Introduction

CRISPR-based genome engineering allows researchers to make a variety of specific changes to a cell’s DNA. While the basic concept of CRISPR engineering is relatively straightforward, the variety of different systems, formats and delivery methods may seem overwhelming. Here we aim to deconvolute CRISPR engineering by focusing on.

CRISPR Components

The most basic CRISPR gene editing experiment, gene knockout, requires two components be delivered to the cell’s nucleus; a targeted guide RNA and a Cas nuclease. These components form a complex that binds DNA at the target sequence and generates a double strand break, creating an opportunity for knockout.

Engineering Workflows

The most common CRISPR engineering workflows include:

Workflow sgRNA Cas Recommended Delivery Description Ideal for Benefits
RNP synthetic protein Transfection (electroporation, lipofection) Synthetic CRISPR sgRNA and Cas protein are complexed as ribonucleoprotein (RNP) prior to delivery. ex vivo editing, embryonic microinjection DNA-free/transgene-free. Editing begins immediately.
mRNA synthetic mRNA Co-transfection Synthetic CRISPR sgRNA and Cas mRNA are co-delivered. in vivo editing DNA-free/transgene-free. Editing begins post-Cas9 expression.
Co-plasmid plasmid plasmid Viral packaging Separate viral packaged plasmids containing CRISPR sgRNA and Cas are co-delivered, and randomly integrate for constitutive expression. Difficult-to-transfect cell lines Simple delivery and selection/enrichment.
All-in-one single plasmid Viral packaging Single viral packaged plasmid containing CRISPR sgRNA and Cas is delivered, and randomly integrates for constitutive expression. Difficult-to-transfect cell lines Single viral transduction delivery step
Stable Cas9 cell line synthetic plasmid Viral Cas plasmid, transfected sgRNA Viral packaged Cas plasmid is delivered to generate stable Cas-expressing cell line. Then synthetic sgRNA is delivered for editing. Screening Easily test stable Cas9 expressing line with various gRNA

CRISPR Toolbox

The “CRISPR toolbox” offers a diverse set of gene manipulation tools, including gene knockout and knock-in, base and prime editing, as well as transcriptional regulation of protein expression.

CRISPR Applications

Disease Modeling
Editing Immune Cells
Screening & Hit Validation
AgBio

GenCRISPR Services

CRISPR Resources

Featured Webinar | CRISPR Based CAR-T Cell Editing: Large gene knock-ins in human T cells using non-viral HDR templates

Dr. Theo Roth from Marson Lab, UCSF introduces the advantages of a new CRISPR based method that allows for the insertion of large DNA sequences (>1Kb) in primary T cells without a virus.

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