BENG183 Lecture 14: Advanced CRISPR Technologies, Perturbation Screening, and MPRA

Vocabulary practice flashcards covering CRISPR-Cas tools, repair mechanisms, base and prime editing, pooled and single-cell screening modalities, and high-throughput reporter assays (MPRA and STARR-seq) from Lecture 14.

ClinVar

A public repository of human genomic variations and their interpretations, in which >99% of identified single nucleotide variants remain unstudied, and among clinically classified variants, 40.5% are classified as variants of uncertain significance and 16.8% as pathogenic or likely pathogenic.

Genome-Wide Association Study (GWAS)

An observational study design that compares DNA microarrays across individuals with and without a complex trait to identify statistically linked susceptibility loci across chromosomes.

Regulatory Noncoding Variants

Genomic sequence changes located outside of protein-coding exons that constitute approximately $88.5\%$ of all NHGRI-EBI GWAS catalog variants, altering promoters, enhancers, silencers, or noncoding RNAs rather than directly modifying amino acid sequences.

Protospacer Adjacent Motif (PAM)

A short, essential DNA sequence (such as $5'\text{-NGG-}3'$ for native SpCas9) situated directly adjacent to the $3'$ end of the protospacer target site that is recognized by Cas9 to permit target search, DNA unwinding, and R-loop stabilization.

HNH Domain

The catalytic nuclease domain of Cas9 that cuts the target DNA strand complementary to the single-guide RNA protospacer sequence, specifically cleaving between protospacer positions 17 and 18.

RuvC Domain

The catalytic nuclease domain of Cas9 that cuts the non-target DNA strand approximately 3 base pairs upstream of the protospacer adjacent motif.

Non-Homologous End Joining (NHEJ)

An endogenous cellular DNA double-strand break repair pathway that directly ligates broken DNA ends, typically resulting in random insertions and deletions (indels) or targeted deletions between two Cas9 cut sites to generate gene knockouts.

Homology-Directed Repair (HDR)

A precise DNA repair mechanism that uses an exogenous single-stranded oligodeoxynucleotide (ssODN) or double-stranded DNA donor template with sequence homology arms to achieve targeted gene insertion or scarless target alteration.

Cytosine Base Editor (CBE)

A precision gene editing construct composed of a Cas9 nickase (nCas9) fused to a cytidine deaminase and a uracil glycosylase inhibitor (UGI), converting a $C \cdot G$ base pair to a $T \cdot A$ base pair within an edit window (protospacer positions 4 to 8) without generating double-strand breaks.

Adenine Base Editor (ABE)

A precision gene editing tool composed of a Cas9 nickase (nCas9) fused to an evolved TadA adenosine deaminase that deaminates adenosine into inosine within an editing window, converting an $A \cdot T$ base pair to a $G \cdot C$ base pair without creating double-strand breaks.

Uracil Glycosylase Inhibitor (UGI)

A protein domain appended to cytosine base editors (such as BE2, BE3, and BE4) that inhibits endogenous uracil DNA glycosylase from excising deaminated cytidine (uracil), thereby suppressing base excision repair and increasing transition editing efficiency.

CRISPR Interference (CRISPRi)

A transcriptional silencing platform that uses catalytically dead Cas9 (dCas9) fused to a transcriptional repressor domain, such as KRAB or SID, to inhibit transcription initiation or elongation at promoters, enhancers, or $5'\text{ UTRs}$.

CRISPR Activation (CRISPRa)

A transcriptional upregulation platform that uses catalytically dead Cas9 (dCas9) fused to transcriptional activation domains (such as VP64, VP16, p65, RTA, or HSF) targeted to upstream regulatory elements to induce endogenous gene transcription.

Prime Editing (PE)

A search-and-replace precision gene editing technology combining a Cas9 nickase (nCas9) fused to an engineered Moloney murine leukemia virus (M-MLV) reverse transcriptase and a pegRNA, capable of generating targeted small indels, transitions, and transversions without requiring double-strand breaks or donor DNA templates.

pegRNA (Prime Editing Guide RNA)

A specialized, extended guide RNA that specifies the genomic target site with its protospacer and carries an extension containing both a primer binding site and a reverse transcriptase template that encodes the desired edit.

eSpCas9

An engineered Streptococcus pyogenes Cas9 variant containing structure-guided alanine mutations (K810A, K1003A, R1060A) that weaken non-specific interactions with the non-target DNA strand to dramatically reduce off-target cleavage while retaining high on-target activity.

Pooled CRISPR Screen

A high-throughput screening approach wherein a complex library of thousands of sgRNAs is cloned into plasmids, packaged into lentivirus, and transduced en masse into cells to evaluate fitness or survival under selection by sequencing sgRNA abundance shifts.

Arrayed CRISPR Screen

A screening format in which cells are partitioned into individual multiwell plate wells where each well receives a unique perturbation, enabling quantitative high-content imaging and multiparametric cellular phenotype quantification.

Single-Cell Functional Genomics (scFG / Perturb-seq)

A screening modality combining pooled CRISPR perturbations with droplet-based single-cell RNA sequencing to simultaneously capture individual cellular transcriptomes and their corresponding sgRNA barcodes, linking perturbations to genome-wide expression changes.

FACS-Based CRISPR Screen

A pooled functional screening modality that sorts transduced cells displaying top and bottom percentiles of a flow-cytometry-detectable marker (e.g., protein expression or RNA-FISH signal) followed by deep sequencing to identify sgRNAs that regulate that marker.

CRISPRi-FlowFISH

A high-density screening strategy that tiles CRISPRi guide RNAs across candidate noncoding GWAS SNPs, uses RNA fluorescence in situ hybridization to label transcripts of a target gene, and sorts cells by flow cytometry to functionally connect distal regulatory variants to target gene expression.

Massively Parallel Reporter Assay (MPRA)

A high-throughput molecular assay that measures the regulatory activity of tens of thousands of synthesized candidate cis-regulatory elements (CREs) simultaneously by cloning them into plasmids with unique barcodes and calculating the ratio of transcribed RNA barcodes to plasmid DNA barcodes.

STARR-seq (Self-Transcribing Active Regulatory Region Sequencing)

A genome-wide reporter assay in which sheared genomic DNA fragments or open chromatin regions are cloned downstream of a minimal promoter into the $3'\text{ UTR}$ of a reporter gene, allowing active enhancers to transcribe themselves as their own quantitative barcodes.

Saturation Mutagenesis MPRA

A high-throughput reporter strategy where all possible single-nucleotide substitutions across a defined cis-regulatory element are synthesized and quantified to evaluate single-base functional contributions and reconstruct transcription factor binding motifs.

lentiMPRA

A massively parallel reporter assay platform that delivers synthesized cis-regulatory elements and $5'\text{ UTR}$ barcodes using lentiviral transduction, measuring regulatory activity within a native, chromatinized genomic context rather than on transient episomes.

SuRE-seq (Survey of Regulatory Elements)

A promoterless, high-throughput reporter assay in which sheared genomic DNA is cloned upstream of an open reading frame with a $5'\text{ UTR}$ barcode to measure autonomous promoter and enhancer activities across the genome.

VAMP-seq (Variant Abundance by Massively Parallel Sequencing)

A plasmid-based screening assay designed for coding variants that fuses variant coding sequences to eGFP, bins cells by fluorescence intensity via FACS, and uses deep sequencing to quantify the effect of each variant on steady-state protein stability and abundance.

Optogenetic CRISPR Control

A light-inducible genome engineering system that uses blue light illumination to drive the heterodimerization of photosensitive proteins (such as CRY2 and CIB1) to conditionally recruit transcriptional effectors (like VP64) to dCas9.