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 F1 progeny heterozygous for the dominant marker mutation, F2 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 F1 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.

Keywords: CRISPR/Cas9; coconversion; dpy-10; oligonucleotide-mediated homologous recombination; rde-1.

Figure 1

Strategy to track effective gene conversion using dominant point mutations. (A) A Cas9 expression plasmid [pDD162 (Dickinson et al. 2013)] is co-injected with a target-specific gRNA plasmid and a template oligonucleotide bearing the desired mutations. The F₁ of the injected animal is screened for phenotypically affected animals. (B) Schematic of the coding strand of the rol-6(su1006) locus. The su1006 mutation (Arg→Cys) is indicated in red, with wild-type Arg boxed (Kramer and Johnson 1993). The gRNA sequence is indicated with an arrow, with PAM underlined. Additional silent mutations conferred by the donor single-stranded DNA (ssDNA) creating a BbvI restriction site (italics) are highlighted in blue. The expected Cas9 cleavage site is indicated with arrow and scissors. Twenty-eight animals were injected. (C) Schematic for unc-58(e665) DdeI site in italics and causative lesion (e665, Leu→Phe) noted (Phil S. Hartman, James Barry, Whitney Finstad, Numan Khan, S. Sato, Naoaki Ishii, and Kayo Yasuda, unpublished results). Twenty-one animals were injected. (D) Schematic for unc-109(n499). PciI site is in italics (n499 sequence from Chen and Jorgensen 2013). Twenty-one animals were injected. (E) Schematic for dpy-10(cn64). SphI site is in italics. See text for description of Dpy, Dpy Rol, and Rol animals. Twenty-one animals were injected. (F) Schematic for sqt-1(e1350), with BbvI site in italics, using injection of two gRNAs. Thirty animals were injected. Injections were performed with day-to-day variability in injection efficiency, and the number of phenotypically affected progeny should not be interpreted as a statement on the efficiency of HR at that locus. All plasmids are 50 ng/μl, and ssDNA is ∼20 ng/μl.

Figure 2

Coconversion strategy for induction of designated point mutations. (A) The coconversion strategy. A gRNA and donor oligonucleotide to create the rol-6(su1006) mutation are co-injected with a gRNA and donor oligonucleotide to create a point mutation in the desired gene (in this case rde-1). F₁ progeny were screened for the Rol phenotype. Rol animals were singled and, after laying eggs, were screened by single-worm PCR and characterization of the designated mutational target (rde-1 for this experiment). Nonroller F₂ progeny of appropriate F₁ animals were singled and after laying eggs were screened for homozygosity of the rde-1 mutation. (B) Schematic of the rde-1(H974A) locus. Ala mutations are shown in red and the BbvI site in italics. All plasmids were 50 ng/μl. Two additional animals at this stage rolled but failed single-worm PCR; these were not included in the 23 count. (C) Schematic for rde-1(D801A) locus. NaeI site is in italics. Two of the seven D801A events contained only the D801A mutation and lacked a complete NaeI site (blue C). gRNA plasmids were 25 ng/μl. An additional animal rolled but failed single-worm PCR and was not included in the 14 count.

Figure 3

Oligonucleotide-mediated homologous recombination is local. Schematic of the rde-1(D718A) locus is shown. Silent mutations are in blue, the SnaBI site is in italics, and the D718A mutation is in red. For all injections, both gRNA plasmids were 25 ng/μl. For both rol-6 injections, the rde-1(D718A) donor DNA was 20 ng/μl. For unc-58 and dpy-10 the rde-1(D718A) donor DNA was ∼17.25 ng/μl. *From the sequencing trace it was unclear whether an additional animal was full or partial HR. **From the sequencing trace it was unclear whether two additional animals were full or partial HR.

Figure 4

Coconversion from nonoligonucleotide substrates as the second-site donor DNA. (A) Coconversion strategy using plasmid DNA as the donor for the rde-1 mutations. The rde-1(AAA) plasmid was included in the injection mix at ∼593 ng/μl. F₁ Rol animals that tested positive for rde-1(AAA) DNA were subsequently progeny tested and homozygotes isolated, to discern transgenesis from true integration events. (B) Coconversion strategy using a PCR product as the donor for the rde-1 mutations. The rde-1(AAA) PCR product was included in the injection mix at ∼287 ng/μl. The PCR product also included three silent mutations upstream of D718A (not shown). F₁ Unc animals were tested for integration, using one primer inside the PCR product and another outside of it. F₁ Unc animals testing positive for rde-1(AAA) DNA were progeny tested as in A. Each of the eight rde-1 conversion events occurred in an independent animal.

Figure 5

Oligonucleotide-templated HR as a marker for integration of GFP at a second locus. (A) L7969 is a derivative of VT333G (Hong et al. 2000) and encodes a C-terminal lin-14::GFP fusion (exons 4 through the C terminus of lin-14) and was included in the injection mix at 20 ng/μl. Guide RNA plasmids were 25 ng/μl, pDD162 was 50 ng/μl, and dpy-10 donor DNA was 500 nM. Transgenic-marking reporter fusions were included in the injection mix: Pmyo-2::mCherry::unc-54 (pCFJ90, 2.5 ng/μl), Pmyo-3::mCherry::unc-54 (pCFJ104, 5 ng/μl), and Prab-3::mCherry::unc-54 (pGH8, 10 ng/μl). Forty-four animals were injected and 23 yielded Rol progeny. A large number of Rol and non-Rol progeny were observed, only a fraction of which were scored here. The HR category includes F₁ animals screened by a combination of single-worm PCR and examination of their young progeny for characteristic lin-14::GFP expression patterns. The transgene array category includes animals yielding heritable GFP and mCherry coexpression. Of the 16 lin-14::GFP integration events among F₁ Rol, 3 were from F₁ mCherry-positive parents. (B) LIN-14::GFP expression pattern in a newly hatched L₁ larva, similar to that reported in Hong et al. (2000). (C) Close-up of LIN-14::GFP expression pattern showing punctate nuclear GFP signal, consistent with that in Hong et al. (2000). Also note lack of expression in L4 larvae (bottom half). The faint yellowish signal in the L4 larvae is autofluorescence from the gut.