Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

Lo, Te‐Wen; Pickle, Catherine S.; Lin, Steven; Ralston, Edward J.; Gurling, Mark; Schartner, Caitlin; Bian, Qian; Doudna, Jennifer A.; Meyer, Barbara J

doi:10.1534/genetics.113.155382

Cited by 178 publications

(160 citation statements)

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“…The initial report describing the successful use of Cas9 in C. elegans demonstrated a range of editing frequencies from 0.5 to 80%, with only two targets exceeding 4% . Subsequent publications also reported variably low mutagenesis rates that required molecular screening of hundreds of animals or required the desired mutation to cause an easily detectable mutant phenotype or to be introduced in tandem with a coselection marker (Chiu et al 2013;Cho et al 2013;Dickinson et al 2013;Katic and Grosshans 2013;Lo et al 2013;Tzur et al 2013;Waaijers et al 2013;Chen et al 2014;Liu et al 2014;Paix et al 2014;Shen et al 2014;Zhao et al 2014). More recent studies greatly improved the odds of detecting a targeted mutation through the simultaneous co-conversion of a mutation in an unrelated target that causes a visible phenotype (Arribere et al 2014;Kim et al 2014;Ward 2014).…”

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Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Farboud¹,

2015

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Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 39 end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 39 GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome. KEYWORDS CRISPR; Cas9; genome editing; co-conversion; C. elegans T HE use of site-specific nucleases with programmable target specificity has transformed the art of genome editing and thereby revolutionized the dissection and manipulation of genome function (reviewed in Mali et al. 2013;Carroll 2014;Doudna and Charpentier 2014;Hsu et al. 2014). Most widely used is the CRISPR-associated nuclease Cas9, whose RNA-programmed DNA cleaving activities create DNA double-strand breaks (DSBs). These DSBs can be repaired imprecisely by nonhomologous end joining (NHEJ) to generate random insertions and deletions or repaired precisely by homology-directed repair (HDR) templated from exogenous DNA to generate custom-designed insertions, deletions, or substitutions (Gasiunas et al. 2012;Jinek et al. 2012;Cong et al. 2013;Jinek et al. 2013;Mali et al. 2013). Modified variants of Cas9 that lack DNA cleaving activity have also been utilized to regulate transcription of designated gene targets and to cytologically mark and track genomic loci in living cells (Bikard et al. 2013;Chen et al. 2013;Larson et al. 2013;Maeder et al. 2013;Perez-Pinera et al. 2013;Qi et al. 2013;Gilbert et al. 2014;Tanenbaum et al. 2014).The Cas9 protein is targeted to a specific genomic locus by a guide RNA that encodes a 20-nt region of homology to the DNA target ( Figure 1A) (Mojica et al. 2009;Garneau et al. 2010;Jinek et al. 2012). The most commonly used guide RNAs are chimeric fusions between the CRISPR RNA (crRNA), which encodes the 20-nt target-specific sequence, and the tracer RNA (trRNA), which enables the formation of active Cas9-guide RNA complexes ( Figure 1A) (Jinek et al. 2012). Few constraints are known for Cas9 ...

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Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Farboud¹,

2015

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“…Compared with the zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), which have been used for genome editing [1], the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR-associated (Cas) system has emerged as a new powerful tool for genome modifications. It has recently been adopted for genome editing in human cell lines [2][3][4], mouse [5], zebrafish [6], C. elegans [7][8][9][10][11][12], and plants [13].…”

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“…The DSDBs can be repaired either through non-homologous end joining (NHEJ), which leads to generation of random deletions, insertions, or both (InDels) [2-5, 7-9, 11, 13], or through homologous recombination (HR), which could generate specific and precise nucleotide or sequence replacements [3,5,9,10,12] when a plasmid or a single-stranded oligonucleotide (oligo) template is also present. The use of oligonucleotides as donor templates, which can be rapidly synthesized through commercial sources, to achieve Cas9-mediated knock-ins has not yet been reported in C. elegans.…”

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“…Sequencing analysis of homozygous progeny from this heterozygous F1 animal confirmed correct editing at the mec-4(u231) locus ( Figure 1G and Supplementary information, Figure S6B-S6D). Therefore, this oligo-based gene editing method can be broadly used to generate precise nucleotide changes at sites where an SNP can be identified by restriction digestion, and potentially, can be used at any sgRNA-targetable site in the genome, when combined with a mismatch-specific endonuclease such as the CEL-1 endonuclease [9].…”

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Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system

Zhao

Zhang

et al. 2014

Cell Res

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“…The development of multiplex genome engineering provides the ability to simultaneously introduce DNA mutations into several genomic loci (Wang et al, 2009(Wang et al, , 2012Esvelt & Wang, 2013). Further, Cas9-dependent and TALE-dependent nicking, cleavage, and mutagenesis have expanded site-directed genome engineering to diverse organisms (Miller et al, 2010;Bassett et al, 2013;Chang et al, 2013;Lo et al, 2013;Mali et al, 2013;Ran et al, 2013). Although techniques are readily available to construct or modify large genetic systems of interest, we currently cannot predict the DNA sequences that will achieve an optimal behavior, particularly when the actions of multiple proteins are responsible for a system's function.…”

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Efficient Search, Mapping, and Optimization of Multi-protein Genetic Systems in Diverse Bacteria

Farasat

Kushwaha

Collens

et al. 2013

Preprint

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Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs.

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Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

Cited by 178 publications

References 64 publications

Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system

Efficient Search, Mapping, and Optimization of Multi-protein Genetic Systems in Diverse Bacteria

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