https://www.alphaknockout.com

Mouse Prkar1b Knockout Project (CRISPR/Cas9)

Objective: To create a Prkar1b knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Prkar1b gene (NCBI Reference Sequence: NM_008923 ; Ensembl: ENSMUSG00000025855 ) is located on Mouse chromosome 5. 11 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000026973). Exon 3~4 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: Homozygous null mice have reduced LTD and LTP in specific CNS tracts, but normal neuroanatomy and behavior. Response to pain after inflammation is reduced, concurrent with decreased plasma extravasation during the inflammatory response.

Exon 3 starts from about 15.57% of the coding region. Exon 3~4 covers 23.01% of the coding region. The size of effective KO region: ~2034 bp. The KO 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 11

Legends Exon of mouse Prkar1b Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(21.9% 438) | C(24.45% 489) | T(25.35% 507) | G(28.3% 566)

Note: The 2000 bp section upstream of Exon 3 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(25.4% 508) | C(23.05% 461) | T(26.0% 520) | G(25.55% 511)

Note: The 2000 bp section downstream 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr5 - 139108655 139110654 2000 browser details YourSeq 377 1441 1913 2000 95.7% chr5 + 139937100 140012386 75287 browser details YourSeq 374 1441 1896 2000 96.4% chr7 - 28202181 28478462 276282 browser details YourSeq 350 1441 1903 2000 95.9% chr7 - 6201316 6202066 751 browser details YourSeq 345 1441 1836 2000 96.3% chr5 - 144637766 144638171 406 browser details YourSeq 342 1440 1836 2000 95.3% chr7 + 36024837 36025247 411 browser details YourSeq 340 1441 1836 2000 96.3% chr5 - 144637819 144638223 405 browser details YourSeq 338 1442 1836 2000 95.8% chr7 - 34922629 34923142 514 browser details YourSeq 338 1441 1836 2000 96.5% chr7 - 30304231 30305115 885 browser details YourSeq 338 1441 1836 2000 96.5% chr7 - 30304442 30305062 621 browser details YourSeq 337 1441 1870 2000 94.7% chr5 + 139640142 139937800 297659 browser details YourSeq 336 1441 1836 2000 95.2% chr5 - 139680332 139898400 218069 browser details YourSeq 335 1441 1836 2000 96.5% chr7 - 28478052 28706156 228105 browser details YourSeq 334 1441 1836 2000 96.4% chr7 - 19351171 19398737 47567 browser details YourSeq 334 1441 1829 2000 96.0% chr7 - 18945981 19351474 405494 browser details YourSeq 334 1441 1837 2000 97.0% chr5 + 141782725 141783242 518 browser details YourSeq 333 1441 1836 2000 95.2% chr7 + 25111347 25111752 406 browser details YourSeq 332 1441 1830 2000 95.7% chr5 - 144637719 144638119 401 browser details YourSeq 332 1441 1836 2000 94.7% chr5 - 142720678 142721185 508 browser details YourSeq 331 1441 1836 2000 95.9% chr7 - 19908014 19908901 888

Note: The 2000 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 2000 1 2000 2000 100.0% chr5 - 139104621 139106620 2000 browser details YourSeq 317 891 1663 2000 88.8% chr1 + 164516976 164556329 39354 browser details YourSeq 264 1334 1663 2000 92.1% chr10 - 122892216 122892555 340 browser details YourSeq 263 1350 1663 2000 92.3% chr10 + 61287688 61288000 313 browser details YourSeq 259 1350 1663 2000 91.7% chr6 + 146278841 146704599 425759 browser details YourSeq 259 1352 1663 2000 92.3% chr10 + 116876056 116882182 6127 browser details YourSeq 257 1352 1663 2000 91.4% chr13 - 35958089 35958402 314 browser details YourSeq 257 1349 1663 2000 91.1% chr4 + 32362689 32363005 317 browser details YourSeq 254 1348 1663 2000 90.5% chr16 - 37006258 37006575 318 browser details YourSeq 252 1350 1661 2000 90.7% chr7 - 127811738 127812051 314 browser details YourSeq 252 1350 1663 2000 91.5% chr11 - 82689669 82689983 315 browser details YourSeq 250 1350 1664 2000 91.0% chr17 - 13031560 13031880 321 browser details YourSeq 250 1355 1663 2000 90.9% chr17 + 84061366 84061675 310 browser details YourSeq 248 1354 1663 2000 90.3% chr14 - 118829992 118830303 312 browser details YourSeq 246 1353 1670 2000 90.0% chr10 - 69629457 69629780 324 browser details YourSeq 246 925 1663 2000 87.6% chr5 + 113228629 113229265 637 browser details YourSeq 246 1350 1663 2000 90.7% chr2 + 91992237 91992552 316 browser details YourSeq 245 1350 1661 2000 89.5% chr8 - 125736799 125737114 316 browser details YourSeq 245 1350 1663 2000 91.1% chr17 - 13169071 13169390 320 browser details YourSeq 245 1350 1661 2000 89.3% chr17 - 5624767 5625077 311

Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Prkar1b protein kinase, cAMP dependent regulatory, type I beta [ Mus musculus (house mouse) ] Gene ID: 19085, updated on 24-Oct-2019

Gene summary

Official Symbol Prkar1b provided by MGI Official Full Name protein kinase, cAMP dependent regulatory, type I beta provided by MGI Primary source MGI:MGI:97759 See related Ensembl:ENSMUSG00000025855 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 RIbeta; AI385716 Expression Biased expression in cortex adult (RPKM 158.4), frontal lobe adult (RPKM 140.4) and 6 other tissues See more Orthologs human all

Genomic context

Location: 5 G2; 5 77.74 cM See Prkar1b in Genome Data Viewer Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (139017304..139150703, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (139493258..139605706, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 11 transcripts

Gene: Prkar1b ENSMUSG00000025855

Description protein kinase, cAMP dependent regulatory, type I beta [Source:MGI Symbol;Acc:MGI:97759] Gene Synonyms RIbeta Location Chromosome 5: 139,017,306-139,150,001 reverse strand. GRCm38:CM000998.2 About this gene This gene has 11 transcripts (splice variants), 205 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Prkar1b-201 ENSMUST00000026973.13 2637 381aa ENSMUSP00000026973.7 Protein coding CCDS19803 P12849 TSL:1 GENCODE basic APPRIS P1

Prkar1b-203 ENSMUST00000110890.7 2626 381aa ENSMUSP00000106515.1 Protein coding CCDS19803 P12849 TSL:1 GENCODE basic APPRIS P1

Prkar1b-202 ENSMUST00000110889.7 2457 257aa ENSMUSP00000106514.1 Protein coding - D3Z0V6 TSL:5 GENCODE basic

Prkar1b-204 ENSMUST00000129851.7 918 234aa ENSMUSP00000121093.1 Protein coding - D3YTM5 CDS 3' incomplete TSL:2

Prkar1b-210 ENSMUST00000148002.7 795 251aa ENSMUSP00000123286.1 Protein coding - D3Z068 CDS 3' incomplete TSL:2

Prkar1b-209 ENSMUST00000147505.7 440 116aa ENSMUSP00000116727.1 Protein coding - D3Z406 CDS 3' incomplete TSL:3

Prkar1b-211 ENSMUST00000155833.7 426 107aa ENSMUSP00000122693.1 Protein coding - D3Z4A5 CDS 3' incomplete TSL:3

Prkar1b-208 ENSMUST00000141985.1 402 45aa ENSMUSP00000117412.1 Protein coding - D3Z4L4 CDS 3' incomplete TSL:5

Prkar1b-206 ENSMUST00000134762.1 367 85aa ENSMUSP00000116022.1 Protein coding - D3Z7E4 CDS 3' incomplete TSL:3

Prkar1b-207 ENSMUST00000139415.1 390 No protein - lncRNA - - TSL:2

Prkar1b-205 ENSMUST00000130180.1 380 No protein - lncRNA - - TSL:3

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152.70 kb Forward strand 139.02Mb 139.04Mb 139.06Mb 139.08Mb 139.10Mb 139.12Mb 139.14Mb 139.16Mb Genes Dnaaf5-201 >protein coding (Comprehensive set...

Dnaaf5-203 >protein coding

Contigs < AC117698.19 AC125065.3 > Genes (Comprehensive set... < Prkar1b-201protein coding < Prkar1b-205lncRNA

< Prkar1b-203protein coding < Gm15672-201lncRNA

< Prkar1b-202protein coding

< Prkar1b-207lncRNA < Prkar1b-210protein coding

< Prkar1b-204protein coding

< Prkar1b-208protein coding

< Prkar1b-209protein coding

< Prkar1b-211protein coding

< Prkar1b-206protein coding

Regulatory Build

139.02Mb 139.04Mb 139.06Mb 139.08Mb 139.10Mb 139.12Mb 139.14Mb 139.16Mb Reverse strand 152.70 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000026973

< Prkar1b-201protein coding

Reverse strand 112.45 kb

ENSMUSP00000026... MobiDB lite Superfamily SSF47391 Cyclic nucleotide-binding-like SMART cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain

Cyclic nucleotide-binding domain Prints PR00103 Pfam Cyclic nucleotide-binding domain

cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain PROSITE profiles Cyclic nucleotide-binding domain PROSITE patterns Cyclic nucleotide-binding, conserved site

Cyclic nucleotide-binding, conserved site PIRSF cAMP-dependent protein kinase regulatory subunit

PANTHER PTHR11635:SF126

PTHR11635 Gene3D 1.20.890.10 RmlC-like jelly roll fold CDD cd12102 Cyclic nucleotide-binding domain

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 381

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

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