https://www.alphaknockout.com

Mouse Mrps24 Knockout Project (CRISPR/Cas9)

Objective: To create a Mrps24 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mrps24 (NCBI Reference Sequence: NM_026080 ; Ensembl: ENSMUSG00000020477 ) is located on Mouse 11. 4 exons are identified, with the ATG in exon 1 and the TAA in exon 4 (Transcript: ENSMUST00000154330). Exon 1~4 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 0.2% of the coding region. Exon 1~4 covers 100.0% of the coding region. The size of effective KO region: ~3209 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4

Legends Exon of mouse Mrps24 Knockout region

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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. 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 stop codon 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(25.7% 514) | C(25.35% 507) | T(27.35% 547) | G(21.6% 432)

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(28.7% 574) | C(20.65% 413) | T(26.15% 523) | G(24.5% 490)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr11 - 5707663 5709662 2000 browser details YourSeq 247 1419 1770 2000 92.8% chr15 + 8510665 8511181 517 browser details YourSeq 240 1419 1769 2000 95.9% chr16 - 3996576 3997122 547 browser details YourSeq 234 1448 1770 2000 93.7% chr16 + 4893216 4893527 312 browser details YourSeq 233 1448 1784 2000 91.3% chr5 + 146269978 146270300 323 browser details YourSeq 226 1516 1770 2000 98.3% chr3 + 137874864 137875465 602 browser details YourSeq 218 1494 1771 2000 93.0% chr5 - 140405556 140405795 240 browser details YourSeq 216 1490 1769 2000 95.8% chr12 + 21448209 21448528 320 browser details YourSeq 215 1490 1769 2000 96.6% chr1 - 192219522 192220074 553 browser details YourSeq 212 1519 1769 2000 98.7% chr15 - 31488528 31488972 445 browser details YourSeq 211 1518 1769 2000 96.9% chr12 + 111753956 111754316 361 browser details YourSeq 209 1516 1769 2000 97.3% chr15 + 98783681 98783933 253 browser details YourSeq 208 1557 1770 2000 98.6% chr6 - 93552386 93552599 214 browser details YourSeq 208 1557 1769 2000 99.1% chr17 - 29977257 29977479 223 browser details YourSeq 205 1557 1769 2000 97.2% chr8 - 107553594 107553805 212 browser details YourSeq 205 1565 1773 2000 99.6% chr11 + 106126917 106127146 230 browser details YourSeq 204 1532 1771 2000 97.3% chr15 - 39222415 39222716 302 browser details YourSeq 203 1563 1769 2000 99.1% chr4 + 128847634 128847840 207 browser details YourSeq 202 1563 1771 2000 97.2% chr5 - 37465181 37465387 207 browser details YourSeq 201 1570 1770 2000 100.0% chr7 - 127955578 127955778 201

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% chr11 - 5702452 5704451 2000 browser details YourSeq 513 1303 1983 2000 89.4% chr6 - 141591048 142141636 550589 browser details YourSeq 489 1312 1987 2000 89.2% chr1 - 41845053 41845779 727 browser details YourSeq 476 1368 2000 2000 90.3% chr6 + 41330755 41331436 682 browser details YourSeq 469 1341 1987 2000 90.6% chr6 + 103156720 103157414 695 browser details YourSeq 468 1310 1987 2000 90.4% chr9 + 119857302 119858003 702 browser details YourSeq 459 1382 2000 2000 90.5% chr6 - 11678738 11679412 675 browser details YourSeq 458 1412 1985 2000 92.9% chr19 - 55169492 55170112 621 browser details YourSeq 458 1318 1984 2000 88.0% chr1 + 100806468 100807186 719 browser details YourSeq 454 1370 1999 2000 89.0% chr16 + 66128936 66129619 684 browser details YourSeq 454 1317 1966 2000 89.7% chr1 + 110155905 110156589 685 browser details YourSeq 452 1303 1987 2000 90.7% chr2 + 43494378 43495132 755 browser details YourSeq 451 1371 2000 2000 88.8% chr4 + 94295171 94430664 135494 browser details YourSeq 450 1397 2000 2000 89.8% chr3 - 103071854 103072509 656 browser details YourSeq 443 1412 1987 2000 90.9% chr3 - 21747540 21748177 638 browser details YourSeq 442 1303 2000 2000 89.1% chr10 + 45057813 45058575 763 browser details YourSeq 441 1367 2000 2000 89.8% chr12 + 111522142 111522823 682 browser details YourSeq 440 1428 1983 2000 93.0% chr1 + 88015006 88015614 609 browser details YourSeq 439 1303 1983 2000 91.9% chr13 - 13842955 13843660 706 browser details YourSeq 438 1383 1980 2000 90.0% chr19 - 17544222 17544863 642

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

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Gene and information: Mrps24 mitochondrial S24 [ Mus musculus (house mouse) ] Gene ID: 64660, updated on 12-Aug-2019

Gene summary

Official Symbol Mrps24 provided by MGI Official Full Name mitochondrial ribosomal protein S24 provided by MGI Primary source MGI:MGI:1928142 See related Ensembl:ENSMUSG00000020477 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 S24mt; Rpms24; MRP-S24; AI414579; 3110030K20Rik Expression Ubiquitous expression in adrenal adult (RPKM 188.3), stomach adult (RPKM 152.2) and 27 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 A1 See Mrps24 in Genome Data Viewer Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (5703982..5707701, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (5603986..5607702, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 5 transcripts

Gene: Mrps24 ENSMUSG00000020477

Description mitochondrial ribosomal protein S24 [Source:MGI Symbol;Acc:MGI:1928142] Gene Synonyms 3110030K20Rik, Rpms24 Location Chromosome 11: 5,703,983-5,715,680 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 200 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein ID Biotype CCDS UniProt Flags

Mrps24-205 ENSMUST00000154330.1 995 167aa ENSMUSP00000119535.1 Protein coding CCDS24402 Q9CQV5 TSL:1 GENCODE basic APPRIS P1

Mrps24-201 ENSMUST00000020770.10 700 No protein - Retained intron - - TSL:2

Mrps24-203 ENSMUST00000132874.1 503 No protein - Retained intron - - TSL:3

Mrps24-202 ENSMUST00000123697.1 418 No protein - Retained intron - - TSL:2

Mrps24-204 ENSMUST00000149980.1 714 No protein - lncRNA - - TSL:3

31.70 kb Forward strand 5.70Mb 5.71Mb 5.72Mb Contigs AL627069.10 > (Comprehensive set... < Mrps24-205protein coding < Urgcp-201protein coding

< Mrps24-201retained intron < Urgcp-206protein coding

< Mrps24-204lncRNA < Urgcp-205protein coding

< Mrps24-203retained intron < Urgcp-202protein coding

< Mrps24-202retained intron < Urgcp-204protein coding

< Urgcp-203protein coding

Regulatory Build

5.70Mb 5.71Mb 5.72Mb Reverse strand 31.70 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000154330

< Mrps24-205protein coding

Reverse strand 3.71 kb

ENSMUSP00000119... Low complexity (Seg) Pfam 28S ribosomal protein S24, mitochondrial PANTHER 28S ribosomal protein S24, mitochondrial

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

Variant Legend stop gained frameshift variant missense variant splice region variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 167

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

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