https://www.alphaknockout.com Mouse Hnrnpa3 Knockout Project (CRISPR/Cas9) Objective: To create a Hnrnpa3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Hnrnpa3 gene (NCBI Reference Sequence: NM_146130 ; Ensembl: ENSMUSG00000059005 ) is located on Mouse chromosome 2. 11 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000111964). Exon 2~8 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 2 starts from about 6.42% of the coding region. Exon 2~8 covers 78.45% of the coding region. The size of effective KO region: ~2249 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 7 8 11 Legends Exon of mouse Hnrnpa3 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 2 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 1313 bp section downstream of Exon 8 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(22.5% 450) | C(20.65% 413) | T(28.55% 571) | G(28.3% 566) Note: The 2000 bp section upstream of Exon 2 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(1313bp) | A(30.46% 400) | C(16.53% 217) | T(33.89% 445) | G(19.12% 251) Note: The 1313 bp section downstream of Exon 8 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% chr2 + 75659467 75661466 2000 browser details YourSeq 33 1247 1292 2000 94.5% chr1 - 105910030 105910260 231 browser details YourSeq 31 1290 1359 2000 94.2% chr8 + 107276036 107276105 70 browser details YourSeq 31 1291 1363 2000 97.0% chr10 + 12110920 12110994 75 browser details YourSeq 26 1332 1359 2000 96.5% chr17 - 46224439 46224466 28 browser details YourSeq 26 1302 1359 2000 72.5% chr19 + 4730876 4730933 58 browser details YourSeq 25 1326 1355 2000 93.2% chr5 + 86509145 86509176 32 browser details YourSeq 24 1314 1337 2000 100.0% chr11 + 77212288 77212311 24 browser details YourSeq 22 869 890 2000 100.0% chr6 - 139555896 139555917 22 browser details YourSeq 21 1558 1578 2000 100.0% chr9 + 39281366 39281386 21 browser details YourSeq 21 1809 1829 2000 100.0% chr14 + 40895586 40895606 21 browser details YourSeq 20 1317 1336 2000 100.0% chr1 - 119723172 119723191 20 browser details YourSeq 20 285 304 2000 100.0% chr1 - 64533451 64533470 20 browser details YourSeq 20 776 795 2000 100.0% chr1 + 44984590 44984609 20 Note: The 2000 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 1313 1 1313 1313 100.0% chr2 + 75663716 75665028 1313 browser details YourSeq 105 310 477 1313 93.6% chr14 - 20352253 20352446 194 browser details YourSeq 95 357 484 1313 95.4% chr1 + 12818929 13178437 359509 browser details YourSeq 94 314 480 1313 90.6% chr17 - 62050923 62051110 188 browser details YourSeq 92 357 478 1313 95.2% chr5 - 66267227 66267373 147 browser details YourSeq 89 362 482 1313 92.5% chr8 - 110800444 110800579 136 browser details YourSeq 86 315 483 1313 92.3% chr7 - 80378153 80378338 186 browser details YourSeq 86 363 480 1313 91.4% chr15 - 40117186 40117326 141 browser details YourSeq 85 369 477 1313 93.0% chr8 + 123843937 123844066 130 browser details YourSeq 85 305 450 1313 94.0% chr4 + 119159600 119159750 151 browser details YourSeq 84 362 477 1313 94.7% chr15 + 12159334 12159449 116 browser details YourSeq 82 315 450 1313 91.2% chr5 + 99095704 99095839 136 browser details YourSeq 81 362 477 1313 94.6% chr2 - 166061527 166061665 139 browser details YourSeq 80 312 453 1313 92.8% chr19 - 10223921 10224063 143 browser details YourSeq 80 363 473 1313 91.8% chr16 + 49019854 49019992 139 browser details YourSeq 79 357 477 1313 90.9% chr2 + 92499229 92499372 144 browser details YourSeq 76 362 481 1313 89.0% chr2 - 132699063 132699205 143 browser details YourSeq 74 310 450 1313 93.2% chr19 - 36858168 36858308 141 browser details YourSeq 73 307 477 1313 95.2% chr6 - 122506701 122507125 425 browser details YourSeq 72 362 477 1313 91.0% chr3 + 82532981 82533117 137 Note: The 1313 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Hnrnpa3 heterogeneous nuclear ribonucleoprotein A3 [ Mus musculus (house mouse) ] Gene ID: 229279, updated on 14-Aug-2019 Gene summary Official Symbol Hnrnpa3 provided by MGI Official Full Name heterogeneous nuclear ribonucleoprotein A3 provided by MGI Primary source MGI:MGI:1917171 See related Ensembl:ENSMUSG00000059005 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 Hnrpa3; 2410013L13Rik; 2610209F03Rik; 2610510D13Rik Expression Broad expression in CNS E11.5 (RPKM 167.7), liver E14 (RPKM 103.2) and 15 other tissues See more Orthologs human all Genomic context Location: 2; 2 C3 See Hnrnpa3 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (75659105..75669407) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (75497316..75507464) Chromosome 2 - NC_000068.7 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Hnrnpa3 ENSMUSG00000059005 Description heterogeneous nuclear ribonucleoprotein A3 [Source:MGI Symbol;Acc:MGI:1917171] Gene Synonyms 2410013L13Rik, 2610209F03Rik, 2610510D13Rik, Hnrpa3 Location Chromosome 2: 75,659,261-75,669,407 forward strand. GRCm38:CM000995.2 About this gene This gene has 7 transcripts (splice variants), 122 orthologues, 41 paralogues and is a member of 2 Ensembl protein families. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Hnrnpa3- ENSMUST00000111962.7 5150 357aa ENSMUSP00000107593.1 Protein coding CCDS50610 Q0VG47 TSL:1 203 Q8BG05 GENCODE basic APPRIS ALT2 Hnrnpa3- ENSMUST00000111964.7 1654 379aa ENSMUSP00000107595.1 Protein coding CCDS16149 Q5FB19 TSL:1 204 Q8BG05 GENCODE basic APPRIS P3 Hnrnpa3- ENSMUST00000090792.10 1140 379aa ENSMUSP00000088298.4 Protein coding CCDS16149 Q5FB19 TSL:5 201 Q8BG05 GENCODE basic APPRIS P3 Hnrnpa3- ENSMUST00000164947.8 1074 357aa ENSMUSP00000126069.2 Protein coding CCDS50610 Q0VG47 TSL:5 207 Q8BG05 GENCODE basic APPRIS ALT2 Hnrnpa3- ENSMUST00000111961.7 1734 318aa ENSMUSP00000107592.1 Protein coding - A2AL12 TSL:1 202 GENCODE basic APPRIS ALT2 Hnrnpa3- ENSMUST00000141974.1 754 194aa ENSMUSP00000116125.1 Protein coding - A2AL13 CDS 5' 206 incomplete TSL:2 Hnrnpa3- ENSMUST00000132392.1 596 No - Retained - - TSL:2 205 protein intron Page 7 of 9 https://www.alphaknockout.com 30.15 kb Forward strand 75.65Mb 75.66Mb 75.67Mb Genes Gm24574-201 >miRNA (Comprehensive set... Hnrnpa3-204 >protein coding Hnrnpa3-203 >protein coding Hnrnpa3-202 >protein coding Hnrnpa3-205 >retained intron Hnrnpa3-201 >protein coding Hnrnpa3-207 >protein coding Hnrnpa3-206 >protein coding Contigs AL772214.3 > AL772404.5 > Genes < Nfe2l2-201protein coding (Comprehensive set... Regulatory Build 75.65Mb 75.66Mb 75.67Mb Reverse strand 30.15 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000111964 6.58 kb Forward strand Hnrnpa3-204 >protein coding ENSMUSP00000107... MobiDB lite Low complexity (Seg) Superfamily RNA-binding domain superfamily SMART RNA recognition motif domain Pfam RNA recognition motif domain PROSITE profiles RNA recognition motif domain PANTHER PTHR15241 PTHR15241:SF252 Gene3D Nucleotide-binding alpha-beta plait domain superfamily CDD hnRNP A3, RNA recognition motif 2 hnRNP A3, RNA recognition motif 1 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 379 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.