scholarly journals Knock-In of a 25-Kilobase Pair BAC-Derived Donor Molecule by Traditional and CRISPR/Cas9-Stimulated Homologous Recombination

2016 ◽  
Author(s):  
Tiffany Leidy-Davis ◽  
Kai Cheng ◽  
Leslie O. Goodwin ◽  
Judith L. Morgan ◽  
Wen Chun Juan ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Traditional Approach ◽  
Artificial Chromosome ◽  
Human Exon ◽  
Short Palindromic Repeat ◽  
Orthologous Locus ◽  
Mouse Zygotes ◽  
Living Organisms ◽  
Intron 2 ◽  
Dna Size

ABSTRACTHere, we describe an expansion of the DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus. Driving our efforts was the desire to create a whole animal model that would replace 17 kbp of the mouseBcl2l11gene with the corresponding 25-kbp segment of humanBCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3′ of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic ES cell targeting, dual selection, and recombinase-driven cassette removal (traditional approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with CRISPR RNA guides andCas9into mouse zygotes (CRISPR approach). In both instances we were able to achieve humanization ofBcl2l11to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable,loxP-flanked, 2.9-kbp intronic segment.AUTHOR SUMMARYClustered regularly interspaced short palindromic repeat (CRISPR) technology can be used to place DNA sequences (designed in the laboratory) into the genomes of living organisms. Here, we describe a new method, whereby we have replaced an exceptionally large segment of the mouseBcl2l11gene with the corresponding segment of humanBCL2L11gene. The method represents an expansion of the DNA size limitations typically associated with the introduction of DNA sequences through traditional CRISPR methods.

scholarly journals Modified HuffBit Compress Algorithm – An Application of R

2018 ◽  
Vol 15 (3) ◽  
Author(s):  
Nahida Habib ◽  
Kawsar Ahmed ◽  
Iffat Jabin ◽  
Mohammad Motiur Rahman
Keyword(s):  
Dna Sequences ◽  
Binary Tree ◽  
Deoxyribonucleic Acid ◽  
Repetitive Sequences ◽  
Huffman Codes ◽  
R Language ◽  
Living Organisms ◽  
Encoding Method ◽  
Dna Sequence Compression ◽  
Sequence Compression

Abstract The databases of genomic sequences are growing at an explicative rate because of the increasing growth of living organisms. Compressing deoxyribonucleic acid (DNA) sequences is a momentous task as the databases are getting closest to its threshold. Various compression algorithms are developed for DNA sequence compression. An efficient DNA compression algorithm that works on both repetitive and non-repetitive sequences known as “HuffBit Compress” is based on the concept of Extended Binary Tree. In this paper, here is proposed and developed a modified version of “HuffBit Compress” algorithm to compress and decompress DNA sequences using the R language which will always give the Best Case of the compression ratio but it uses extra 6 bits to compress than best case of “HuffBit Compress” algorithm and can be named as the “Modified HuffBit Compress Algorithm”. The algorithm makes an extended binary tree based on the Huffman Codes and the maximum occurring bases (A, C, G, T). Experimenting with 6 sequences the proposed algorithm gives approximately 16.18 % improvement in compression ration over the “HuffBit Compress” algorithm and 11.12 % improvement in compression ration over the “2-Bits Encoding Method”.

scholarly journals CRISPR base editors: genome editing without double-stranded breaks

2018 ◽  
Vol 475 (11) ◽  
pp. 1955-1964 ◽  
Author(s):  
Ayman Eid ◽  
Sahar Alshareef ◽  
Magdy M. Mahfouz
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genetic Diseases ◽  
Great Promise ◽  
Cytidine Deaminases ◽  
Strand Breaks ◽  
Base Editing ◽  
Potential Applications ◽  
Short Palindromic Repeat ◽  
Basic Biology

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2)

Genome ◽  
2001 ◽  
Vol 44 (2) ◽  
pp. 154-162 ◽  
Author(s):  
Meizhong Luo ◽  
Yi-Hong Wang ◽  
David Frisch ◽  
Tarek Joobeur ◽  
Rod A Wing ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Fusarium Wilt ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Microtiter Plates ◽  
Bac Libraries ◽  
Average Size ◽  
Improved Methods

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.Key words: bacterial artificial chromosome (BAC) library, Fusarium wilt, melon, pCUGIBAC1, resistant gene.

Transgenic modification of cows milk for value-added processing

1998 ◽  
Vol 10 (8) ◽  
pp. 671 ◽  
Author(s):  
Kurt A. Zuelke
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Dairy Cattle ◽  
Dna Sequences ◽  
Gene Locus ◽  
Bac Library ◽  
Value Added ◽  
Artificial Chromosome ◽  
Coordinated Expression ◽  
Casein Genes ◽  
Transgenic Technologies

The application of transgenic technologies in dairy cattle has been restricted largely to producing potential pharmaceutical or nutriceutical products in the mammary gland. Broader application of transgenesis in dairy cattle production will require identifying target traits that are both amenable to transgenic modification and economically important to the dairy industry. The casein proteins are the most valuable component of cows milk destined for value-added processing. The four bovine casein genes lie within a single, multi-gene locus of approximately 200 kb in length. The working hypothesis is that this multi-gene locus contains all of the DNA sequences required to regulate the coordinated expression of all four individual casein genes (i.e. a locus control region or LCR). The initial research aim is to clone the entire casein locus into a bacterial artificial chromosome (BAC) vector, thus preserving the extended 5′and 3′ regions that flank the locus, as well as maintaining the spatial integrity of the four individual casein genes that comprise the locus. The author's laboratory has prepared a bacterial artificial chromosome (BAC) library of genomic DNA from elite dairy cattle. Partial, non-elite BAC clones of the casein gene locus are being tested in transgenic mice to establish proof of concept. Advances in nuclear transfer of transfected somatic cells should improve the efficiency of producing transgenic calves that possess a BAC casein construct introduced into an elite genetic background.

Genomics and homeostasis

2003 ◽  
Vol 284 (3) ◽  
pp. R611-R627 ◽  
Author(s):  
Allen W. Cowley
Keyword(s):  
Dna Sequences ◽  
Positional Cloning ◽  
Regulatory Pathways ◽  
Complex Functions ◽  
Challenges And Opportunities ◽  
The Galaxy ◽  
Systems Physiology ◽  
Living Organisms ◽  
New Hypothesis ◽  
Statistical Results

The Cannon lecture this year illustrates how knowledge of DNA sequences of complex living organisms is beginning to shape the landscape of physiology in the 21st century. Enormous challenges and opportunities now exist for physiologists to relate the galaxy of genes to normal and pathological functions. The first extensive genomic systems biology map for cardiovascular and renal function was completed last year as well as a new hypothesis-generating tool (“physiological profiling”) that enables us to hypothesize relationships between specific genes responsible for the regulation of regulatory pathways. Techniques of chromosomal substitution (consomic and congenic rats) are beginning to confirm statistical results from linkage analysis studies, narrow the regions of genetic interest for positional cloning, and provide genetically well-defined control strains for physiological studies. Patterns of gene expression identified by microarray and mapping of expressed genes to chromosomal sites are adding to the understanding of systems physiology. The previously unimaginable goal of connecting ∼36,000 genes to the complex functions of mammalian systems is indeed well underway.

scholarly journals Molecular data indicate a cryptic species in Neotoma albigula (Cricetidae: Neotominae) from northwestern México

2015 ◽  
Vol 97 (1) ◽  
pp. 187-199
Author(s):  
Robert D. Bradley ◽  
Matthew R. Mauldin
Keyword(s):  
Dna Sequences ◽  
Phylogenetic Analyses ◽  
Nuclear Gene ◽  
Molecular Data ◽  
Genetic Distances ◽  
Dehydrogenase Gene ◽  
Mitochondrial Cytochrome B ◽  
Northwestern Mexico ◽  
Morphological Differences ◽  
Intron 2

Abstract DNA sequences from the mitochondrial cytochrome- b gene were obtained from 41 specimens of Neotoma albigula from the southwestern United States and northwestern México. Phylogenetic analyses depicted that samples of N. a. melanura from southern Sonora and northern Sinaloa formed a clade separate from representatives of the other sampled subspecies of N. albigula ( albigula , laplataensis , mearnsi , seri , sheldoni , and venusta ). Genetic distances detected between these clades (7.41%) approached divergence levels reported for other sister species of woodrats and indicated that N. a. melanura is presumably a cryptic and genetically differentiated species relative to N . albigula . Analyses of DNA sequences from a nuclear gene (intron 2 of the alcohol dehydrogenase gene, Adh 1-I2) indicated that samples of N. a. melanura formed a separate, monophyletic clade relative to the remainder of N. albigula . Further, habitat, geographic distinctions, and morphological differences were apparent between members of the 2 clades. Together, those data support the elevation of N. a. melanura to species status. Secuencias de ADN del gen mitocondrial citocromo- b se obtuvieron de 41 especímenes de Neotoma albigula provenientes del suroeste de los Estados Unidos y del noroeste de México. Análisis filogenéticos revelaron que las muestras de N. a. melanura del sureste de Sonora y del norte de Sinaloa formaron un clado separado de especímenes que representan a las otras subspecies de N. albigula (albigula, laplataensis, mearnsi, seri, sheldoni , y venusta ). Las distancias genéticas detectadas entre estos clados (7.41%) se aproximan a los valores de divergencia reportados para otras especies de ratas magueyeras, indicando que N. a. melanura es presumiblemente una especie críptica y relativamente diferenciada genéticamente de N. albigula . Los análisis de secuencias de ADN de un gen nuclear (intron 2 del gen alcohol deshidrogenasa, Adh -I2) indicaron que las muestras de N. a. melanura formaron un clado separado relativamente a especímenes de N. albigula . Adicionalmente, hábitat, distinciones geográficas, y diferencias morfológicas fueron aparentes entre miembros de los dos clados. Todos estos datos soportan la elevación de N. a. melanura al estatus de especie.

scholarly journals Reversible regulation of Cas12a activities by AcrVA5-mediated acetylation and CobB-mediated deacetylation

2021 ◽  
Author(s):  
Xiaoman Kang ◽  
Lei Yin ◽  
Songkuan Zhuang ◽  
Tianshuai Hu ◽  
Zhile Wu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Wide Spectrum ◽  
Defense System ◽  
Post Translational Modification ◽  
Defense Systems ◽  
Target Dna ◽  
Short Palindromic Repeat ◽  
Type V

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) system protects bacteria and archaea from the invasion of foreign genetic elements. To cope with the host CRISPR systems, phages have evolved many strategies, including the anti-CRISPR (Acr) proteins, to inactivate the Cas nucleases. Recently, it has been reported that the type V-A Cas12a effector can be acetylated and inactivated by AcrVA5, which is a GNAT-family acetyltransferase. However, it is unclear whether the host has any coping strategies to reactivate the defense system. Here we show that the AcrVA5-acetylated Cas12a can be deacetylated by bacterial deacetylase CobB, reactivating Cas12a for both in vitro cleavage of target DNA sequences and in vivo protection of the host from invasion of foreign nucleic acids. Therefore, this study not only shows the reversible regulation of Cas12a activities by post-translational modification but also reveals CobB as a secondary safeguard to bacterial CRISPR defense systems. In addition, we demonstrate that AcrVA5 is a wide-spectrum acetyltransferase, acetylating a large number of target proteins besides Cas12a, and the AcrVA5-acetylated targets can also be deacetylated by CobB.

Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome

1990 ◽  
Vol 10 (8) ◽  
pp. 4163-4169
Author(s):  
W J Pavan ◽  
P Hieter ◽  
R H Reeves
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Embryonal Carcinoma ◽  
Yeast Artificial Chromosome ◽  
Carcinoma Cell ◽  
Artificial Chromosome ◽  
Carcinoma Cell Line ◽  
Yeast Genome ◽  
Embryonal Carcinoma Cell ◽  
Embryonal Carcinoma Cell Line

A neomycin resistance cassette was integrated into the human-derived insert of a 360-kilobase yeast artificial chromosome (YAC) by targeting homologous recombination to Alu repeat sequences. The modified YAC was transferred into an embryonal carcinoma cell line by using polyethylene glycol-mediated spheroplast fusion. A single copy of the human sequence was introduced intact and stably maintained in the absence of selection for over 40 generations. A substantial portion of the yeast genome was retained in hybrids in addition to the YAC. Hybrid cells containing the YAC retained the ability to differentiate when treated with retinoic acid. This approach provides a powerful tool for in vitro analysis because it can be used to modify any human DNA cloned as a YAC and to transfer large fragments of DNA intact into cultured mammalian cells, thereby facilitating functional studies of genes in the context of extensive flanking DNA sequences.

An integrated SSR and RFLP linkage map of Sorghum bicolor (L.) Moench

Genome ◽  
2000 ◽  
Vol 43 (6) ◽  
pp. 988-1002 ◽  
Author(s):  
Dinakar Bhattramakki ◽  
Jianmin Dong ◽  
Ashok K Chhabra ◽  
Gary E Hart
Keyword(s):  
Sorghum Bicolor ◽  
Linkage Map ◽  
Dna Sequences ◽  
Artificial Chromosome ◽  
Polymorphic Loci ◽  
Dna Libraries ◽  
Ssr Loci ◽  
Bac Libraries ◽  
Primer Sets ◽  
Sorghum Bicolor L

We report the development, testing, and use (for genetic mapping) of a large number of polymerase chain reaction (PCR) primer sets that amplify DNA simple sequence repeat (SSR) loci of Sorghum bicolor (L.) Moench. Most of the primer sets were developed from clones isolated from two sorghum bacterial artificial chromosome (BAC) libraries and three enriched sorghum genomic-DNA (gDNA) libraries. A few were developed from sorghum DNA sequences present in public databases. The libraries were probed with radiolabeled di- and trinucleotide oligomers, the BAC libraries with four and six oligomers, respectively, and the enriched gDNA libraries with four and three oligomers, respectively. Both types of libraries were markedly enriched for SSRs relative to a size-fractionated gDNA library studied earlier. However, only 2% of the sequenced clones obtained from the size-fractionated gDNA library lacked a SSR, whereas 13% and 17% of the sequenced clones obtained from the BAC and enriched gDNA libraries, respectively, lacked a SSR. Primer sets were produced for 313 SSR loci. Two-hundred sixty-six (85%) of the loci were amplified and 165 (53%) of the loci were found to be polymorphic in a population composed of 18 diverse sorghum lines. (AG/TC)n and (AC/TG)n repeats comprised 91% of the dinucleotide SSRs and 52% of all of the SSRs at the polymorphic loci, whereas four types of repeats comprised 66% of the trinucleotide SSRs at the loci. Primer sequences are reported for the 165 polymorphic loci and for eight monomorphic loci that have a high degree of homology to genes. Also reported are the genetic map locations of 113 novel SSR loci (including four SSR-containing gene loci) and a linkage map composed of 147 SSR loci and 323 RFLP (restriction fragment length polymorphism) loci. The number of SSR loci per linkage group ranges from 8 to 30. The SSR loci are distributed relatively evenly throughout approximately 75% of the 1406-cM linkage map, but segments of five linkage groups comprising about 25% of the map either lack or contain few SSR loci. Mapping of SSR loci isolated from BAC clones located to these segments is likely to be the most efficient method for placing SSR loci in the segments.Key words: DNA libraries, linkage mapping, Sorghum bicolor, SSRs.

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