Figure 1: A-B. Schematic diagrams of the jump board strategy for engineering the
let-7 locus. To generate the jump board strain (A), hermaphrodites of the strain EG9615(oxSi1091[
mex-5p::Cas9(
smu-2 introns)
unc-119(+)] II;
unc-119(
ed3) III) carrying a transgene expressing the Cas9 protein were injected with a crRNA targeting
let-7 (purple), tracrRNA and dsDNA HDR donor to replace the 64 bp sequence corresponding to the precursor-
let-7 transcript with the 23 bp jump board sequence (blue). To permit recovery of F1 progeny carrying a lethal jump board
let-7 null allele, injected P0 hermaphrodites were crossed to males carrying the mnDp1 [umnIs25] genetic balancer, which contains a wild type
let-7 locus, as well as an integrated
myo-2p::GFP transgene. The resulting jump board strain, VT3742(oxSi1091 II; mnDp1[umnIs25] (X;V)/+ V;
let-7(
ma393) X) was used as a platform for repeated mutagenesis at the
let-7 locus. To use the established jump board strain for mutagenesis (B), the
let-7 jump board allele (
ma393) in VT3742 was targeted by the universal jump board guide INPP4A-crRNA and replaced by HDR with multiplexed dsDNA donors containing the
let-7 locus with engineered mutations (red). The wild type allele of
let-7 contained on mnDp1 is resistant to editing, since INPP4A-crRNA is directed to the jump board sequence, permitting the recovery of lethal
let-7 mutations complemented by mnDp1. C. Screening strategy for (B) using EcoRV restriction analysis. After injection of VT3742, F1 animals with mnDp1 balancer (detected by pharyngeal GFP) were collected after egg laying and subjected to PCR with flanking primers, and the PCR products were digested by EcoRV. For F1 broods with no EcoRV cleavage (homozygous HDR or homozygous InDel), the PCR products were subjected to Sanger sequencing to identify homozygous HDR lines. Note that heterozygous HDR can also be identified by genotyping the F2 without mnDp1 followed by Sanger sequencing. However, we have consistently observed that most F1 animals carrying HDR products were homozygous for the HDR alleles. D. Summary of three mutageneses of
let-7 locus using the jump board platform. Note that more F1 animals with co-CRISPR phenotypes were isolated than were genotyped. E. Aligned sequences of wild type
let-7,
let-7(
ma393), and examples of
let-7 alleles generated by replacement of jump board. Color schemes are consistent with (A-B). F. Aligned sequences of the zinc finger region of
lin-28 representing the application of jump board strategy for protein coding gene. The starting strain VT3711(
lin-28(
ma416) I; maIs105 V) contains a
lin-28 locus (
ma416) expressing functional LIN-28 protein tagged with 3xFLAG::6xHIS. The endogenous zinc finger domain (C144-C181, 114 bp) of
lin-28 was targeted by two crRNAs in Cas9 RNP and replaced with the jump board sequence using a ssDNA HDR donor. The resulting
lin-28 jump board allele
lin-28(
ma416ma487) had severe heterochronic phenotypes (including sterility) and was then crossed with
lin-46(
ma164), a genetic suppressor of
lin-28(lf), to produce the jump board targeting strain, VT3914(
lin-28(
ma416ma487) I;
lin-46(
ma164) V) (Pepper et al., 2004). The
lin-28 jump board allele (
ma487) was subsequently targeted by INPP4A-crRNA and a dsDNA HR donor with 35/35 bp homology to replace
ma487 with a
lin-28 locus carrying a compound mutation (
ma514) in which 8 critical amino acid residues of the two zinc fingers were replaced by alanine residues. The numbers indicate the positions at chr. I according to Wormbase WS277.