scholarly journals Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

2013 ◽  
Vol 41 (20) ◽  
pp. e193-e193 ◽  
Author(s):  
Changchun Chen ◽  
Lorenz A. Fenk ◽  
Mario de Bono
Keyword(s):  
Caenorhabditis Elegans ◽  
Homologous Recombination ◽  
Genome Editing

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Paternal Genome ◽  
Single Nucleotide ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Maternal Genome ◽  
Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

scholarly journals Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans

Genetics ◽  
2016 ◽  
Vol 203 (1) ◽  
pp. 133-145 ◽  
Author(s):  
T. Brooke McClendon ◽  
Meghan R. Sullivan ◽  
Kara A. Bernstein ◽  
Judith L. Yanowitz
Keyword(s):  
Caenorhabditis Elegans ◽  
Homologous Recombination

scholarly journals Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Patricia L. Baker ◽  
Gregory S. Orf ◽  
Kimberly Kevershan ◽  
Michael E. Pyne ◽  
Taner Bicer ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Reaction Center ◽  
Photochemical Reaction ◽  
Genetic Locus ◽  
Gene Replacement ◽  
Type I ◽  
Content Type ◽  
Heliobacterium Modesticaldum

ABSTRACT In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome. IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum. Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

scholarly journals Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans

Genetics ◽  
2018 ◽  
Vol 210 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Single Stranded Dna

scholarly journals Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans

Genetics ◽  
2014 ◽  
Vol 198 (4) ◽  
pp. 1347-1356 ◽  
Author(s):  
Alexandre Paix ◽  
Yuemeng Wang ◽  
Harold E. Smith ◽  
Chih-Yung S. Lee ◽  
Deepika Calidas ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing

Melting dsDNA donor molecules potentiates precision genome editing in C. elegans

2020 ◽  
Author(s):  
Krishna S. Ghanta ◽  
Craig C. Mello
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Germ Cells ◽  
Multiple Injections ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Selection Strategies ◽  
Double Stranded Dna ◽  
Adult Hermaphrodite

ABSTRACTCRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.

scholarly journals Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system

2017 ◽  
Vol 28 (7) ◽  
pp. 898-906 ◽  
Author(s):  
Yohei Katoh ◽  
Saki Michisaka ◽  
Shohei Nozaki ◽  
Teruki Funabashi ◽  
Tomoaki Hirano ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Protein Trafficking ◽  
Target Genes ◽  
Practical Method ◽  
Cell Types ◽  
Recombination Activity ◽  
Ciliary Protein ◽  
Genes Encoding ◽  
Universal Donor

The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.

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