ABSTRACT
Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20-40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1-20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by 'cold shocking' cells at 32 °C for 24-48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or 'perfect HDR', as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated ('partial HDR'). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing 'perfect HDR' compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.
Subject(s)
Gene Editing/methods , Induced Pluripotent Stem Cells/metabolism , Recombinational DNA Repair , CRISPR-Cas Systems , Cold Temperature , HEK293 Cells , HumansSubject(s)
Chromosomes, Human, Pair 1 , Deafness/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Retinitis Pigmentosa/genetics , Animals , Brain/embryology , Chromosomes, Artificial, Yeast , Cosmids , DNA, Complementary , Deafness/congenital , Exons , Gene Expression , Genes, Recessive , Humans , Mice , Multigene Family , Receptors, Estrogen/genetics , Syndrome , Tissue DistributionABSTRACT
Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.
Subject(s)
Extracellular Matrix Proteins/genetics , Hearing Loss, Sensorineural/genetics , Retinitis Pigmentosa/genetics , Amino Acid Sequence , Animals , Cell Adhesion Molecules/chemistry , Chromosome Mapping , Chromosomes, Human, Pair 1 , Cochlea/chemistry , Epidermal Growth Factor/chemistry , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/physiology , Female , Fibronectins/chemistry , Frameshift Mutation , Gene Expression , Genes, Recessive , Glycosylation , Humans , Laminin/chemistry , Male , Molecular Sequence Data , Pedigree , Retina/chemistry , Syndrome , Tumor Cells, CulturedABSTRACT
Usher syndrome type 1 (USH1) is an autosomal recessive, genetically heterogeneous disorder causing severe congenital deafness, retinitis pigmentosa, and vestibular dysfunction. The USHla locus located on 14q32 has been linked to the genetic markers D14S250 and D14S78. Using D14S250 and D14S78, we have isolated two nonchimeric YACs, 878g10 and 844g2, and a single BAC (135i20) and PAC (194e17) clone and have arranged them into a contig spanning over the D14S250 and D14S78 markers. The analysis of the YACs, BAC, and PAC revealed that the physical distance between D14S250 and D14S78 is less than 25 kb. Iterative cDNA library screening initiated with the EST 219670 found in the vicinity of the D14S78 marker yielded a cDNA contig. The nucleotide sequence of the cDNA encodes a protein of 717 amino acids in length, showing a high level of homology to the Echinoderm 77-kDa microtubule-associated protein (EMAP). The human homologue of Echinoderm microtubule-associated protein defines a novel human gene. We propose that the human EMAP is a strong candidate for the USH1a gene based on its genomic location and the proposed function of the protein.
Subject(s)
Chromosomes, Human, Pair 14/genetics , Deafness/genetics , Echinodermata/genetics , Microtubule-Associated Proteins/genetics , Animals , Base Sequence , Chromosome Mapping , DNA, Complementary/genetics , Deafness/congenital , Genetic Linkage , Genetic Markers , Humans , Molecular Sequence Data , Retinitis Pigmentosa/genetics , Sequence Tagged Sites , Species Specificity , Syndrome , Vestibular Diseases/geneticsABSTRACT
The gene for Usher syndrome type II (USH2A), an autosomal recessive syndromic deafness, has been mapped to a region of 1q41 flanked proximally by D1S217 and distally by D1S439. Using sequence-tagged sites (STSs) within the region, a total of 21 yeast artificial chromosome (YAC) clones were isolated and ordered into a single contig that spans approximately 11.0 Mb. The order of microsatellite and STS markers in this region was established as D1S505-D1S425-DXS217-D1S556-D1S237-D1S4 74-EB1-EB2-KB6-AFM144XF2-KB1-K B4-D1S229-D1S490-D1S227-TGFbeta2-D1S439. Analysis of newly positioned polymorphic markers in recombinant individuals in two Usher syndrome type IIa families has enabled us to identify DXS474 and AFM144XF2 as two flanking markers for the Usher type IIa locus. The physical distance between the two markers is 1.0 Mb. This region is covered by eight YACs from the CEPH library: 945f7, 867g9, 762a6, 919h3, 794b8, 785h4, 848b9, and 841g2. A long-range physical map of the Usher type IIa critical region, using MluI, BssHII, NotI, EagI, and SacII, has been developed.
