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Mouse Nars Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Nars (NCBI Reference Sequence: NM_001142950 ; Ensembl: ENSMUSG00000024587 ) is located on Mouse 18. 15 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000237400). Exon 3~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nars gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-76J14 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 3 starts from about 7.57% of the coding region. The knockout of Exon 3~6 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 3276 bp, and the size of intron 6 for 3'-loxP site insertion: 1137 bp. The size of effective cKO region: ~2097 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 3 4 5 6 7 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Nars 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(8597bp) | A(25.22% 2168) | C(19.95% 1715) | T(31.22% 2684) | G(23.61% 2030)

Note: The sequence of homologous arms and cKO region 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 3000 1 3000 3000 100.0% chr18 - 64512300 64515299 3000 browser details YourSeq 247 2449 2747 3000 92.3% chr2 - 170875812 170876120 309 browser details YourSeq 239 2446 2725 3000 93.9% chr11 + 114353063 114353383 321 browser details YourSeq 238 2449 2746 3000 90.9% chr11 + 112695726 112696044 319 browser details YourSeq 238 2449 2747 3000 91.5% chr11 + 57447127 57447458 332 browser details YourSeq 237 2449 2747 3000 91.1% chr8 + 71703326 71703648 323 browser details YourSeq 237 2449 2747 3000 91.7% chr5 + 96928137 96928445 309 browser details YourSeq 236 2449 2746 3000 90.6% chr13 - 95174973 95175311 339 browser details YourSeq 236 2458 2747 3000 92.5% chr15 + 75668108 75668407 300 browser details YourSeq 235 2453 2747 3000 92.5% chr14 + 118811236 118830300 19065 browser details YourSeq 234 2450 2732 3000 92.5% chr17 - 31881226 31881538 313 browser details YourSeq 232 2449 2741 3000 90.4% chr18 - 64494976 64495280 305 browser details YourSeq 232 2449 2747 3000 92.2% chr5 + 112127850 112128179 330 browser details YourSeq 231 2439 2747 3000 90.6% chr5 - 142789071 142789379 309 browser details YourSeq 231 2449 2741 3000 91.8% chr3 + 41507156 41507461 306 browser details YourSeq 231 2452 2747 3000 91.5% chr10 + 83685619 83685944 326 browser details YourSeq 230 2448 2747 3000 90.1% chr2 - 21350212 21350537 326 browser details YourSeq 229 2450 2744 3000 92.9% chr10 - 60328371 60328670 300 browser details YourSeq 228 2450 2747 3000 90.2% chr3 - 36159712 36160017 306 browser details YourSeq 228 2449 2730 3000 92.0% chr13 - 19401171 19401490 320

Note: The 3000 bp section upstream of Exon 3 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 - 64507203 64510202 3000 browser details YourSeq 132 2825 2999 3000 91.9% chr6 + 34286288 34286477 190 browser details YourSeq 130 2834 2997 3000 90.3% chr8 + 41209092 41209257 166 browser details YourSeq 128 2837 2997 3000 90.1% chr2 - 147173846 147174007 162 browser details YourSeq 124 2837 2999 3000 88.3% chr6 + 117714400 117714563 164 browser details YourSeq 123 2847 2999 3000 90.2% chr10 + 89720279 89720431 153 browser details YourSeq 122 2858 2996 3000 94.3% chr4 - 125986844 125986983 140 browser details YourSeq 121 2858 2999 3000 93.0% chr7 - 142078854 142078996 143 browser details YourSeq 120 2841 2991 3000 90.0% chr1 - 164847612 164847763 152 browser details YourSeq 120 2842 2997 3000 89.5% chr10 + 95691283 95691438 156 browser details YourSeq 119 2838 2997 3000 87.5% chr13 - 30056481 30056641 161 browser details YourSeq 119 2843 2997 3000 90.6% chr14 + 31598667 31598831 165 browser details YourSeq 118 2847 3000 3000 91.0% chr8 + 95318078 95318232 155 browser details YourSeq 118 2858 2999 3000 90.8% chr15 + 76153768 76153908 141 browser details YourSeq 117 2858 2999 3000 91.6% chr5 + 150885637 150885779 143 browser details YourSeq 116 2837 2993 3000 89.2% chr16 - 90734650 90734807 158 browser details YourSeq 116 2846 2999 3000 86.0% chr12 - 76357716 76357867 152 browser details YourSeq 116 2842 2993 3000 87.4% chr10 - 110312560 110312710 151 browser details YourSeq 116 2839 2997 3000 86.8% chr1 - 57194730 57194889 160 browser details YourSeq 116 2867 2999 3000 94.0% chr7 + 122959478 122959611 134

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Nars asparaginyl-tRNA synthetase [ Mus musculus (house mouse) ] Gene ID: 70223, updated on 24-Oct-2019

Gene summary

Official Symbol Nars provided by MGI Official Full Name asparaginyl-tRNA synthetase provided by MGI Primary source MGI:MGI:1917473 See related Ensembl:ENSMUSG00000024587 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 ASNRS; C78150; AA960128; 3010001M15Rik Expression Ubiquitous expression in liver E14 (RPKM 51.7), placenta adult (RPKM 44.5) and 28 other tissues See more Orthologs human all

Genomic context

Location: 18; 18 E1 See Nars in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (64499647..64516557, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (64659301..64676211, complement)

Chromosome 18 - NC_000084.6

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Transcript information: This gene has 16 transcripts

Gene: Nars ENSMUSG00000024587

Description asparaginyl-tRNA synthetase [Source:MGI Symbol;Acc:MGI:1917473] Gene Synonyms ASNRS Location : 64,499,647-64,516,652 reverse strand. GRCm38:CM001011.2 About this gene This gene has 16 transcripts (splice variants), 236 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nars- ENSMUST00000237400.1 2745 559aa ENSMUSP00000157402.1 Protein coding CCDS50308 Q8BP47 GENCODE 213 basic APPRIS P2

Nars- ENSMUST00000025483.10 2687 558aa ENSMUSP00000025483.10 Protein coding - - TSL:1 201 GENCODE basic APPRIS ALT1

Nars- ENSMUST00000236186.1 1784 517aa ENSMUSP00000158356.1 Protein coding - A0A494BB89 GENCODE 205 basic

Nars- ENSMUST00000237351.1 1492 497aa ENSMUSP00000158278.1 Protein coding - A0A494BAX5 CDS 5' 211 incomplete

Nars- ENSMUST00000235325.1 761 210aa ENSMUSP00000157468.1 Protein coding - A0A494B927 CDS 3' 202 incomplete

Nars- ENSMUST00000236873.1 2482 127aa ENSMUSP00000157502.1 Nonsense mediated - A0A494B996 - 209 decay

Nars- ENSMUST00000237369.1 2103 70aa ENSMUSP00000157488.1 Nonsense mediated - A0A494B949 - 212 decay

Nars- ENSMUST00000236392.1 935 164aa ENSMUSP00000158211.1 Nonsense mediated - A0A494BAW6 - 206 decay

Nars- ENSMUST00000236463.1 894 43aa ENSMUSP00000158367.1 Nonsense mediated - A0A494BB92 - 207 decay

Nars- ENSMUST00000235647.1 874 69aa ENSMUSP00000157626.1 Nonsense mediated - A0A494B9G0 - 203 decay

Nars- ENSMUST00000236583.1 845 35aa ENSMUSP00000158408.1 Nonsense mediated - A0A494BB81 - 208 decay

Nars- ENSMUST00000237585.1 2268 No - Retained intron - - - 215 protein

Nars- ENSMUST00000237503.1 1648 No - Retained intron - - - 214 protein

Nars- ENSMUST00000237027.1 848 No - Retained intron - - - 210 protein

Nars- ENSMUST00000237831.1 774 No - Retained intron - - - 216 protein

Nars- ENSMUST00000235887.1 434 No - Retained intron - - - 204 protein

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37.01 kb Forward strand

64.49Mb 64.50Mb 64.51Mb 64.52Mb Contigs AC102268.11 > (Comprehensive set... < Fech-209protein coding < Nars-213protein coding

< Nars-209nonsense mediated decay

< Nars-214retained intron< Nars-206nonsense mediated decay

< Nars-201protein coding

< Nars-212nonsense mediated decay

< Nars-215retained intron

< Nars-211protein coding

< Nars-205protein coding

< Nars-204retained intron < Nars-210retained intron

< Nars-216retained intron

< Nars-208nonsense mediated decay

< Nars-203nonsense mediated decay

< Nars-207nonsense mediated decay

< Nars-202protein coding

Regulatory Build

64.49Mb 64.50Mb 64.51Mb 64.52Mb Reverse strand 37.01 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

processed transcript

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Transcript: ENSMUST00000237400

< Nars-213protein coding

Reverse strand 17.01 kb

ENSMUSP00000157... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) TIGRFAM Asparagine-tRNA ligase Superfamily Nucleic acid-binding, OB-fold SSF55681

Prints Aspartyl/Asparaginyl-tRNA synthetase, class IIb Pfam Aminoacyl-tRNA synthetase, class II (D/K/N)

OB-fold nucleic acid binding domain, AA-tRNA synthetase-type PROSITE profiles Aminoacyl-tRNA synthetase, class II PANTHER PTHR22594

PTHR22594:SF16 Gene3D 1.10.10.1430 2.40.50.140 3.30.930.10

CDD cd04323 cd00776

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

Variant Legend missense variant splice region variant synonymous variant stop retained variant

Scale bar 0 60 120 180 240 300 360 420 480 559

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

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