Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans

Abstract

Facilitated by recent advances using CRISPR/Cas9, genome editing technologies now permit custom genetic modifications in a wide variety of organisms. Ideally, modified animals could be both efficiently made and easily identified with minimal initial screening and without introducing exogenous sequence at the locus of interest or marker mutations elsewhere. To this end, we describe a coconversion strategy, using CRISPR/Cas9 in which screening for a dominant phenotypic oligonucleotide-templated conversion event at one locus can be used to enrich for custom modifications at another unlinked locus. After the desired mutation is identified among the F 1 progeny heterozygous for the dominant marker mutation, F 2 animals that have lost the marker mutation are picked to obtain the desired mutation in an unmarked genetic background. We have developed such a coconversion strategy for Caenorhabditis elegans, using a number of dominant phenotypic markers. Examining the coconversion at a second (unselected) locus of interest in the marked F 1 animals, we observed that 14-84% of screened animals showed homologous recombination. By reconstituting the unmarked background through segregation of the dominant marker mutation at each step, we show that custom modification events can be carried out recursively, enabling multiple mutant animals to be made. While our initial choice of a coconversion marker [rol-6(su1006)] was readily applicable in a single round of coconversion, the genetic properties of this locus were not optimal in that CRISPR-mediated deletion mutations at the unselected rol-6 locus can render a fraction of coconverted strains recalcitrant to further rounds of similar mutagenesis. An optimal marker in this sense would provide phenotypic distinctions between the desired mutant/+ class and alternative +/+, mutant/null, null/null, and null/+ genotypes. Reviewing dominant alleles from classical C. elegans genetics, we identified one mutation in dpy-10 and one mutation in sqt-1 that meet these criteria and demonstrate that these too can be used as effective conversion markers. Coconversion was observed using a variety of donor molecules at the second (unselected) locus, including oligonucleotides, PCR products, and plasmids. We note that the coconversion approach described here could be applied in any of the variety of systems where suitable coconversion markers can be identified from previous intensive genetic analyses of gain-of-function alleles.T YPE II CRISPR/Cas9 bacterial immunity systems provide programmable DNA endonuclease activities that have recently revolutionized genome editing in a wide range of organisms (Wang et al. 1999; Chiu et al. 2013;Cho et al. 2013;Dicarlo et al. 2013;Friedland et al. 2013;Gratz et al. 2013;Jiang et al. 2013;Katic and Großhans 2013;Li et al. 2013;Lo et al. 2013;Nekrasov et al. 2013;Kim et al. 2014;Zhao et al. 2014). Recognition by the Cas9 protein entails two sequence elements in the target: a protospacer adjacent motif (PAM) (NGG for Streptococcus pyogenes Cas9) and a re...

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

“…The median frequency of editing at all targets was 51% without any coselectable markers, a 10-fold increase above previous studies that also reported numerous guide RNAs in the nonfunctional class Waaijers et al 2013;Kim et al 2014). Combining our already effective guide RNA design with the co-conversion/ co-CRISPR strategy of others (Arribere et al 2014;Kim et al 2014;Ward 2014) enhanced the ease of mutant recovery and boosted our median for both precise and imprecise genome editing to 86%. Our strategy for guide RNA design should be widely applicable to diverse organisms and cell lines.…”

“…For the guide RNA vector pRB1017, which utilizes the R07E5.16 snRNA promoter, complementary oligos encoding the desired guide RNA sequences were annealed and ligated into BsaI-digested pRB1017, as described in Arribere et al (2014).…”

“…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). Nevertheless, the overall frequency of editing at most targets remained relatively low.…”

Decreased function of survival motor neuron protein impairs endocytic pathways

“…1B) (31,32). To confirm that the defects described here are caused by smn-1 loss, we generated a new smn-1 allele, smn-1(rt248), using CRISPR/Cas9-targeted mutagenesis (34,35). The rt248 allele results in a premature truncation at the 19th amino acid (D19fs) of the SMN-1 protein; disrupts RNA binding, Tudor, and oligomerization domains; and likely eliminates SMN-1 protein function (Fig.…”

“…The small guide RNA (sgRNA) plasmid targeting the smn-1 gene (pHA#730) for CRISPR/Cas9-mediated genome editing was generated by amplification of PU6::klp-12 (35) and subsequent ligation of the PCR product obtained by the following primers: 5′-AACATCGTCTAAACATTTAGATTTGCAATTCAATTATATAGGGACC-3′ and 5′-TGGGATGATAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC-3′. The resulting plasmid was injected at 50 ng/μL into wild-type animals following the dpy-10 (cn64) coconversion protocol from Arribere et al (34). After backcross, the resulting smn-1(rt248) allele was balanced over the hT2 chromosomal translocation.…”