https://www.alphaknockout.com Mouse Elp2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Elp2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Elp2 gene (NCBI Reference Sequence: NM_021448 ; Ensembl: ENSMUSG00000024271 ) is located on Mouse chromosome 18. 22 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 22 (Transcript: ENSMUST00000234266). 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 Elp2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-193O18 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: Exon 2 starts from about 5.58% 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: 2724 bp, and the size of intron 2 for 3'-loxP site insertion: 2694 bp. The size of effective cKO region: ~579 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 22 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Elp2 Homology arm cKO region loxP site Page 2 of 8 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(7079bp) | A(25.78% 1825) | C(22.22% 1573) | T(29.33% 2076) | G(22.67% 1605) 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 8 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% chr18 + 24603608 24606607 3000 browser details YourSeq 145 1242 1427 3000 91.4% chr5 + 146269906 146270093 188 browser details YourSeq 145 1240 1427 3000 88.9% chr16 + 4893148 4893331 184 browser details YourSeq 143 1245 1424 3000 89.8% chrX - 104663011 104663189 179 browser details YourSeq 136 354 523 3000 90.0% chr18 + 28432853 28433022 170 browser details YourSeq 132 1256 1415 3000 89.3% chr8 + 110763064 110763221 158 browser details YourSeq 131 1262 1415 3000 92.9% chr2 - 149756665 149756840 176 browser details YourSeq 131 1245 1412 3000 89.5% chr3 + 88724502 88724663 162 browser details YourSeq 129 1244 1404 3000 90.2% chr19 + 7237096 7237254 159 browser details YourSeq 129 2721 2894 3000 87.9% chr12 + 17441080 17660766 219687 browser details YourSeq 128 2722 2894 3000 87.3% chr9 + 104784656 104784829 174 browser details YourSeq 126 2722 2899 3000 85.9% chr10 + 91166200 91331107 164908 browser details YourSeq 125 2725 2894 3000 87.0% chr3 + 33988688 33988858 171 browser details YourSeq 124 2720 2886 3000 87.5% chr17 - 28704084 28704251 168 browser details YourSeq 123 1269 1416 3000 89.8% chr5 + 29795877 29796022 146 browser details YourSeq 123 1269 1411 3000 91.6% chr1 + 47417675 47417816 142 browser details YourSeq 121 2722 2891 3000 88.1% chr13 - 63866284 63866454 171 browser details YourSeq 121 2720 2894 3000 85.1% chr11 + 50188205 50188381 177 browser details YourSeq 120 2722 2873 3000 90.2% chr12 - 31980025 31980178 154 browser details YourSeq 120 2722 2894 3000 86.3% chr6 + 88606982 88607154 173 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% chr18 + 24607187 24610186 3000 browser details YourSeq 311 398 967 3000 86.6% chr16 + 97941228 97941805 578 browser details YourSeq 261 405 787 3000 86.2% chr5 - 113935013 113973736 38724 browser details YourSeq 258 418 1000 3000 88.3% chr12 + 112863736 112864435 700 browser details YourSeq 231 421 790 3000 82.6% chr8 - 126996967 126997351 385 browser details YourSeq 229 421 788 3000 84.3% chr4 - 8925769 8926152 384 browser details YourSeq 224 561 1001 3000 82.3% chr15 - 84870062 84870435 374 browser details YourSeq 223 405 782 3000 82.2% chr5 - 125624509 125624882 374 browser details YourSeq 222 405 746 3000 84.8% chr11 + 106390807 106391157 351 browser details YourSeq 220 396 746 3000 85.7% chr7 + 118675250 118675612 363 browser details YourSeq 217 390 747 3000 83.0% chr8 - 18981639 18981995 357 browser details YourSeq 217 405 741 3000 85.0% chr15 + 57558338 57558683 346 browser details YourSeq 213 400 747 3000 82.4% chr10 - 77659538 77659893 356 browser details YourSeq 209 421 776 3000 86.3% chr8 - 124402300 124402662 363 browser details YourSeq 209 436 774 3000 83.0% chr15 + 51689261 51689627 367 browser details YourSeq 206 400 710 3000 84.2% chr15 - 101739778 101740094 317 browser details YourSeq 206 397 741 3000 82.5% chr8 + 36048977 36049312 336 browser details YourSeq 205 399 747 3000 84.4% chr17 + 84719958 84720307 350 browser details YourSeq 204 396 747 3000 87.3% chr6 - 73000015 73000369 355 browser details YourSeq 203 396 777 3000 86.0% chr4 + 98194476 98194864 389 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Elp2 elongator acetyltransferase complex subunit 2 [ Mus musculus (house mouse) ] Gene ID: 58523, updated on 10-Oct-2019 Gene summary Official Symbol Elp2 provided by MGI Official Full Name elongator acetyltransferase complex subunit 2 provided by MGI Primary source MGI:MGI:1889642 See related Ensembl:ENSMUSG00000024271 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 Epl2; StIP1; Statip1; AU023723 Expression Ubiquitous expression in CNS E11.5 (RPKM 45.7), CNS E14 (RPKM 36.0) and 25 other tissues See more Orthologs human all Genomic context Location: 18; 18 A2 See Elp2 in Genome Data Viewer Exon count: 22 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (24603961..24638830) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (24762462..24797331) Chromosome 18 - NC_000084.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Elp2 ENSMUSG00000024271 Description elongator acetyltransferase complex subunit 2 [Source:MGI Symbol;Acc:MGI:1889642] Gene Synonyms StIP1, Stat3-interacting protein, Statip1 Location Chromosome 18: 24,602,866-24,639,507 forward strand. GRCm38:CM001011.2 About this gene This gene has 8 transcripts (splice variants), 199 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Elp2-204 ENSMUST00000234266.1 3466 831aa ENSMUSP00000157109.1 Protein coding CCDS29103 Q91WG4 GENCODE basic APPRIS P1 Elp2-201 ENSMUST00000025120.7 2497 741aa ENSMUSP00000025120.7 Protein coding - Q91WG4 TSL:1 GENCODE basic Elp2-207 ENSMUST00000234847.1 2527 No protein - Retained intron - - - Elp2-202 ENSMUST00000234014.1 757 No protein - Retained intron - - - Elp2-203 ENSMUST00000234164.1 737 No protein - Retained intron - - - Elp2-206 ENSMUST00000234484.1 673 No protein - Retained intron - - - Elp2-205 ENSMUST00000234351.1 627 No protein - Retained intron - - - Elp2-208 ENSMUST00000234903.1 429 No protein - lncRNA - - - Page 6 of 8 https://www.alphaknockout.com 56.64 kb Forward strand 24.60Mb 24.61Mb 24.62Mb 24.63Mb 24.64Mb Genes (Comprehensive set... Elp2-208 >lncRNA Elp2-203 >retained intron Elp2-205 >retained intron Elp2-204 >protein coding Elp2-201 >protein coding Elp2-206 >retained intron Elp2-202 >retained intron Elp2-207 >retained intron Contigs AC131713.3 > Genes < Slc39a6-201protein coding (Comprehensive set... < Slc39a6-204protein coding < Slc39a6-202retained intron < Slc39a6-203protein coding Regulatory Build 24.60Mb 24.61Mb 24.62Mb 24.63Mb 24.64Mb Reverse strand 56.64 kb Regulation Legend Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000234266 35.55 kb Forward strand Elp2-204 >protein coding ENSMUSP00000157... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily WD40-repeat-containing domain superfamily SMART WD40 repeat Pfam WD40 repeat PROSITE profiles WD40 repeat WD40-repeat-containing domain PANTHER Elongator complex protein 2 Gene3D WD40/YVTN repeat-like-containing domain superfamily CDD cd00200 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe insertion missense variant splice region variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 831 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.