Theory to Action: Applying CRISPR in Research
Traditional Gene Knockout
As described above, the CRISPR-Cas complex generates a targeted double-strand
creating an opportunity for gene editing.
Gene knockout (KO) experiments exploit flaws in the cell’s native repair mechanism,
joining (NHEJ). Imperfectly repaired genes with insertions or deletions of base pairs will
frameshift mutations, rendering the gene and its corresponding protein non-functional. KO
algorithmically designed and ranked for both specificity (low homology with any other
and functionality (targeting a portion of the gene most likely to result in functional
CRISPR KO can be applied in many research scenarios, from basic screening, functional
studies of a gene,
bioengineering, agricultural biotechnology, to developing therapeutic gene and cell
Increasing CRISPR/Cas9 Editing Specificity
CRISPR Cas9 based gene knockout relies on the specificity of both the sgRNA and
protein. Poorly designed sgRNA and traditional wildtype Cas9 would lead to editing at
locations, which are considered as off-target edits. Many Cas9 variants have been developed
specificity. For example, the eSpCas9(1.1), also referred to as SpCas9
structurally engineered from Feng Zhang lab contains alanine mutations that weaken the
the HNH/RuvC groove and the none targeting DNA strand, reducing off-target effects by over
maintaining robust on-target genome editing efficiency (Slaymaker et al. 2016).
Researchers have also engineered Cas9 variants to “nick” a single DNA strand
creating double stranded DNA breaks. Two individual nickases targeting opposite DNA strands
necessary to generate a break site for gene editing, which significantly increases targeting
specificity. For example, SpCas9 nickase (Cas9n D10A) contains a mutation allowing the
create single-strand nicks, as opposed to DSBs. Pairing two opposite facing gRNA sequences
nickase can be an efficient method of gene editing that prevents unwanted indels from
Novel Editing Technologies Without DSBs (Prime Editing/Base
Cas9 nickases also could enable novel single-strand CRISPR gene editing
avoid the potential for unintended genomic changes posed by double-strand DNA breaks.
utilizes a Cas nickase protein fused to a reverse transcriptase (RT) to write a new sequence
target DNA site directly. Prime editing guide RNAs (pegRNA) contain both a DNA targeting
sequence and an
RT template so that the pegRNA-dCas9 nickase complex can identify the target site, cut a
and write the new sequence. Multiple versions of the prime editing system have been
developed to improve
Base editing utilizes a Cas9 nickase, or alternatively a catalytically dead
(dCas9) which only
binds to target DNA without cutting, fused to a nucleobase deaminase enzyme and a DNA
inhibitor to make targeted point mutations. For example, cytosine base editors (CBEs) and
editors (ABEs), by fusing Cas9 nickase or dCas9 to a cytidine deaminase like APOBEC or
deaminases, can convert C to T (or G to A) and A to G (or T to C).
Traditional Gene Knockin
Gene knock-ins have various applications in biotechnology, including disease
gene and cell therapy applications. CRISPR based gene insertion relies on a pathway
different than NHEJ,
it is called Homology directed repair (HDR) pathway. HDR process is very precise allowing
gene knockin, but HDR occurs in less frequency compared to NHEJ leading to less editing
compared to knockout. KI experiments also require the introduction of a Donor DNA HDR templates,
could be either single- or double-stranded DNA designed with overlapping homology arms to
To increase the efficiency of HDR, experiment optimization by testing different
format, delivery approaches, culture conditions, as well as using small molecules for cell
synchronization, enhance HDR or inhibit NHEJ have been used to overcome this challenge.
Read more on HDR
formats available and design.
CRISPR Transcriptional Regulation
Additional Cas9 variants have been developed for transcriptional regulation of
expression. Rather than editing DNA, these deactivated Cas9 proteins simply bind the target
activate or inhibit expression.
CRISPR activation (CRISPRa) systems utilize a dCas9 fused to transcriptional
up regulate endogenous gene expression, whereas CRISPR interference (CRISPRi) systems use
dCas9 fused to
transcriptional repressors to downregulate gene expression. Multiple CRISPRa and CRISPRi
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
epigentically modify and silence genes without editing the underlying genome. The epigenetic
persists through cell differentiation and is heritably passed to future cell generations.
can be re-activated using a different fusion protein called CRISPRon.
||CRISPRa guide RNA + dCas9-SAM
|Overexpression screening and hit analysis
|| CRISPRi guide RNA + dCas9-KRAB
|Transient loss-of-function, siRNA validation
|CRISPRoff guide RNA + CRISPRoff
||Heritable gene silencing without DNA editing