Coupling endonucleases with DNA end–processing enzymes to drive gene disruption

Certo, Michael; Gwiazda, Kamila S.; Kuhar, Ryan; Sather, Blythe; Curinga, Gabrielle; Mandt, Tyler; Brault, Michelle; Lambert, Abigail R.; Baxter, Sarah K.; Jacoby, Kyle; Ryu, Byoung Y.; Kiem, Hans Peter; Gouble, Agnès; Pâques, Frédéric; Rawlings, David J.; Scharenberg, Andrew M.

doi:10.1038/nmeth.2177

Cited by 86 publications

(75 citation statements)

References 15 publications

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“…The ability of MNs to induce TM was assessed by cotransforming Pt with a MN-encoding plasmid, an autonomous selection cassette plasmid (NAT gene to select for nourseothricin (NAT) resistance), and a plasmid encoding the DNA processing enzyme scTrex2, which has previously been shown to increase about 10-fold the TM frequency induced by MNs in mammalian cells 27,28 . Twelve NAT-resistant colonies transformed with Mn17181 were analysed for the presence of mutations using locus-specific PCR followed by deep sequencing.…”

Section: Resultsmentioning

confidence: 99%

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

et al. 2014

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Section: Resultsmentioning

confidence: 99%

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

et al. 2014

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“…For example, indel frequencies have been increased by coupling SSNs with exonucleases (Certo et al, 2012;Kwon et al, 2012), altering guide RNA (gRNA) architecture or using specialized promoters Yan et al, 2015). Increased specificity has been achieved using paired TALEN or CRISPR/Cas9 nickases , truncated gRNAs (Fu et al, 2014), or dimeric CRISPR/Cas9 nucleases (Guilinger et al, 2014;Tsai et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

et al. 2017

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We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

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“…If so, increasing endonuclease expression levels, persistence, or specific activity may result in higher mutagenesis, as does the inclusion of DNA end-processing enzymes such as Trex2. 16,17 However, such efforts may yield only incremental improvements, and targeting a single site for non-homologous end joininginduced mutation may be incapable of achieving the required >99% disruption needed for HIV cure.…”

Section: Maximizing the Efficacy Of Targeted Endonuclease Activitymentioning

confidence: 99%

Disruption or Excision of Provirus as an Approach to HIV Cure

Jerome

2016

AIDS Patient Care and STDs

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An effective approach to HIV cure will almost certainly require a combination of strategies, including some means of reducing the latent HIV reservoir. Because the integrated HIV provirus represents the major source of viral persistence and reactivation, one attractive approach is the direct targeting of provirus for disruption or excision using targeted endonucleases, such as CRISPR/Cas9, zinc finger nucleases, TAL effector nucleases, or meganucleases (homing endonucleases). This article highlights some of the challenges for successful endonuclease therapy for HIV, including optimization of enzyme activity and specificity, the possible emergence of viral resistance, and most importantly, efficient in vivo delivery of the enzymes to a sufficient portion of the latent reservoir.

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Coupling endonucleases with DNA end–processing enzymes to drive gene disruption

Cited by 86 publications

References 15 publications

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

Disruption or Excision of Provirus as an Approach to HIV Cure

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