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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.
Review the components required for basic CRISPR gene editing
Compare CRISPR workflows and the reagent formats required
Outline CRISPR experiment types and the systems required
Explore the many applications in which CRISPR engineering is used
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.
CRISPR guide RNAs are designed to target specific DNA sequences near a PAM site, and may be used as single guide RNA (sgRNA) or as a duplex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
GenCRISPR guide RNAs are available as synthetic sgRNA or plasmid crRNA .
Cas nucleases are RNA-guided proteins capable of cleaving the phosphodiester bonds of a DNA backbone to create a double-strand break. The most commonly used Cas for CRISPR engineering is Cas9 from S. pyogenes (SpCas9). Cas9 orthologs have also been discovered from S. aureus (SaCas9) and C. jejuni (CjCas9). Additional naturally occurring Cas nucleases include Cas12a, Cas12c, Cas12e, Cas13a and CasX. Variations in PAM specificity among these Cas variants will affect guide design flexibility and the potential for incidental off-target editing.
In addition to the wild type Cas nucleases, researchers have engineered variants for specific uses, including improved editing specificity (eSpCas9) and for relaxed PAM specificity (SpCas9-NG). Researchers have also altered Cas9 nuclease activity for single-strand cutting (nCas9), or deactivated it altogether (dCas9) for use with transcriptional activators or repressors. For more information on these alternative CRISPR-Cas systems, please see the CRISPR Toolboxsection.
Both CRISPR guide RNA and Cas nuclease must be delivered into the cell nucleus for successful editing to occur. CRISPR/Cas delivery strategies may be separated into two types; non-viral transfection and viral vector transduction. The chosen strategy will largely determine which reagent formats are suitable. For more information, see the Engineering Workflows section.
| Non-viral transfection | Viral vector transduction |
|---|---|
| Deliver CRISPR components as RNA or protein directly into cell nucleus |
Deliver virus packaged plasmids encoding CRISPR components for integration |
| Electroporation, lipofection, microinjection, biolistics |
Lentivirus, AAV |
Multiple delivery strategies may also be used in series, for example by first transducing lentiviral packaged Cas9 plasmid to generate a stable Cas9 expressing cell line, and then electroporating guide RNAs for editing.
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 |
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.
As described above, the CRISPR-Cas complex generates a targeted double strand break, creating an opportunity for gene editing.
Gene knockout (KO) experiments exploit flaws in the cell’s native repair mechanism, non-homologous end joining. Imperfectly repaired genes with insertions or deletions of base pairs will result in frameshift mutations, rendering the gene and its corresponding protein non-functional. KO guides are algorithmically designed and ranked for both specificity (low homology with any other genomic sequence) and functionality (targeting a portion of the gene most likely to result in functional protein KO).
Homology Directed Repair (HDR) mediated knock-in (KI) experiments utilize the same CRISPR-Cas machinery as KO experiments to create a double-strand break. However, KI experiments also introduce a DNA template and rely on the HDR repair pathway for insertion. HDR templates may be either single- or double-stranded DNA designed with overlapping homology arms to the specific cut site.
| Technique | Components Required | Common Experiments | Common Applications |
|---|---|---|---|
| Knockout | Guide RNA + Cas nuclease | Loss-of-function screening and hit analysis | Drug discovery, disease modeling |
| Knock-in | Guide RNA + Cas nuclease + HDR template | Insert a protein tag, restriction site, sequence change or gene | Gene and cell therapy |
Researchers have also engineered Cas9 variants to “nick” a single DNA strand. These Cas9 nickases enable novel single-strand CRISPR gene editing techniques that avoid the potential for unintended genomic changes posed by double-strand DNA breaks.
Prime editing utilizes a catalytically disabled Cas9 (dCas9) fused to a reverse transcriptase (RT) to directly write a new sequence into a target DNA site. Prime editing guide RNAs (pegRNA) contain both a DNA targeting sequence and a RT template, so the pegRNA-dCas9 nickase complex can identify the target site, cut a single strand and write the new sequence. Multiple versions of the prime editing system have been developed to improve editing efficiency.
Base editing utilizes a dCas9 fused to a nucleobase deaminase enzyme and a DNA glycosylase inhibitor to make targeted point mutations.
Cas9 nickase is currently available in plasmid format.
| Technique | Components Required | Common Experiments | Common Applications |
|---|---|---|---|
| Prime Editing | pegRNA + Cas H840A nickase (or other PE variant) | In situ gene editing | Gene therapy |
| Base Editing | Guide RNA + Cas nickase | Introduce single nucleotide variants | Gene therapy |
Additional Cas9 variants have been developed for transcriptional regulation of gene expression. Rather than editing DNA, these deactivated Cas9 proteins simply bind the target site and activate or inhibit expression.
CRISPR activation (CRISPRa) systems utilize a dCas9 fused to transcriptional activators to up regulate endogenous gene expression, whereas CRISPR interference (CRISPRi) systems use dCas9 fused to transcriptional repressors to downregulate gene expression. Multiple CRISPRa and CRISPRi systems have been developed using different activators or repressors. CRISPRa gRNAs for the SAM system are currently available in plasmid formats.
Researchers have also recently developed a Cas fusion protein, CRISPRoff, that can epigentically modify and silence genes without editing the underlying genome. The epigenetic memory persists through cell differentiation and is heritably passed to future cell generations. Gene activity can be re-activated using a different fusion protein called CRISPRon.
| Technique | Components Required | Common Experiments | Common Applications |
|---|---|---|---|
| CRISPRa | CRISPRa guide RNA + dCas9-SAM (or other) | Overexpression screening and hit analysis | Drug discovery |
| CRISPRi | CRISPRi guide RNA + dCas9-KRAB (or other) | Transient loss-of-function, siRNA validation | Drug discovery |
| CRISPRoff/ CRISPRon | CRISPRoff guide RNA + CRISPRoff | Heritable gene silencing without DNA editing | Gene therapy |
CRISPR/Cas9 sgRNA
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CRISPR/Cas12a crRNA
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Prime Editing Guide RNA
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Base Editing Guide RNA
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gRNA + HDR Template Design Tools
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cGMP Guide RNA
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cGMP HDR Templates
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CRISPR gRNA Plasmid Libraries
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CRISPR Cell Lines
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CRISPR/Cas Proteins
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CRISPR Plasmids
Learn MoreFeatured 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.
