Gene editing positive cell screening

NegotiableUpdate on 03/04

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Nature of the Manufacturer: Producers

Product Category

Place of Origin

Online Consult

Overview

Gene editing positive cell screening refers to the identification and isolation of cells that have successfully undergone target gene modification through specific methods after gene editing experiments (such as CRISPR/Cas9). Common screening methods include antibiotic screening (such as using resistance gene markers), fluorescent protein marker screening, PCR or sequencing validation, etc. This process is crucial for obtaining homozygous or heterozygous mutant cell lines, studying gene function, or constructing disease models.

Product Details

Gene editing positive cell screeningTechnical Strategy and Optimization Process

Positive cell screening is the core process of gene editing, and its efficiency directly affects the experimental cycle and success rate.

1、 Screening Objectives and Challenges

core objective

Isolation of successfully edited cells from mixed cell populations (such as gene knockout/KO, knock in/KI).
Exclude interference from wild-type cells (high proportion of unedited cells can easily lead to false negatives).

key challenges

cell damageLong term screening leads to cell aging (decreased proliferation ability of pig fibroblasts after passage).
False positive riskPartial editing did not completely destroy gene function (such as frameshift mutations not causing loss of function).
Flow bottleneckTraditional monoclonal culture requires more than 22 days and the positivity rate is less than 20%.

2、 Mainstream screening techniques and operational processes

（1） Enrichment technology based on reporting system

Using repair mechanisms to trigger fluorescence/resistance marker expression, achieving visualization and rapid sorting:

Repair mechanism	Report system design	screening method	advantage	case
NUMBER	Targeted destruction of fluorescent protein terminators → restoration of expression	FACS sorting GFP ⁺ cells	Intuitive and efficient, suitable for KO	CRISPR-DIY Vector
HDR	Homologous recombination insertion of resistance genes (such as Puro ᵣ)	Antibiotic stress screening	Suitable for KI, low cost	Biyun Tian CRISPR plasmid
SSA	Fracture induced single chain annealing repair → fluorescent protein reconstruction	flow cytometry	High sensitivity, low off target rate	Multiple gene editing validation

operating procedure(Taking NHEJ-GFP system as an example):

Build withGFP-TAA terminatorThe editing vector (sgRNA targeting TAA region).

After transfection of cells, NHEJ repairs and destroys the terminator → GFP expression.

Use after 72 hoursFlow cytometry (such as iQue) ®） Sorting GFP ⁺ cells.

（2） Rapid identification method for trace cells

Breakthrough planOnly 50 cells are needed to complete genotype identification, shortening the cycle by more than 15 days.

key parameters：

Cell quantity≥ 50 (insufficient detection rate in the 20 cell group, P<0.01).
Enzyme sensitivityT7E1 can detect editing efficiency of ≥ 5%.
Verification stepsSanger sequencing confirms the type of mutation (such as insertion/deletion).

（3） Enzyme digestion and sequencing validation technology

method	principle	Applicable scenarios	limitation
T7E1 enzyme digestion	Mismatch cutting generates heterologous double chains	Initial screening (low-cost)	Low sensitivity (≥ 5% editing rate)
Sanger sequencing	Directly read sequence variations	Monoclonal validation	Low flux
high-throughput sequencing	Deep coverage of target site mutations	Multi gene/off target analysis	成本高

Operation optimization：

Mixed clone pre screeningFA-PCR detects the population editing rate, and when it exceeds 30%, single clones are sorted.

Dual verification strategyT7E1 initial screening positive clones → Sanger sequencing confirmation (to avoid false positives).

3、 Technology selection and scene adaptation

（1） Select by cell type

cell type	Recommended methods	reason
primary cells	Microcellular identification method	Avoid aging caused by long-term cultivation (pig fibroblasts)
Tumor cell line	Antibiotic screening+FACS	Rapid proliferation and stable drug resistance
iPSCs	HDR reporting system+monoclonal sequencing	Maintaining pluripotency requires high-precision editing

（2） Select by editing type

Edit Type	Screening techniques	key metrics
Gene knockout (KO)	NHEJ-GFP reporting system	GFP ⁺ cell proportion (flow cytometry quantification)
Gene Knockin (KI)	Antibiotic screening+PCR validation	Resistance survival rate+flanking sequence amplification
Point mutation (PM)	T7E1+Deep Sequencing	Mutation frequency>90%

Gene editing positive cell screening