CRISPR Plasmids: Mammalian Expression
The following CRISPR plasmids have been designed for use in mammalian expression systems.
Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles.
To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.
Catalytically dead dCas9 fused to a cytidine deaminase protein becomes a specific base editor that can alter DNA bases without inducing a DNA break. Base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the gRNA. Adenine base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite strand) mutations. CRISPR-X, a base-editing system from the Bassik lab, uses an MS2 hairpin-tagged gRNA to recruit a cytidine deaminase and induce somatic hypermutation in a 100 bp window. This technique creates diverse populations of mutants for directed evolution.
CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two gRNAs that target opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted off-target effects. Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed repair (HDR).
Cas9 H840A nickase fused to a reverse transcriptase (RT) is capable of installing targeted insertions, deletions, and all possible base-to-base conversions using a prime editing guide RNA (pegRNA). The pegRNA directs the nickase to the target site by homology to a genomic DNA locus and encodes a primer binding site and the desired edits on an RT template.
Catalytically dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene of interest. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-activator to your specific locus.
Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression by interfering with transcription. Design your gRNA to target your gene of interest’s promoter/enhancer or the beginning of the coding sequence. If the plasmid you’re using does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-repressor to your specific locus.
To make targeted epigenetic modifications, researchers have fused catalytically dead dCas9 to epigenetic modifiers. Design your gRNA to target a specific promoter or enhancer for your gene of interest. Available modifications include histone acetylation by p300, histone demethylation by LSD1, cytosine methylation by DNMT3A or MQ1, and cytosine demethylation by Tet1. These modifications persist over time and are potentially heritable in dividing cells.
Type VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. Type VI enzymes that function in mammalian cells can be used to attentuate RNA levels. In mammalian systems, Cas13a does not exhibit the collateral RNA degradation seen in bacteria.
Type VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. Fusing the catalytic domain of ADAR2(E488Q) adenosine deaminase to catalytically dead Cas13b creates a programmable RNA editor that converts adenosine to inosine in RNA. Since inosine is functionally equivalent to guanosine, the result is an A->G change in RNA. dPspCas13b does not require a Protospacer Flanking Sequence (PFS), making it a very flexible editing system.
A catalytically inactive Cas9 (dCas9) can be used to purify a region of genomic DNA and its associated proteins, RNA, and DNA. The enCHIP system uses an anti-FLAG antibody to immunoprecipitate FLAG-tagged Cas9. In the CAPTURE system, Cas9 is tagged with a biotin acceptor site and co-expressed with BirA biotin ligase. The locus is isolated using streptavidin affinity purification.
For both enCHIP and CAPTURE, design your gRNA sequence to direct dCas9 to a specific locus, avoiding known transcription factor and other protein binding sites.
A catalytically inactive Cas9 (dCas9) fused to a fluorescent protein (FP) can help visualize specific genomic loci using fluorescent microscopy in living cells. Design your gRNA sequence to direct the dCas9-FP fusion to a specific genomic sequence - potential target locations can include both unique and repetitive regions. Fluorophores may also be fused to gRNA sequences to produce fluorescence at a target region, as in the CRISPRainbow kit.
Empty gRNA Expression Vectors
You can use the tables on Addgene's Empty gRNA Vectors page to search based on factors such as selectable marker or cloning method. To isolate mammalian vectors, simply type "Mammalian" into the search bar. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single category (cut, nick, etc.) On the other hand, gRNA plasmids that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids.
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