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Mouse Nsfl1c Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Nsfl1c conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Nsfl1c gene (NCBI Reference Sequence: NM_198326 ; Ensembl: ENSMUSG00000027455 ) is located on Mouse chromosome 2. 10 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000089140). Exon 3~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nsfl1c gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-112E9 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 18.28% of the coding region. The knockout of Exon 3~5 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 4033 bp, and the size of intron 5 for 3'-loxP site insertion: 931 bp. The size of effective cKO region: ~2965 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 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Nsfl1c 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(9465bp) | A(26.1% 2470) | C(21.43% 2028) | T(30.99% 2933) | G(21.49% 2034)

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% chr2 + 151497466 151500465 3000 browser details YourSeq 97 2806 2960 3000 82.0% chr1 - 190767584 190989909 222326 browser details YourSeq 93 2818 2985 3000 78.0% chr1 + 178387571 178387739 169 browser details YourSeq 89 2748 2898 3000 84.5% chr11 - 32029271 32029407 137 browser details YourSeq 84 2776 2941 3000 81.2% chr4 + 150067939 150068100 162 browser details YourSeq 81 2834 2961 3000 84.1% chr11 + 94021855 94022255 401 browser details YourSeq 77 2812 2940 3000 77.4% chr12 - 16596890 16597009 120 browser details YourSeq 75 2758 2919 3000 75.9% chr15 - 38353216 38353378 163 browser details YourSeq 72 2834 2942 3000 83.5% chr8 - 110567759 110567868 110 browser details YourSeq 72 2810 2960 3000 77.3% chr7 + 24336936 24337086 151 browser details YourSeq 72 2758 2945 3000 88.3% chr3 + 34760679 34760867 189 browser details YourSeq 72 2803 2943 3000 82.4% chr11 + 62626091 62626231 141 browser details YourSeq 71 2812 2940 3000 78.9% chr17 - 26436735 26436864 130 browser details YourSeq 70 2814 2959 3000 75.4% chr14 - 50842321 50842467 147 browser details YourSeq 69 2764 2884 3000 76.7% chr11 - 63703609 63703704 96 browser details YourSeq 69 2808 2918 3000 81.9% chr11 + 83806371 83806483 113 browser details YourSeq 68 2753 2922 3000 86.1% chr3 - 144429470 144429639 170 browser details YourSeq 68 2819 2945 3000 77.2% chr1 - 183746554 183746681 128 browser details YourSeq 68 2810 2925 3000 78.8% chr9 + 53401903 53402016 114 browser details YourSeq 67 2814 2923 3000 81.7% chr15 - 79634894 79635006 113

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% chr2 + 151503431 151506430 3000 browser details YourSeq 322 681 2954 3000 90.3% chr5 + 49552602 49552942 341 browser details YourSeq 171 4 332 3000 92.1% chr12 - 50039361 50039965 605 browser details YourSeq 151 12 317 3000 92.3% chr8 - 83337846 83338497 652 browser details YourSeq 150 9 214 3000 93.7% chr1 - 96960044 96960430 387 browser details YourSeq 150 1 173 3000 93.6% chr14 + 24719097 24719270 174 browser details YourSeq 149 6 173 3000 93.4% chr19 - 42851959 42852124 166 browser details YourSeq 149 6 193 3000 92.1% chr10 + 39859951 39860184 234 browser details YourSeq 148 12 173 3000 95.7% chrX - 39648135 39648296 162 browser details YourSeq 148 6 192 3000 92.5% chr3 + 121004068 121004297 230 browser details YourSeq 146 6 173 3000 93.5% chr2 - 148662032 148662199 168 browser details YourSeq 146 1 171 3000 93.5% chr19 - 39928327 39928497 171 browser details YourSeq 146 1 173 3000 93.0% chr17 - 77816281 77816453 173 browser details YourSeq 146 11 173 3000 95.1% chr9 + 90145061 90145226 166 browser details YourSeq 145 6 173 3000 93.5% chr7 - 92460154 92460321 168 browser details YourSeq 145 11 173 3000 94.5% chr5 - 68952060 68952222 163 browser details YourSeq 145 11 173 3000 94.5% chr12 - 35094459 35094621 163 browser details YourSeq 145 1 171 3000 92.9% chr6 + 22977565 22977738 174 browser details YourSeq 145 11 225 3000 91.5% chr12 + 29431218 29431771 554 browser details YourSeq 144 4 184 3000 90.6% chr3 - 139484190 139484476 287

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Nsfl1c NSFL1 (p97) cofactor (p47) [ Mus musculus (house mouse) ] Gene ID: 386649, updated on 12-Aug-2019

Gene summary

Official Symbol Nsfl1c provided by MGI Official Full Name NSFL1 (p97) cofactor (p47) provided by MGI Primary source MGI:MGI:3042273 See related Ensembl:ENSMUSG00000027455 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 p47; Stxbp3a; Munc-18c Expression Ubiquitous expression in CNS E11.5 (RPKM 67.7), CNS E14 (RPKM 54.4) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 G3 See Nsfl1c in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (151494182..151511310)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (151320044..151337041)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 10 transcripts

Gene: Nsfl1c ENSMUSG00000027455

Description NSFL1 (p97) cofactor (p47) [Source:MGI Symbol;Acc:MGI:3042273] Gene Synonyms p47 Location Chromosome 2: 151,494,182-151,511,414 forward strand. GRCm38:CM000995.2 About this gene This gene has 10 transcripts (splice variants), 218 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nsfl1c- ENSMUST00000089140.12 1486 372aa ENSMUSP00000086542.6 Protein coding CCDS16868 Q3UVN5 TSL:5 202 Q9CZ44 GENCODE basic APPRIS P3

Nsfl1c- ENSMUST00000028949.15 1364 370aa ENSMUSP00000028949.9 Protein coding CCDS71160 Q9CZ44 TSL:1 201 GENCODE basic APPRIS ALT1

Nsfl1c- ENSMUST00000103160.4 1369 339aa ENSMUSP00000099449.4 Protein coding - A2AT02 TSL:5 203 GENCODE basic

Nsfl1c- ENSMUST00000153333.7 1631 No - Retained - - TSL:5 209 protein intron

Nsfl1c- ENSMUST00000139229.1 930 No - Retained - - TSL:2 207 protein intron

Nsfl1c- ENSMUST00000134081.1 899 No - Retained - - TSL:2 206 protein intron

Nsfl1c- ENSMUST00000146695.7 789 No - Retained - - TSL:2 208 protein intron

Nsfl1c- ENSMUST00000125270.7 744 No - Retained - - TSL:2 204 protein intron

Nsfl1c- ENSMUST00000133197.1 685 No - Retained - - TSL:2 205 protein intron

Nsfl1c- ENSMUST00000156182.1 447 No - lncRNA - - TSL:3 210 protein

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37.23 kb Forward strand

151.49Mb 151.50Mb 151.51Mb 151.52Mb Genes (Comprehensive set... Nsfl1c-202 >protein coding

Nsfl1c-209 >retained intron Nsfl1c-207 >retained intron

Nsfl1c-204 >retained intron Nsfl1c-210 >lncRNA

Nsfl1c-201 >protein coding

Nsfl1c-203 >protein coding

Nsfl1c-205 >retained intron Nsfl1c-206 >retained intron

Nsfl1c-208 >retained intron

Contigs AL928719.6 > Regulatory Build

151.49Mb 151.50Mb 151.51Mb 151.52Mb Reverse strand 37.23 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: ENSMUST00000089140

17.13 kb Forward strand

Nsfl1c-202 >protein coding

ENSMUSP00000086... MobiDB lite Low complexity (Seg) Superfamily UBA-like superfamily NSFL1 cofactor p47, SEP domain superfamily

Ubiquitin-like domain superfamily SMART SEP domain UBX domain

Pfam PF14555 SEP domain UBX domain

PROSITE profiles SEP domain UBX domain

PANTHER PTHR23333:SF24

PTHR23333 Gene3D 1.10.8.10 NSFL1 cofactor p47, SEP domain superfamily

3.10.20.90 CDD cd14348 cd17162

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

Variant Legend start lost missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 372

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

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