https://www.alphaknockout.com Mouse Hspb7 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Hspb7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Hspb7 gene (NCBI Reference Sequence: NM_013868 ; Ensembl: ENSMUSG00000006221 ) is located on Mouse chromosome 4. 3 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000102486). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Hspb7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-240G5 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 knock-out allele show embryonic lethality during organogenesis and defects in heart development associated with increased thin filament length and formation of atypical actin filament bundles in cardiomyocytes. Exon 2 starts from about 38.86% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 520 bp, and the size of intron 2 for 3'-loxP site insertion: 1244 bp. The size of effective cKO region: ~634 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Hspb7 cKO region loxP site Page 2 of 7 https://www.alphaknockout.com 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(7134bp) | A(21.7% 1548) | C(29.42% 2099) | T(21.47% 1532) | G(27.4% 1955) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr4 + 141419252 141422251 3000 browser details YourSeq 166 1 215 3000 91.5% chr2 + 166772037 166772253 217 browser details YourSeq 159 1 210 3000 96.0% chr8 + 107111188 107111774 587 browser details YourSeq 158 52 529 3000 85.4% chr8 + 111269066 111269329 264 browser details YourSeq 157 43 225 3000 94.3% chr10 - 85922653 85922843 191 browser details YourSeq 154 2 230 3000 91.3% chr10 - 6878478 6878903 426 browser details YourSeq 153 54 230 3000 91.0% chr10 + 128170220 128170385 166 browser details YourSeq 152 55 529 3000 82.8% chr1 - 86077050 86077244 195 browser details YourSeq 151 43 210 3000 95.2% chr17 - 36287648 36287816 169 browser details YourSeq 149 52 215 3000 93.2% chr7 - 113315392 113315552 161 browser details YourSeq 149 52 215 3000 93.2% chr10 - 20877610 20877770 161 browser details YourSeq 149 48 210 3000 96.3% chr4 + 40743551 40743722 172 browser details YourSeq 148 48 229 3000 90.9% chr11 - 20087632 20087811 180 browser details YourSeq 147 54 215 3000 93.1% chr9 - 73079599 73079757 159 browser details YourSeq 147 52 209 3000 94.2% chr7 + 126402788 126402942 155 browser details YourSeq 147 51 210 3000 92.9% chr6 + 48040320 48040473 154 browser details YourSeq 147 52 215 3000 92.6% chr1 + 87682433 87682593 161 browser details YourSeq 146 52 210 3000 93.6% chr16 - 33023127 33023282 156 browser details YourSeq 146 52 210 3000 94.3% chr13 - 28941649 28941805 157 browser details YourSeq 146 52 210 3000 93.6% chr10 - 62485304 62485459 156 Note: The 3000 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 3000 1 3000 3000 100.0% chr4 + 141422886 141425885 3000 browser details YourSeq 58 1861 1918 3000 100.0% chr18 - 42319352 42319409 58 browser details YourSeq 57 1828 1910 3000 91.1% chr7 - 80769431 80769512 82 browser details YourSeq 34 2459 2494 3000 100.0% chr10 - 76115696 76115881 186 browser details YourSeq 25 1614 1638 3000 100.0% chr8 + 74620333 74620357 25 browser details YourSeq 24 2462 2490 3000 81.5% chr17 + 71119941 71119967 27 browser details YourSeq 22 30 51 3000 100.0% chr10 + 82186251 82186272 22 browser details YourSeq 21 2744 2764 3000 100.0% chr7 - 40004416 40004436 21 browser details YourSeq 21 927 947 3000 100.0% chr19 - 37848621 37848641 21 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Hspb7 heat shock protein family, member 7 (cardiovascular) [ Mus musculus (house mouse) ] Gene ID: 29818, updated on 3-Sep-2019 Gene summary Official Symbol Hspb7 provided by MGI Official Full Name heat shock protein family, member 7 (cardiovascular) provided by MGI Primary source MGI:MGI:1352494 See related Ensembl:ENSMUSG00000006221 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 27kDa; cvHsp; Hsp25-2 Expression Biased expression in heart adult (RPKM 366.5) and stomach adult (RPKM 18.0) See more Orthologs human all Genomic context Location: 4; 4 D3 See Hspb7 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (141420779..141425310) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (140976694..140981225) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Hspb7 ENSMUSG00000006221 Description heat shock protein family, member 7 (cardiovascular) [Source:MGI Symbol;Acc:MGI:1352494] Gene Synonyms Hsp25-2, cvHsp Location Chromosome 4: 141,420,779-141,425,311 forward strand. GRCm38:CM000997.2 About this gene This gene has 1 transcript (splice variant), 238 orthologues, 8 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Hspb7-201 ENSMUST00000102486.4 2769 169aa ENSMUSP00000099544.4 Protein coding CCDS18873 P35385 TSL:1 GENCODE basic APPRIS P1 24.53 kb Forward strand 141.415Mb 141.420Mb 141.425Mb 141.430Mb 141.435Mb Genes (Comprehensive set... Gm13075-202 >lncRNA Hspb7-201 >protein coding Contigs AL670285.10 > Genes < Clcnkb-202protein coding < Srarp-201protein coding (Comprehensive set... < Clcnkb-201protein coding < Clcnkb-204lncRNA Regulatory Build 141.415Mb 141.420Mb 141.425Mb 141.430Mb 141.435Mb Reverse strand 24.53 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 Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000102486 4.53 kb Forward strand Hspb7-201 >protein coding ENSMUSP00000099... MobiDB lite Low complexity (Seg) Superfamily HSP20-like chaperone Prints Alpha crystallin/Heat shock protein Pfam Alpha crystallin/Hsp20 domain PROSITE profiles Alpha crystallin/Hsp20 domain PANTHER PTHR46907:SF2 PTHR46907 Gene3D HSP20-like chaperone CDD Heat shock protein beta-7, ACD domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion synonymous variant Scale bar 0 20 40 60 80 100 120 140 169 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.