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Mouse A2ml1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a A2ml1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The A2ml1 gene (NCBI Reference Sequence: NM_001001179.3 ; Ensembl: ENSMUSG00000047228 ) is located on Mouse chromosome 6. 36 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 35 (Transcript: ENSMUST00000060574). Exon 8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse A2ml1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-376K22 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 8 starts from about 16.83% of the coding region. The knockout of Exon 8 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 3456 bp, and the size of intron 8 for 3'-loxP site insertion: 1044 bp. The size of effective cKO region: ~618 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 8 9 10 11 36 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse A2ml1 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(7118bp) | A(29.53% 2102) | C(21.2% 1509) | T(29.12% 2073) | G(20.15% 1434)

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% chr6 - 128572334 128575333 3000 browser details YourSeq 437 1705 2201 3000 95.2% chr11 + 72375369 72375999 631 browser details YourSeq 434 1704 2208 3000 94.4% chr8 + 65007756 65008289 534 browser details YourSeq 432 1703 2196 3000 94.9% chr9 - 113477472 113478001 530 browser details YourSeq 425 1705 2207 3000 93.4% chr5 + 126317508 126318049 542 browser details YourSeq 416 1709 2209 3000 93.8% chr7 + 30501978 30502519 542 browser details YourSeq 416 1705 2201 3000 93.6% chr6 + 148146875 148147412 538 browser details YourSeq 413 1705 2207 3000 93.4% chr5 + 46126942 46555069 428128 browser details YourSeq 412 1709 2207 3000 93.2% chr7 + 30495431 30496227 797 browser details YourSeq 412 1701 2210 3000 92.8% chr1 + 33276998 33277509 512 browser details YourSeq 410 1704 2201 3000 93.5% chr19 - 47740539 47741042 504 browser details YourSeq 407 1707 2208 3000 93.7% chrX - 48735435 48735938 504 browser details YourSeq 406 1705 2207 3000 91.7% chr7 - 4352245 4352727 483 browser details YourSeq 405 1707 2208 3000 93.1% chr3 - 16879641 16880147 507 browser details YourSeq 403 1704 2208 3000 92.7% chr14 - 65804367 65804898 532 browser details YourSeq 400 1705 2210 3000 91.3% chr14 + 63003598 63004092 495 browser details YourSeq 398 1705 2210 3000 90.9% chrX + 136118779 136119265 487 browser details YourSeq 397 1705 2201 3000 92.8% chr17 - 20398261 20398786 526 browser details YourSeq 397 1709 2207 3000 92.7% chr4 + 124050475 124050964 490 browser details YourSeq 395 1705 2207 3000 91.8% chr16 + 22781859 22782335 477

Note: The 3000 bp section upstream of Exon 8 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% chr6 - 128568716 128571715 3000 browser details YourSeq 147 1950 2107 3000 94.2% chr2 + 70736928 70737080 153 browser details YourSeq 141 1950 2101 3000 96.7% chr1 + 74205511 74205964 454 browser details YourSeq 140 1944 2099 3000 95.5% chrX - 169481561 169481728 168 browser details YourSeq 140 1950 2099 3000 97.4% chr3 + 40967054 40967207 154 browser details YourSeq 140 1950 2099 3000 97.4% chr15 + 82253905 82254058 154 browser details YourSeq 139 1876 2101 3000 87.2% chr4 - 136037481 136037651 171 browser details YourSeq 139 1950 2099 3000 94.6% chr1 + 176798401 176798548 148 browser details YourSeq 138 1950 2206 3000 93.8% chr17 - 78529722 78530085 364 browser details YourSeq 138 1950 2099 3000 97.4% chr5 + 117496161 117496316 156 browser details YourSeq 137 1951 2101 3000 94.7% chr12 - 3919895 3920044 150 browser details YourSeq 137 1950 2097 3000 96.7% chr1 - 134519196 134519344 149 browser details YourSeq 137 1950 2101 3000 93.4% chr1 - 114982618 114982767 150 browser details YourSeq 137 1950 2093 3000 98.0% chr1 - 85987007 85987151 145 browser details YourSeq 136 1956 2099 3000 98.0% chr2 - 74167561 74167713 153 browser details YourSeq 136 1950 2093 3000 95.9% chr17 - 42931594 42931736 143 browser details YourSeq 136 1954 2099 3000 94.5% chr10 - 7188379 7188522 144 browser details YourSeq 136 1950 2101 3000 93.3% chr1 - 4795237 4795386 150 browser details YourSeq 136 1950 2101 3000 96.1% chr8 + 25857072 25857228 157 browser details YourSeq 135 1950 2099 3000 95.4% chr7 - 80239813 80239967 155

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: A2ml1 alpha-2-macroglobulin like 1 [ Mus musculus (house mouse) ] Gene ID: 232400, updated on 26-Jun-2020

Gene summary

Official Symbol A2ml1 provided by MGI Official Full Name alpha-2-macroglobulin like 1 provided by MGI Primary source MGI:MGI:3039594 See related Ensembl:ENSMUSG00000047228 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 Ovos; Ovos2 Expression Biased expression in ovary adult (RPKM 19.2), liver E18 (RPKM 5.0) and 1 other tissue See more

Genomic context

Location: 6; 6 F3 See A2ml1 in Genome Data Viewer Exon count: 36

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (128539822..128581606, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (128489840..128531624, complement)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 5 transcripts

Gene: A2ml1 ENSMUSG00000047228

Description alpha-2-macroglobulin like 1 [Source:MGI Symbol;Acc:MGI:3039594] Gene Synonyms BC048546, Ovos2 Location Chromosome 6: 128,539,821-128,581,608 reverse strand. GRCm38:CM000999.2 About this gene This gene has 5 transcripts (splice variants), 744 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

A2ml1-201 ENSMUST00000060574.8 4695 1456aa ENSMUSP00000059426.7 Protein coding CCDS39651 Q3UU35 TSL:1 GENCODE basic APPRIS P1

A2ml1-202 ENSMUST00000203129.1 2563 No protein - Retained intron - - TSL:NA

A2ml1-204 ENSMUST00000203889.1 748 No protein - Retained intron - - TSL:2

A2ml1-203 ENSMUST00000203291.1 700 No protein - Retained intron - - TSL:1

A2ml1-205 ENSMUST00000205167.1 670 No protein - Retained intron - - TSL:3

61.79 kb Forward strand Contigs AC123060.4 > Genes (Comprehensive set... < Gm8724-201processed pseudogene < A2ml1-205retained intron < A2ml1-204retained intron < A2ml1-202retained intron < Gm44009-201lincRNA

< A2ml1-203retained intron

< A2ml1-201protein coding

Regulatory Build

Reverse strand 61.79 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

pseudogene processed transcript RNA gene

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Transcript: ENSMUST00000060574

< A2ml1-201protein coding

Reverse strand 41.78 kb

ENSMUSP00000059... Low complexity (Seg) Cleavage site (Sign... Superfamily Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid

Immunoglobulin E-set Alpha-macroglobulin, receptor-binding domain superfamily SMART Alpha-2-macroglobulin, bait region domain Alpha-macroglobulin, receptor-binding

Alpha-2-macroglobulin Pfam Macroglobulin domain Alpha-2-macroglobulin Alpha-macroglobulin, receptor-binding

Alpha-2-macroglobulin, bait region domain Alpha-macroglobulin-like, TED domain

Macroglobulin domain MG4

Macroglobulin domain MG3 PROSITE patterns Alpha-2-macroglobulin, conserved site

PANTHER PTHR11412

PTHR11412:SF152 Gene3D 2.60.40.1930 2.20.130.20 1.50.10.20 Alpha-macroglobulin, receptor-binding domain superfamily

2.60.40.1940 2.60.120.1540

Immunoglobulin-like fold CDD Alpha-2-macroglobulin, TED domain

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1456

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

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