https://www.alphaknockout.com

Mouse Mbnl3 Knockout Project (CRISPR/Cas9)

Objective: To create a Mbnl3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mbnl3 gene (NCBI Reference Sequence: NM_134163 ; Ensembl: ENSMUSG00000036109 ) is located on Mouse chromosome X. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000114876). Exon 4~6 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: Mice homozygous for an allele lacking exon 2 exhibit impaired muscle regeneration.

Exon 4 starts from about 33.43% of the coding region. Exon 4~6 covers 56.53% of the coding region. The size of effective KO region: ~2700 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 4 5 6 8

Legends Exon of mouse Mbnl3 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 4 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 Exon 6 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(33.05% 661) | C(17.05% 341) | T(29.55% 591) | G(20.35% 407)

Note: The 2000 bp section upstream of Exon 4 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(29.75% 595) | C(19.35% 387) | T(33.25% 665) | G(17.65% 353)

Note: The 2000 bp section downstream of Exon 6 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% chrX - 51131621 51133620 2000 browser details YourSeq 39 1809 1866 2000 73.5% chr14 - 105065263 105065311 49 browser details YourSeq 34 1795 1838 2000 97.3% chr14 - 117412570 117412615 46 browser details YourSeq 33 1714 1879 2000 65.8% chr14 - 55359975 55360102 128 browser details YourSeq 31 1385 1422 2000 94.5% chr4 + 128973530 128973574 45 browser details YourSeq 27 1860 1886 2000 100.0% chr5 + 50597836 50597862 27 browser details YourSeq 27 1382 1410 2000 89.3% chr3 + 97374454 97374481 28 browser details YourSeq 26 1723 1758 2000 96.5% chr12 - 60190775 60190811 37 browser details YourSeq 26 1722 1753 2000 96.5% chr12 + 37980688 37980721 34 browser details YourSeq 23 1387 1412 2000 96.0% chr13 - 53893945 53893972 28 browser details YourSeq 23 1386 1413 2000 88.0% chr17 + 3839986 3840012 27 browser details YourSeq 23 1387 1412 2000 96.0% chr14 + 50009475 50009502 28 browser details YourSeq 23 1258 1285 2000 80.8% chr10 + 85231456 85231481 26 browser details YourSeq 22 1862 1886 2000 95.9% chr18 - 30720866 30720891 26 browser details YourSeq 22 1861 1882 2000 100.0% chr3 + 25955591 25955612 22

Note: The 2000 bp section upstream of Exon 4 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% chrX - 51126921 51128920 2000 browser details YourSeq 311 1356 2000 2000 87.4% chr16 + 42915559 42916170 612 browser details YourSeq 305 1620 2000 2000 91.4% chr9 + 24950272 24950654 383 browser details YourSeq 304 1617 2000 2000 90.4% chr5 - 69396136 69637552 241417 browser details YourSeq 303 1620 2000 2000 90.9% chr2 - 155324955 155325342 388 browser details YourSeq 301 1617 2000 2000 90.9% chr17 + 71145630 71146021 392 browser details YourSeq 299 1620 2000 2000 89.5% chr10 + 76678833 76679219 387 browser details YourSeq 298 1616 2000 2000 90.1% chrX + 73008786 73009172 387 browser details YourSeq 297 1617 2000 2000 90.4% chrX - 40457992 40458374 383 browser details YourSeq 297 1620 2000 2000 90.5% chr17 - 44933427 44933809 383 browser details YourSeq 296 1620 2000 2000 90.3% chr12 + 26172243 26172629 387 browser details YourSeq 295 1620 2000 2000 89.8% chr11 - 112048013 112048390 378 browser details YourSeq 295 1622 2000 2000 90.5% chr4 + 91474352 91474738 387 browser details YourSeq 294 1621 1988 2000 91.6% chr3 + 122896921 123088105 191185 browser details YourSeq 294 1621 1998 2000 91.1% chr2 + 22724711 22725091 381 browser details YourSeq 292 1621 1999 2000 91.6% chr8 - 63950889 64055454 104566 browser details YourSeq 292 1617 2000 2000 90.6% chr10 + 31237772 31238155 384 browser details YourSeq 291 1620 2000 2000 89.2% chr2 - 172023452 172023831 380 browser details YourSeq 291 1616 2000 2000 89.9% chr13 - 116032968 116033359 392 browser details YourSeq 291 1619 2000 2000 88.4% chr2 + 127019506 127019895 390

Note: The 2000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 https://www.alphaknockout.com

Gene and protein information: Mbnl3 muscleblind like splicing factor 3 [ Mus musculus (house mouse) ] Gene ID: 171170, updated on 12-Aug-2019

Gene summary

Official Symbol Mbnl3 provided by MGI Official Full Name muscleblind like splicing factor 3 provided by MGI Primary source MGI:MGI:2444912 See related Ensembl:ENSMUSG00000036109 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 CHCR; MBLX; MBXL; MBLX39; AI661274; A530038J18Rik; E430034C16Rik Expression Biased expression in placenta adult (RPKM 12.8), genital fat pad adult (RPKM 8.0) and 9 other tissues See more Orthologs human all

Genomic context

Location: X; X A5 See Mbnl3 in Genome Data Viewer Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (51113494..51205990, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (48466671..48559009, complement)

Chromosome X - NC_000086.7

Page 6 of 9 https://www.alphaknockout.com

Transcript information: This gene has 7 transcripts

Gene: Mbnl3 ENSMUSG00000036109

Description muscleblind like splicing factor 3 [Source:MGI Symbol;Acc:MGI:2444912] Gene Synonyms A530038J18Rik, CHCR, E430034C16Rik Location Chromosome X: 51,117,269-51,206,532 reverse strand. GRCm38:CM001013.2 About this gene This gene has 7 transcripts (splice variants), 198 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mbnl3- ENSMUST00000114875.7 4649 246aa ENSMUSP00000110525.1 Protein coding CCDS81126 Q3TJQ3 TSL:1 202 GENCODE basic

Mbnl3- ENSMUST00000114876.8 2243 342aa ENSMUSP00000110526.2 Protein coding CCDS40969 Q542D8 Q8R003 TSL:1 203 GENCODE basic APPRIS P1

Mbnl3- ENSMUST00000041495.13 1803 246aa ENSMUSP00000046036.7 Protein coding CCDS81126 Q3TJQ3 TSL:1 201 GENCODE basic

Mbnl3- ENSMUST00000136404.2 1632 332aa ENSMUSP00000138520.1 Protein coding - S4R267 TSL:5 204 GENCODE basic

Mbnl3- ENSMUST00000150014.1 4121 No protein - Retained intron - - TSL:1 206

Mbnl3- ENSMUST00000148116.1 1082 No protein - lncRNA - - TSL:5 205

Mbnl3- ENSMUST00000156028.1 356 No protein - lncRNA - - TSL:3 207

Page 7 of 9 https://www.alphaknockout.com

109.26 kb Forward strand 51.12Mb 51.14Mb 51.16Mb 51.18Mb 51.20Mb Contigs AL844490.4 > AL671848.7 > Genes (Comprehensive set... < Mbnl3-202protein coding

< Mbnl3-203protein coding

< Mbnl3-201protein coding < Mbnl3-207lncRNA

< Mbnl3-206retained intron < Gm7834-201processed pseudogene

< Mbnl3-204protein coding

< Mbnl3-205lncRNA

Regulatory Build

51.12Mb 51.14Mb 51.16Mb 51.18Mb 51.20Mb Reverse strand 109.26 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene pseudogene

Page 8 of 9 https://www.alphaknockout.com

Transcript: ENSMUST00000114876

< Mbnl3-203protein coding

Reverse strand 85.91 kb

ENSMUSP00000110... MobiDB lite Low complexity (Seg) SMART Zinc finger, CCCH-type Pfam PF14608

E3 ligase, CCCH-type zinc finger PROSITE profiles Zinc finger, CCCH-type PANTHER PTHR12675:SF3

PTHR12675 Gene3D 1.10.150.840

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

Variant Legend inframe deletion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 342

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

Page 9 of 9