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Mouse Rbm14 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rbm14 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rbm14 (NCBI Reference Sequence: NM_019869 ; Ensembl: ENSMUSG00000006456 ) is located on Mouse 19. 3 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 3 (Transcript: ENSMUST00000006625). 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 Rbm14 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-63O23 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 16.84% 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: 7156 bp, and the size of intron 2 for 3'-loxP site insertion: 745 bp. The size of effective cKO region: ~2005 bp. The cKO 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 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rbm14 Homology arm cKO region loxP site

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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(8465bp) | A(23.33% 1975) | C(23.37% 1978) | T(29.37% 2486) | G(23.93% 2026)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr19 - 4804266 4807265 3000 browser details YourSeq 191 383 972 3000 84.1% chr11 + 105319677 105320009 333 browser details YourSeq 177 392 976 3000 84.1% chr15 - 80524266 80524642 377 browser details YourSeq 175 392 976 3000 83.5% chr3 + 95104198 95104511 314 browser details YourSeq 169 382 966 3000 83.3% chr11 + 50165760 50166078 319 browser details YourSeq 167 409 1083 3000 87.7% chr13 + 24829168 24829814 647 browser details YourSeq 161 380 954 3000 91.0% chr12 - 69072782 69073407 626 browser details YourSeq 160 387 960 3000 91.2% chr14 - 25992640 26272041 279402 browser details YourSeq 156 407 966 3000 91.5% chr1 - 84828565 84829196 632 browser details YourSeq 155 387 977 3000 81.1% chr1 - 191535443 191535746 304 browser details YourSeq 147 812 983 3000 93.6% chr12 + 54143424 54143604 181 browser details YourSeq 146 409 971 3000 83.0% chrX - 125424300 125424784 485 browser details YourSeq 146 409 971 3000 83.0% chrX - 124489300 124489784 485 browser details YourSeq 146 806 976 3000 91.5% chr5 + 125461622 125461787 166 browser details YourSeq 141 811 993 3000 89.1% chr13 - 94591241 94591433 193 browser details YourSeq 141 801 983 3000 92.3% chr1 + 103432968 103433397 430 browser details YourSeq 139 799 976 3000 88.6% chr13 - 91440937 91441112 176 browser details YourSeq 139 382 977 3000 83.3% chr11 + 117046368 117046525 158 browser details YourSeq 138 816 976 3000 93.2% chr13 - 43428873 43429043 171 browser details YourSeq 138 774 976 3000 91.6% chr1 - 4795245 4795563 319

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% chr19 - 4799301 4802300 3000 browser details YourSeq 57 2353 2455 3000 95.4% chr18 - 44798756 44798950 195 browser details YourSeq 48 2352 2418 3000 91.3% chr16 + 69750018 69750116 99 browser details YourSeq 43 2357 2458 3000 72.4% chr1 + 42252203 42252268 66 browser details YourSeq 42 2349 2413 3000 86.3% chr18 + 62361626 62361688 63 browser details YourSeq 33 2369 2451 3000 78.4% chr1 - 131862364 131862440 77 browser details YourSeq 31 2351 2407 3000 97.0% chr7 + 49990088 49990377 290 browser details YourSeq 27 2371 2411 3000 83.0% chr10 - 116052015 116052055 41 browser details YourSeq 26 2357 2390 3000 86.3% chr7 - 67795981 67796013 33 browser details YourSeq 25 1125 1156 3000 96.3% chr12 - 103806791 103806823 33 browser details YourSeq 24 2403 2426 3000 100.0% chr12 - 38196246 38196269 24

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Rbm14 RNA binding motif protein 14 [ Mus musculus (house mouse) ] Gene ID: 56275, updated on 12-Aug-2019

Gene summary

Official Symbol Rbm14 provided by MGI Official Full Name RNA binding motif protein 14 provided by MGI Primary source MGI:MGI:1929092 See related Ensembl:ENSMUSG00000006456 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 p16; PSP2; p16K; Sytip; 1300007E16Rik Expression Ubiquitous expression in thymus adult (RPKM 59.4), ovary adult (RPKM 56.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 19; 19 A See Rbm14 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (4800566..4811634, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (4800925..4811634, complement)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 3 transcripts

Gene: Rbm14 ENSMUSG00000006456

Description RNA binding motif protein 14 [Source:MGI Symbol;Acc:MGI:1929092] Gene Synonyms 1300007E16Rik, PSP2, p16 Location Chromosome 19: 4,800,569-4,811,634 reverse strand. GRCm38:CM001012.2 About this gene This gene has 3 transcripts (splice variants), 245 orthologues, 23 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rbm14-201 ENSMUST00000006625.7 3165 669aa ENSMUSP00000006625.7 Protein coding CCDS29437 Q8C2Q3 TSL:1 GENCODE basic APPRIS P1

Rbm14-202 ENSMUST00000113793.3 3508 614aa ENSMUSP00000109424.3 Protein coding - E9QL13 TSL:1 GENCODE basic

Rbm14-203 ENSMUST00000180008.1 370 119aa ENSMUSP00000137466.1 Protein coding - J3QPT3 TSL:5 GENCODE basic

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31.07 kb Forward strand 4.80Mb 4.81Mb 4.82Mb Gm21844-201 >lncRNA (Comprehensive set...

Contigs < AC147618.3 < AC141437.4 Genes (Comprehensive set... < Rbm4-206protein coding < Rbm14-201protein coding

< Rbm4-203protein coding < Rbm14-202protein coding

< Rbm4-207protein coding < Rbm14-203protein coding

< Rbm4-201protein coding

< Rbm4-204protein coding

< Rbm4-202protein coding

< Rbm4-205retained intron

< Gm21992-203nonsense mediated decay

< Gm21992-206protein coding

< Gm21992-204protein coding

< Gm21992-201protein coding

< Gm21992-205retained intron

< Gm21992-202lncRNA

Regulatory Build

4.80Mb 4.81Mb 4.82Mb Reverse strand 31.07 kb

Regulation Legend CTCF Enhancer 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: ENSMUST00000006625

< Rbm14-201protein coding

Reverse strand 11.07 kb

ENSMUSP00000006... 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

PS51257 PANTHER PTHR23147

PTHR23147:SF53 Gene3D Nucleotide-binding alpha-beta plait domain superfamily CDD RBM14, RNA recognition motif 2

RBM14, RNA recognition motif 1

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 669

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

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