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Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

Published on Sep 1, 2018in Nature Biotechnology31.864
· DOI :10.1038/nbt.4192
Michael Kosicki3
Estimated H-index: 3
,
Kärt Tomberg6
Estimated H-index: 6
,
Allan Bradley116
Estimated H-index: 116
Sources
Abstract
Cas9-induced double stranded breaks can cause large deletions near the target site and more complex genomic rearrangements in mouse and human stem cells.
  • References (38)
  • Citations (245)
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References38
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#1Katharina Boroviak (Wellcome Trust Sanger Institute)H-Index: 5
#2Beiyuan Fu (Wellcome Trust Sanger Institute)H-Index: 29
Last. Allan Bradley (Wellcome Trust Sanger Institute)H-Index: 116
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Modelling human diseases caused by large genomic rearrangements has become more accessible since the utilization of CRISPR/Cas9 in mammalian systems. In a previous study, we showed that genomic rearrangements of up to one million base pairs can be generated by direct injection of CRISPR/Cas9 reagents into mouse zygotes. Although these rearrangements are ascertained by junction PCR, we describe here a variety of anticipated structural changes often involving reintegration of the region demarcated...
12 CitationsSource
#1Molly Gasperini (UW: University of Washington)H-Index: 7
#2Gregory M. Findlay (UW: University of Washington)H-Index: 8
Last. Jay Shendure (UW: University of Washington)H-Index: 101
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The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1 , the housekeeping gene underlying Lesch-Nyhan syndrome, a...
46 CitationsSource
#1Ha Youn Shin (Konkuk University)H-Index: 6
#2Chaochen Wang (NIH: National Institutes of Health)H-Index: 20
Last. Lothar Hennighausen (NIH: National Institutes of Health)H-Index: 89
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CRISPR/Cas9 gene editing has been used to generate mutations in several mouse genes. Here, the authors show that targeting events using single guide RNAs cause large deletions at 17 sites in the mouse genome, suggesting that careful genotyping is needed and sequential targeting may avoid such deletions.
61 CitationsSource
#1Supriya Sinha (University of Texas Health Science Center at San Antonio)H-Index: 2
#2Fuyang Li (University of Texas Health Science Center at San Antonio)H-Index: 17
Last. Sang Eun Lee (University of Texas Health Science Center at San Antonio)H-Index: 32
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Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more...
17 CitationsSource
#1Tatjana I. Cornu (University of Freiburg)H-Index: 2
#2Claudio Mussolino (University of Freiburg)H-Index: 15
Last. Toni Cathomen (University of Freiburg)H-Index: 38
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In this Review, Cathomen and colleagues present the latest advances, including improvements in nuclease specificity and delivery, that will expedite the clinical translation of genome editing.
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#1Megan van OverbeekH-Index: 6
#2Daniel CapursoH-Index: 2
Last. Andrew P. MayH-Index: 20
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Summary The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent acr...
135 CitationsSource
#1Alexis C. KomorH-Index: 13
#2Yongjoo KimH-Index: 7
Last. David R. LiuH-Index: 73
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CRISPR/Cas9 DNA editing creates a double-stranded break in the target DNA, which can frequently generate random insertion or deletion of bases (indels); a new genome editing approach combining Cas9 with a cytidine deaminase is described here, which corrects point mutations more efficiently than canonical Cas9, while avoiding double-stranded breaks and indel formation.
866 CitationsSource
#1Katharina Boroviak (Wellcome Trust)H-Index: 5
#2Brendan Doe (Wellcome Trust)H-Index: 9
Last. Allan Bradley (Wellcome Trust)H-Index: 116
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SUMMARY Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9...
35 CitationsSource
#1Benjamin P. Kleinstiver (Harvard University)H-Index: 15
#2Vikram Pattanayak (Harvard University)H-Index: 11
Last. J. Keith Joung (Harvard University)H-Index: 62
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A high-fidelity variant of Streptococcus pyogenes CRISPR–Cas9 is reported that lacks detectable off-target events as assessed by genome-wide break capture and targeted sequencing methods.
947 CitationsSource
#1Ian SlaymakerH-Index: 10
#2Linyi Gao (MIT: Massachusetts Institute of Technology)H-Index: 6
Last. Feng ZhangH-Index: 115
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The RNA-guided endonuclease Cas9 is a versatile genome editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes ...
943 CitationsSource
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Precision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method "Repl...
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Genome editing by Clustered Regularly Inter Spaced Palindromic Repeat (CRISPR) associated (Cas) systems has revolutionized medical research and holds enormous promise for correcting genetic diseases. Understanding how these Cas nucleases work and induce mutations, as well as identifying factors that affect their efficiency and fidelity is key to developing this technology for therapeutic uses. Here, we discuss recent studies that reveal how DNA sequence and chromatin structure influences the dif...
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Abstract Herein, several novel composite films consisting of three-dimensional (3D) Bi5O7I flower-like shaped microsphere and zwitterionic fluorinated polymer (ZFP) were successfully fabricated with the aim of achieving high anti-fouling performance. The prepared Bi5O7I flower-like shaped microsphere particles with diameters in the range of 2∼3 μm were uniformly distributed on the surface and in the internal of ZFP. Benefiting from the hydration layer formed by the ZFP and the efficient photocat...
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ABSTRACT As an alternative and complementary approach to Cas9-based genome editing, Cas12a has not been widely used in mammalian cells largely due to its strict requirement for the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) can efficiently edit the mouse genome based on the TTV PAM sequence with minimal numbers of large on-target deletions or insertions. When TTTV PAM sequence-targeting CRISPR (cr)RNAs of 23 nt spacers are used...
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The efficient use of the yeast Yarrowia lipolytica as a cell factory is hampered by the lack of powerful genetic engineering tools dedicated for the assembly of large DNA fragments and the robust expression of multiple genes. Here we describe the design and construction of artificial chromosomes (ylAC) that allow easy and efficient assembly of genes and chromosomal elements. We show that metabolic pathways can be rapidly constructed by various assembly of multiple genes in vivo into a complete, ...
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