https://www.alphaknockout.com

Mouse Krtdap Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Krtdap gene (NCBI Reference Sequence: NM_001033131 ; Ensembl: ENSMUSG00000074199 ) is located on Mouse chromosome 7. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000098559). Exon 1~6 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:

Exon 1 starts from about 0.33% of the coding region. Exon 1~6 covers 100.0% of the coding region. The size of effective KO region: ~3029 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 2 3 4 5 6

Legends Exon of mouse Krtdap 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 start codon 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 stop codon 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(24.55% 491) | C(22.3% 446) | T(23.15% 463) | G(30.0% 600)

Note: The 2000 bp section upstream of start codon 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(25.35% 507) | C(22.95% 459) | T(26.85% 537) | G(24.85% 497)

Note: The 2000 bp section downstream of stop codon 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% chr7 + 30785928 30787927 2000 browser details YourSeq 73 945 1424 2000 91.1% chr9 - 56821337 56862927 41591 browser details YourSeq 70 1017 1117 2000 87.0% chr4 + 136612554 136612659 106 browser details YourSeq 62 945 1117 2000 70.3% chr13 + 95516688 95516822 135 browser details YourSeq 61 1052 1211 2000 89.8% chr18 - 49356390 49356849 460 browser details YourSeq 60 955 1120 2000 90.6% chr2 - 104834622 104834787 166 browser details YourSeq 59 826 1120 2000 66.7% chr7 - 83959325 83959504 180 browser details YourSeq 55 953 1117 2000 89.8% chr6 + 141426913 141427079 167 browser details YourSeq 54 949 1165 2000 95.0% chr8 - 94075076 94332805 257730 browser details YourSeq 53 938 1116 2000 88.6% chr3 - 119804909 119805110 202 browser details YourSeq 52 952 1118 2000 86.4% chr11 - 55761456 55761621 166 browser details YourSeq 52 1058 1117 2000 93.4% chr5 + 123330848 123330907 60 browser details YourSeq 52 1055 1120 2000 89.4% chr4 + 55717621 55717686 66 browser details YourSeq 52 942 1068 2000 83.2% chr11 + 109688858 109688985 128 browser details YourSeq 52 1052 1116 2000 90.7% chr1 + 72592830 72592894 65 browser details YourSeq 50 1052 1113 2000 90.4% chr18 - 67153463 67153524 62 browser details YourSeq 48 1054 1113 2000 90.0% chr19 - 6930142 6930201 60 browser details YourSeq 48 1052 1113 2000 92.4% chr17 - 45528196 45528256 61 browser details YourSeq 48 1053 1112 2000 90.0% chr13 - 63950742 63950801 60 browser details YourSeq 48 1052 1116 2000 90.0% chr10 - 25115276 25115342 67

Note: The 2000 bp section upstream of start codon 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% chr7 + 30790957 30792956 2000 browser details YourSeq 70 1162 1261 2000 87.4% chr16 - 16966290 16966389 100 browser details YourSeq 69 1184 1304 2000 92.8% chrX - 101378174 101378479 306 browser details YourSeq 61 1162 1248 2000 80.5% chr12 - 110831268 110831349 82 browser details YourSeq 61 1086 1230 2000 90.7% chr10 - 22849239 22849485 247 browser details YourSeq 56 1170 1244 2000 83.6% chr5 + 125434859 125434931 73 browser details YourSeq 55 1177 1249 2000 87.7% chr15 - 93738341 93738413 73 browser details YourSeq 53 1177 1249 2000 86.4% chr11 - 77124904 77124976 73 browser details YourSeq 53 1184 1248 2000 90.8% chr1 - 181245900 181245964 65 browser details YourSeq 52 1162 1249 2000 79.6% chr14 + 55599951 55600038 88 browser details YourSeq 51 1162 1250 2000 78.7% chr11 - 6097600 6097688 89 browser details YourSeq 50 1183 1249 2000 83.1% chr1 + 74619893 74619957 65 browser details YourSeq 48 1162 1239 2000 83.9% chr17 - 13223562 13223637 76 browser details YourSeq 48 1205 1293 2000 91.4% chr10 - 68953591 68953734 144 browser details YourSeq 47 1204 1255 2000 96.2% chr17 + 83176307 83176590 284 browser details YourSeq 46 1192 1247 2000 91.1% chr9 - 71872856 71872911 56 browser details YourSeq 46 1163 1230 2000 88.7% chr5 - 36677074 36677139 66 browser details YourSeq 46 1184 1243 2000 88.4% chr10 - 56509592 56509651 60 browser details YourSeq 45 1162 1244 2000 77.2% chr10 - 58676786 58676868 83 browser details YourSeq 44 1162 1249 2000 75.0% chr13 - 57056264 57056351 88

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

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

Gene and protein information: Krtdap keratinocyte differentiation associated protein [ Mus musculus (house mouse) ] Gene ID: 64661, updated on 24-Oct-2019

Gene summary

Official Symbol Krtdap provided by MGI Official Full Name keratinocyte differentiation associated protein provided by MGI Primary source MGI:MGI:1928282 See related Ensembl:ENSMUSG00000074199 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 Kdap; sk221 Expression Biased expression in stomach adult (RPKM 128.1), limb E14.5 (RPKM 47.2) and 3 other tissues See more Orthologs human all

Genomic context

Location: 7; 7 B1 See Krtdap in Genome Data Viewer Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (30787905..30791089)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (31572924..31576102)

Chromosome 7 - NC_000073.6

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

Transcript information: This gene has 9 transcripts

Gene: Krtdap ENSMUSG00000074199

Description keratinocyte differentiation associated protein [Source:MGI Symbol;Acc:MGI:1928282] Gene Synonyms Kdap, sk221 Location Chromosome 7: 30,787,896-30,791,097 forward strand. GRCm38:CM001000.2 About this gene This gene has 9 transcripts (splice variants), 84 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Krtdap-201 ENSMUST00000098559.2 470 102aa ENSMUSP00000096159.1 Protein coding CCDS21110 Q3V2T4 TSL:1 GENCODE basic APPRIS P1

Krtdap-203 ENSMUST00000187493.6 420 88aa ENSMUSP00000140737.1 Protein coding - A0A087WRR2 TSL:2 GENCODE basic

Krtdap-209 ENSMUST00000190990.6 403 91aa ENSMUSP00000139461.1 Protein coding - Q3V2T4 TSL:1 GENCODE basic

Krtdap-205 ENSMUST00000189673.6 345 77aa ENSMUSP00000139443.1 Protein coding - B7ZNU6 TSL:1 GENCODE basic

Krtdap-206 ENSMUST00000189962.6 323 57aa ENSMUSP00000140489.1 Protein coding - A0A087WR64 TSL:3 GENCODE basic

Krtdap-208 ENSMUST00000190589.6 369 No protein - Retained intron - - TSL:2

Krtdap-204 ENSMUST00000188036.1 295 No protein - Retained intron - - TSL:2

Krtdap-202 ENSMUST00000186077.1 262 No protein - Retained intron - - TSL:2

Krtdap-207 ENSMUST00000190480.1 217 No protein - Retained intron - - TSL:3

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

23.20 kb Forward strand

30.78Mb 30.79Mb 30.80Mb Genes (Comprehensive set... Dmkn-205 >protein coding Krtdap-207 >retained intron

Dmkn-202 >protein coding Krtdap-201 >protein coding

Dmkn-204 >protein coding Krtdap-203 >protein coding

Dmkn-203 >protein coding Krtdap-206 >protein coding

Dmkn-206 >protein coding Krtdap-209 >protein coding

Dmkn-201 >protein coding Krtdap-205 >protein coding

Krtdap-208 >retained intron

Krtdap-202 >retained intron

Krtdap-204 >retained intron

Contigs AC165340.3 > Regulatory Build

30.78Mb 30.79Mb 30.80Mb Reverse strand 23.20 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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

Transcript: ENSMUST00000098559

3.19 kb Forward strand

Krtdap-201 >protein coding

ENSMUSP00000096... Low complexity (Seg) Cleavage site (Sign... Pfam Keratinocyte differentiation-associated protein PANTHER Keratinocyte differentiation-associated protein

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 102

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

Page 9 of 9