R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling

The name of the encoded protein and the length (in bp) of the nucleotide sequence is indicated. RSPO3 (WT), RSPO3 TSP/BR (K/R→E) and RSPO3 ΔTSP/BR were cloned into pHLsec-HA-Tev-Fc-Avi-1D4. RSPO3 ΔTSP/BR HS20, RSPO3 ΔTSP/BR HS20 (GS), RSPO3 ΔTSP/BR HS20 (A), RSPO3 ΔTSP/BR HS20 (R67A/Q72A) and RSPO3 ΔTSP/BR HS20 (F106E/F110E) were cloned into pHLsec-HA-Avi-1D4. Bases in lowercase overlap the sequences upstream of the unique AgeI sites and downstream of the unique KpnI sites in the pHLsec-HA-Tev-Fc-Avi-1D4 and pHLsec-HA-Avi-1D4 vectors, respectively. Bases in uppercase encode RSPO3 WT, mutant and HS20-fusion proteins. For mutant constructs, mutated bases are indicated in red and the resulting altered codons are underlined. For HS20-fusion constructs, bases encoding a codon-optimized glycine/serine linker (STGGSGGSGGSG) are indicated in light blue.

The names and sequences of pairs of oligonucleotides encoding sgRNAs (which were cloned into pX458-mCherry) are shown in the first and second columns, respectively. The names and sequences of pairs of PCR primers used to amplify corresponding genomic regions flanking sgRNA target sites are shown in the third and fourth columns, respectively. The names and sequences of primers used to sequence the amplified target sites are shown in the fifth and sixth columns, respectively.

Clonal cell lines derived from HAP1-7TGP in which multiple genes were targeted using CRISPR/Cas9 (see Materials and methods) are described. The ‘Cell Line Name’ column indicates the generic name used throughout the manuscript to describe the genotype and the ‘Clone #' column identifies the individual clone used. The figures in which each clone was used are also indicated. The ‘CRISPR guide’ column indicates the name of the guide or guides used, which is the same as that of the oligonucleotides encoding sgRNAs (see Materials and methods and Supplementary file 2). The ‘Genomic Sequence’ column shows 80 nucleotides of genomic sequence (5’ relative to the gene is to the left) surrounding the target site; when two adjacent sites within the same gene were targeted, 80 nucleotides of genomic sequence surrounding each target site are shown and the number of intervening bp that are not shown between the two sites is indicated in parenthesis. Each cell line made using a different set of CRISPR guides is separated by a horizontal spacer, under which the reference (WT) genomic sequence (obtained from RefSeq) targeted by each CRISPR guide is indicated. Within this reference genomic sequence, the guide sequence is colored blue and the site of the double strand cut made by Cas9 is between the two underlined bases. Sequencing results for individual mutant clones are indicated below the reference sequence. Mutated, inserted or deleted nucleotides are colored red (dashes represent deleted nucleotides and ellipses are used to indicate that a deletion continues beyond the 80 nucleotides of sequence shown) and the nature of the mutation, the resulting genotype and any pertinent observations are also described. The CRISPR guide or guides used to target different genes, as well as the genomic sequence, mutation, genotype and observations pertaining to each of the targeted genes are designated ‘1’, ‘2’, ‘3’ and ‘4’ in the column headings and are shown under horizontal spacers of different colors.

Genes containing at least one inactivating GT insertion in the population of sorted cells from each of the two genetic screens described in this work are listed in separate spreadsheets (the screen name is indicated on the tab of each spreadsheet), and are ranked based on the significance of inactivating GT insertion enrichment (p-value) in the sorted vs. the unsorted (control) cells. For the unsorted cells, the number of all GT insertions in genes (regardless of orientation) is indicated for the complete dataset and for each gene (column B). For the sorted cells, the total number of inactivating GT insertions in genes (sense and antisense insertions in exons and sense insertions in introns, column C), as well as the number of sense or antisense GT insertions in exons or in introns (columns D-G), is indicated for the complete dataset and for each gene. Three measures of GT insertion enrichment are shown: the p-value and the FDR-corrected p-value (both derived from columns B and C), the latter of which was used to generate the circle plots in Figure 6A and B, and the Intronic GT Insertion Orientation Bias (IGTIOB) score (derived from columns F and G), used to generate the heat map in Figure 6C. See Materials and methods for details.

Genes used to generate the heat map in Figure 6C, comparing the RSPO1 screen in WT HAP1-7TGP cells (Figure 6A) and the RSPO3 screen in LGR4/5/6^KO cells (Figure 6B), are shown. Genes enriched for inactivating GT insertions (FDR-corrected p-value<0.01) in at least one of the two screens are shown, and the FDR-corrected p-value and IGTIOB score for each gene in each screen is indicated. Genes are shown in the same order as in the heat map in Figure 6C, clustered based on their IGTIOB scores (see Materials and methods for details).