2% chr13 - 29708838 29709035 198 browser details YourSeq 138 267 446 3000 91.7% chr4 - 76037351 76037575 225 browser details YourSeq 137 271 440 3000 91.1% chr9 - 65394842 65395027 186 browser details YourSeq 137 270 441 3000 93.2% chr10 - 42492725 42492912 188 browser details YourSeq 135 265 441 3000 90.0% chr8 - 70067423 70067610 188 browser details YourSeq 135 268 441 3000 91.6% chr12 - 78830247 78830435 189 browser details YourSeq 135 270 438 3000 91.0% chr12 - 55731699 55731881 183 browser details YourSeq 135 272 443 3000 91.0% chr8 + 95924264 95924446 183 browser details YourSeq 134 268 444 3000 91.5% chrX + 20923350 20923542 193 browser details YourSeq 133 273 441 3000 94.2% chr14 - 61466477 61466661 185 browser details YourSeq 133 265 444 3000 90.3% chr11 + 104260523 104260715 193 browser details YourSeq 133 263 442 3000 91.1% chr1 + 9832085 9832284 200

Note: The 3000 bp section upstream of Exon 5 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% chr14 - 62711067 62714066 3000 browser details YourSeq 225 893 1830 3000 90.4% chr11 - 104294121 104533702 239582 browser details YourSeq 215 901 1324 3000 85.2% chr7 + 118745208 118745625 418 browser details YourSeq 209 893 1295 3000 86.7% chr6 - 148827471 148827936 466 browser details YourSeq 199 898 1350 3000 87.6% chr11 - 49524958 49525454 497 browser details YourSeq 197 891 1235 3000 87.1% chr13 + 12553512 12553873 362 browser details YourSeq 194 893 1291 3000 85.4% chr10 - 29596829 29597200 372 browser details YourSeq 193 891 1235 3000 85.8% chrX - 85680060 85680428 369 browser details YourSeq 192 893 1324 3000 84.0% chrX - 166984303 166984762 460 browser details YourSeq 189 893 1340 3000 89.3% chr12 - 31137741 31138237 497 browser details YourSeq 189 939 1350 3000 89.3% chr11 + 52154599 52155052 454 browser details YourSeq 179 904 1235 3000 88.9% chr14 - 124446941 124447292 352 browser details YourSeq 178 898 1191 3000 86.8% chr1 - 39440889 39441199 311 browser details YourSeq 177 911 1295 3000 83.2% chr2 - 117483958 117484320 363 browser details YourSeq 177 919 1235 3000 87.2% chr19 - 24252603 24252941 339 browser details YourSeq 173 904 1228 3000 82.3% chr3 - 102177389 102177712 324 browser details YourSeq 171 992 1753 3000 88.7% chr15 - 88878484 89406144 527661 browser details YourSeq 166 901 1235 3000 90.4% chr18 + 5355151 5355501 351 browser details YourSeq 165 938 1233 3000 89.1% chr5 + 20320115 20320470 356 browser details YourSeq 165 893 1262 3000 90.3% chr10 + 94518141 94518553 413

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 http://www.alphaknockout.com/ Gene and information: Ints6 integrator complex subunit 6 [ Mus musculus (house mouse) ] Gene ID: 18130, updated on 12-Aug-2019

Gene summary

Official Symbol Ints6 provided by MGI Official Full Name integrator complex subunit 6 provided by MGI Primary source MGI:MGI:1202397 See related Ensembl:ENSMUSG00000035161 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as HDB; DICE1; Ddx26; Notch2l; AI480962; 2900075H24Rik Expression Ubiquitous expression in testis adult (RPKM 6.7), placenta adult (RPKM 5.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 14; 14 D1 See Ints6 in Genome Data Viewer

Exon count: 21

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (62676325..62761163, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (63295162..63379949, complement)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 7 transcripts

Gene: Ints6 ENSMUSG00000035161

Description integrator complex subunit 6 [Source:MGI Symbol;Acc:MGI:1202397] Gene Synonyms 2900075H24Rik, DICE1, Ddx26, Notch2l Location Chromosome 14: 62,676,330-62,761,169 reverse strand. GRCm38:CM001007.2 About this gene This gene has 7 transcripts (splice variants), 146 orthologues, 2 paralogues, 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

Ints6-202 ENSMUST00000223585.1 10883 883aa ENSMUSP00000152954.1 Protein coding CCDS27191 Q6PCM2 GENCODE basic APPRIS P1

Ints6-201 ENSMUST00000053959.6 4993 883aa ENSMUSP00000086788.4 Protein coding CCDS27191 Q6PCM2 TSL:1 GENCODE basic APPRIS P1

Ints6-206 ENSMUST00000225406.1 2894 No protein - Retained intron - - -

Ints6-204 ENSMUST00000225193.1 2402 No protein - Retained intron - - -

Ints6-203 ENSMUST00000224891.1 639 No protein - Retained intron - - -

Ints6-205 ENSMUST00000225250.1 700 No protein - lncRNA - - -

Ints6-207 ENSMUST00000225700.1 366 No protein - lncRNA - - -

104.84 kb Forward strand 62.68Mb 62.70Mb 62.72Mb 62.74Mb 62.76Mb Serpine3-201 >protein coding (Comprehensive set...

Gm25162-201 >snoRNA

Contigs < CT572983.7

Genes (Comprehensive set... < Ints6-201protein coding

< Ints6-202protein coding

< Ints6-207processed transcript < Ints6-205processed transcript

< Ints6-203retained intron < Ints6-204retained intron

< Ints6-206retained intron

Regulatory Build

62.68Mb 62.70Mb 62.72Mb 62.74Mb 62.76Mb Reverse strand 104.84 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000053959

< Ints6-201protein coding

Reverse strand 84.78 kb

ENSMUSP00000086... Coiled-coils (Ncoils) Superfamily von Willebrand factor A-like domain superfamily Pfam von Willebrand factor, type A INTS6/SAGE1/DDX26B/CT45, C-terminal

PROSITE profiles von Willebrand factor, type A PANTHER PTHR12957:SF23

PTHR12957 Gene3D von Willebrand factor A-like domain superfamily CDD cd00198

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

Variant Legend synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 720 800 883

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

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