Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes

Abstract

Determining the functional impacts of disease-causing genetic variants presents consistent challenges in the genetic disease field. Here, we present a protocol for implementing saturation mutagenesis-reinforced functional assays to generate functional scores for small-sized variants in disease-related genes. We describe procedures for nucleofection to establish cell line platforms, programmed allelic series with common procedures (PALS-C) cloning for saturation mutagenesis, fluorescence-based cell sorting, next-generation sequencing, and functional score generation. This framework holds potential for high-throughput and cost-effective interpretation of unresolved variants in a broad array of disease genes. For complete details on the use and execution of this protocol, please refer to Ma et al.¹.

Keywords: CRISPR; Cell Biology; Flow Cytometry; Genetics; Genomics; High-Throughput Screening; Molecular Biology; Sequencing.

Graphical abstract

Figure 2

Demonstration of Method 2 of isolating single clones from pooled nucleofected cells As described in Step 5c, an alternative method of isolating single clones is to plate 30–3000 nucleofected cells in a 15-cm dish (depending on cell type), grow the cells until they form single colonies, use a microscope object marker to indicate where each colony is located, and isolate single colonies by trypsinizing the colony within a cloning cylinder (2A), resuspending in growth medium (2B), and transferring to 24-well wells (2C).

Figure 1

Experimental setup for Method 1 of isolating single clones from pooled nucleofected cells As described in Step 5b, one method of isolating single clones is to plate the nucleofected cells in a 15-cm dish at 0.2–20 cells/cm² (depending on cell type), grow the cells until they form visible monoclonal clusters, and pick single colonies by viewing the plate under a microscope (placed within a Biosafety Cabinet and UV sterilized beforehand) and pipetting each colony into 24-well wells.

Figure 3

PALS-C cloning for saturation mutagenesis Programmed Allelic Series with Common procedures (PALS-C) is an 8-step cloning strategy to clone plasmid pools from oligonucleotides containing small-sized variants. Schematic representations of each step and their respective products (“pdt”) are depicted. Step 8 (not pictured) uses electrotransformation to deliver the assembled plasmid pools to bacteria for amplification.

Figure 4

Schematic representation of three saturation mutagenesis modes for oligo design Mode 1: Individual nucleotide mutagenesis (each nucleotide mutated to 3 alternatives, generating 3 oligos per position). Mode 2: Degenerate DNA letter code substitution (A to B, C to D, G to H, T to V). Mode 3: Codon-level NNK mutagenesis (only applicable to CDS).

Figure 5

Example design of R1 oligo reverse primer used in Step1 of PALS-C Using the provided Python scripts, small-sized variants can be generated in ssDNA oligonucleotides to use as PCR reverse primers. The primers are designed to also include block-specific sequences for the block-by-block strategy of PALS-C (“Block-specific adaptor”), including an appropriate Type2S recognition sequence (“Recog.”) to enable downstream removal of block-specific adaptors. The Type2S enzyme is selected such that its recognition sequence is not present in the CDS of the GOI. The block-specific insulator prevents generation of strands with more than one variant by ensuring that a strand containing a different variant cannot use the “undesired strand” as a template for extension during PCR reactions in following steps. In the above example from Ma et al., this yielded 64-nucleotide ssDNA oligos containing each variant of all possible FKRP SNVs and the recognition site of the enzyme BsmBI (5′-CGTCTC-3′), with the cleavage site indicated in red.

Figure 6

Schematic representation of the relative locations of the universal forward and reverse primers The universal F1 primer will be utilized to amplify the variant strand in PALS-C Step1 (Figure 3 and protocol Step 16) and should be designed to anneal >100-200 bp upstream of the GOI. The universal F2 and R2 primers will be utilized to amplify the full-length variant strand in PALS-C Step3 (Figure 3 and protocol Step 18) and should be designed to include overlapping sequences with the restriction site of the plasmid backbone into which the variant strands will be inserted.

Figure 7

Example outcome plot from implementing SMuRF assays to resolve small-sized variants in a gene of interest Following the described protocol generates functional scores (SMuRF scores) for each variant in the GOI that are normalized to the mean of synonymous variants such that values below zero indicate deleterious effects of that variant on the assayed function. Plotting variant functional scores by variant category can corroborate the expected functional effect of each type of variant, such as nonsense and start-loss variants exhibiting consistently low SMuRF scores, synonymous variants exhibiting neutral scores near zero, and missense variants exhibiting a wider range of functional scores, though often detrimental in comparison to the synonymous state. In the above example plot, “####” represents the number of variants.