https://www.alphaknockout.com

Mouse Clasp2 Knockout Project (CRISPR/Cas9)

Objective: To create a Clasp2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Clasp2 gene (NCBI Reference Sequence: NM_001286602 ; Ensembl: ENSMUSG00000033392 ) is located on Mouse chromosome 9. 34 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 34 (Transcript: ENSMUST00000163895). Exon 3~5 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: Targeted deletion of this gene leads to impaired formation of stable microtubules in a wound healing assay, and results in a 2-fold reduction of directionally persistent migration in mutant embryonic fibroblasts.

Exon 3 starts from about 4.64% of the coding region. Exon 3~5 covers 8.14% of the coding region. The size of effective KO region: ~9045 bp. The KO region does not have any other known gene.

Page 1 of 9 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 3 4 5 34

Legends Exon of mouse Clasp2 Knockout region

Page 2 of 9 https://www.alphaknockout.com

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

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 5 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.

Page 3 of 9 https://www.alphaknockout.com

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

Sequence 12

Summary: Full Length(2000bp) | A(31.6% 632) | C(19.4% 388) | T(29.15% 583) | G(19.85% 397)

Note: The 2000 bp section upstream 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(32.25% 645) | C(18.7% 374) | T(27.3% 546) | G(21.75% 435)

Note: The 2000 bp section downstream of Exon 5 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.

Page 4 of 9 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr9 + 113833176 113835175 2000 browser details YourSeq 148 1219 1409 2000 89.1% chr14 - 60553349 60553554 206 browser details YourSeq 145 1219 1406 2000 90.1% chr16 - 37693744 37693932 189 browser details YourSeq 144 1219 1411 2000 85.8% chr10 - 40585865 40586054 190 browser details YourSeq 142 1219 1401 2000 89.1% chr9 - 86996337 86996521 185 browser details YourSeq 142 1218 1400 2000 90.9% chr6 + 86408386 86408574 189 browser details YourSeq 142 1219 1411 2000 84.8% chr13 + 96834602 96834791 190 browser details YourSeq 142 1208 1414 2000 84.5% chr1 + 63158373 63158577 205 browser details YourSeq 141 1219 1401 2000 89.1% chr1 - 74581928 74582112 185 browser details YourSeq 139 1216 1557 2000 79.7% chr1 - 93400101 93400288 188 browser details YourSeq 138 1212 1418 2000 84.8% chr1 - 172031929 172032141 213 browser details YourSeq 138 1219 1401 2000 88.0% chr1 - 77362305 77362489 185 browser details YourSeq 137 1222 1400 2000 87.1% chr9 - 119377419 119377596 178 browser details YourSeq 136 1218 1405 2000 84.0% chr15 + 93226379 93226553 175 browser details YourSeq 135 1216 1401 2000 84.7% chr5 - 65541375 65541553 179 browser details YourSeq 135 1249 1595 2000 89.5% chr13 + 58470478 58471085 608 browser details YourSeq 134 1218 1406 2000 84.0% chr18 + 88756007 88756193 187 browser details YourSeq 133 1218 1391 2000 85.9% chr10 - 79839667 79839836 170 browser details YourSeq 133 1218 1400 2000 87.6% chr10 + 117394801 117394984 184 browser details YourSeq 131 1214 1401 2000 84.7% chr19 - 7250042 7250221 180

Note: The 2000 bp section upstream of Exon 3 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% chr9 + 113844221 113846220 2000 browser details YourSeq 235 1131 1594 2000 89.6% chr3 - 95333347 95920492 587146 browser details YourSeq 234 1131 1610 2000 83.9% chr4 - 130689475 130689848 374 browser details YourSeq 234 1155 1610 2000 89.2% chr18 + 56380507 56546623 166117 browser details YourSeq 233 1139 1599 2000 85.3% chr18 - 6335885 6336316 432 browser details YourSeq 226 1134 1603 2000 83.5% chr2 - 179889876 179890248 373 browser details YourSeq 222 1145 1533 2000 90.0% chr12 - 111279696 111280275 580 browser details YourSeq 216 1139 1575 2000 88.5% chrX + 100630820 100631359 540 browser details YourSeq 214 1138 1590 2000 84.1% chr11 - 97430767 97431192 426 browser details YourSeq 213 1139 1588 2000 84.3% chr3 - 141992986 141993280 295 browser details YourSeq 212 1164 1590 2000 86.0% chr13 - 12513122 12513527 406 browser details YourSeq 202 1130 1579 2000 82.3% chr15 - 67340082 67340420 339 browser details YourSeq 199 1170 1599 2000 92.4% chr11 - 62469892 62470532 641 browser details YourSeq 188 1155 1599 2000 81.2% chr13 + 62608109 62608421 313 browser details YourSeq 183 1155 1599 2000 85.1% chr3 + 95864036 95864386 351 browser details YourSeq 182 1134 1605 2000 88.5% chr11 + 80412349 80412821 473 browser details YourSeq 181 1196 1604 2000 88.8% chr14 - 45535397 45536081 685 browser details YourSeq 180 1202 1581 2000 90.9% chr14 + 54593917 54872662 278746 browser details YourSeq 178 1196 1590 2000 89.5% chr5 - 123706343 123706908 566 browser details YourSeq 175 1172 1605 2000 83.4% chr17 - 27658183 27658594 412

Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 https://www.alphaknockout.com

Gene and protein information: Clasp2 CLIP associating protein 2 [ Mus musculus (house mouse) ] Gene ID: 76499, updated on 24-Oct-2019

Gene summary

Official Symbol Clasp2 provided by MGI Official Full Name CLIP associating protein 2 provided by MGI Primary source MGI:MGI:1923749 See related Ensembl:ENSMUSG00000033392 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 C77448; mKIAA0627; CLASP2beta; 1500004F14Rik; 8030404L10Rik Expression Broad expression in CNS E18 (RPKM 23.3), cerebellum adult (RPKM 19.0) and 21 other tissues See more Orthologs human all

Genomic context

Location: 9; 9 F3 See Clasp2 in Genome Data Viewer Exon count: 49

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (113740077..113919697)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (113650591..113828815)

Chromosome 9 - NC_000075.6

Page 6 of 9 https://www.alphaknockout.com

Transcript information: This gene has 11 transcripts

Gene: Clasp2 ENSMUSG00000033392

Description CLIP associating protein 2 [Source:MGI Symbol;Acc:MGI:1923749] Gene Synonyms 1500004F14Rik, 8030404L10Rik, CLASP2, CLASP2alpha, CLASP2beta, CLASP2gamma Location Chromosome 9: 113,741,473-113,919,682 forward strand. GRCm38:CM001002.2 About this gene This gene has 11 transcripts (splice variants), 217 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Clasp2- ENSMUST00000111838.9 5609 1286aa ENSMUSP00000107469.2 Protein coding CCDS52948 E9Q8N5 TSL:1 201 GENCODE basic APPRIS ALT1

Clasp2- ENSMUST00000166734.9 4108 1287aa ENSMUSP00000130201.2 Protein coding CCDS40787 Q08EB6 TSL:5 203 GENCODE basic APPRIS P3

Clasp2- ENSMUST00000214522.1 4159 1304aa ENSMUSP00000149670.1 Protein coding - Q08EB5 TSL:1 207 GENCODE basic APPRIS ALT1

Clasp2- ENSMUST00000163895.2 4112 1307aa ENSMUSP00000128460.2 Protein coding - F7DCH5 TSL:5 202 GENCODE basic APPRIS ALT1

Clasp2- ENSMUST00000213663.1 2727 600aa ENSMUSP00000150145.1 Protein coding - A0A1L1ST22 TSL:5 204 GENCODE basic

Clasp2- ENSMUST00000216817.1 2234 400aa ENSMUSP00000150741.1 Protein coding - Q8BSE7 TSL:1 211 GENCODE basic

Clasp2- ENSMUST00000215022.1 4125 103aa ENSMUSP00000149685.1 Nonsense mediated - A0A1L1SRY3 TSL:5 209 decay

Clasp2- ENSMUST00000213902.1 4655 No - Retained intron - - TSL:1 205 protein

Clasp2- ENSMUST00000214860.1 2773 No - Retained intron - - TSL:1 208 protein

Clasp2- ENSMUST00000216280.1 1927 No - lncRNA - - TSL:1 210 protein

Clasp2- ENSMUST00000213966.1 1487 No - lncRNA - - TSL:1 206 protein

Page 7 of 9 https://www.alphaknockout.com

198.21 kb Forward strand

113.75Mb 113.80Mb 113.85Mb 113.90Mb Genes (Comprehensive set... Clasp2-204 >protein coding

Clasp2-211 >protein coding

Clasp2-210 >lncRNA Clasp2-205 >retained intron

Clasp2-206 >lncRNA

Clasp2-201 >protein coding

Clasp2-208 >retained intron

Clasp2-207 >protein coding

Clasp2-209 >nonsense mediated decay

Clasp2-203 >protein coding

Clasp2-202 >protein coding

Contigs AC167246.2 > AC107803.15 > Genes < Gm25059-201snoRNA (Comprehensive set...

Regulatory Build

113.75Mb 113.80Mb 113.85Mb 113.90Mb Reverse strand 198.21 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

Page 8 of 9 https://www.alphaknockout.com

Transcript: ENSMUST00000163895

105.54 kb Forward strand

Clasp2-202 >protein coding

ENSMUSP00000128... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Armadillo-type fold

SMART TOG domain Pfam CLASP N-terminal domain

PANTHER PTHR21567:SF30

PTHR21567 Gene3D Armadillo-like helical

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1307

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

Page 9 of 9