CRISPR-Cas9 Recombination

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system is a powerful genome editing tool allowing for site-specific genomic targeting. The CRISPR/Cas9 system uses the crRNA (CRISPR RNA):tracrRNA (trans-activating crRNA)-guided Cas9 nuclease to recognize and cleave specific double-stranded DNA (dsDNA) bearing a PAM (protospacer-adjacent motif) and an adjacent sequence complementary to the crRNA.

Cas9 is a dual RNA-guided DNA endonuclease enzyme associated with the CRISPR. The most widely used CRISPR endonuclease is Streptococcus pyogenes Cas9 (SpCas9). However, the large size (~4 kb), the restricted PAM sequence and unwanted off-target effects of SpCas9 are major limitations that hinder its applications. To overcome these limitations, several Cas9 variants with small size / alternative PAMs / high-fidelity, have been developed. For example,
1) SaCas9 is derived from Staphylococcus aureus which requires a different gRNA scaffold sequence and use the PAM sequence NNGRRT.
2) SpCas9-HF1, eSpCas9, HypaCas9 are three versions of Cas9 that have been specifically engineered for reduced off target editing.
3) Cas9 nickase is a D10A mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB.

The combination of crRNA and tracrRNA is also called guide RNA (gRNA). crRNA is a ~20 nt sequence that is complementary to the target DNA, and tracr RNA is a ~70 nt sequence which serves as a binding scaffold for the Cas9 nuclease. PAM is a 2~6-base pair DNA sequence that is not present in the crRNA sequence but needs to be directly downstream of the target sequence in the genomic DNA, on the non-target strand. PAM varies depending on the Cas9 type, for example, SpCas9 recognizes a PAM sequence of NGG.

The Cas9-induced double strand breaks (DSBs) are repaired via non-homologous end joining (NHEJ) or homology directed repair (HDR). Repair via the NHEJ is error-prone resulting in the creation of insertions and deletions (INDELs) at the site of the DSB and thus disrupt gene function. The HDR pathway requires the presence of a donor template allowing insertion of a specific DNA sequence.

The CRISPR/Cas9 system has a wide variety of applications and can be used to generate gene knockouts, gene knockins, point mutations, activation / repression of target genes and epigenetic modifications etc.
1) Gene Knockout: CRISPR-Cas9 system can be used for loss-of-function studies via gene knockouts. It can introduce double-strand break (DSB) in the target genomic region.
NHEJ fixes the produced DSB but results in insertion-deletions (INDELs) during the process. At the end, the introduction of INDELs in exons leads to disrupted protein expression.
2) Gene Knockin: CRISPR-Cas9 system can also be effectively used to insert gene / genetic sequence or substitute nucleotide(s) at specific loci. Instead of NHEJ repair pathway, knockin editing relies on HDR repair pathway which requires a homologous template to repair DSBs and introduce the desired sequence into the target genomic region.
3) CRISPR activation (CRISPRa): CRISPRa is a type of CRISPR-based genetic tool that uses modified versions of endonuclease with added transcriptional activators. The most commonly-used effector is based on Cas9, for example, Cas9 Endonuclease Dead (dead Cas9 or dCas9) which is a mutant form of Cas9 nuclease whose endonuclease activity is removed through point mutations in its endonuclease domains. dCas9 is unable to cleave dsDNA but retains the ability to bind to target specific genes or nucleotides complementary to the gRNA. Thus CRISPRa can be used to target your gene of interest and increase its expression without cutting the DNA backbone.
4) CRISPR interference (CRISPRi): Like for CRISPRa, CRISPRi is a variant of CRISPR genetic tool in which dCas9 is fused with a transcriptional repressor to inhibit target gene expression.

RGBiotech offers a series of plasmids that can be used in CRISPR/Cas9 based recombination. We also provide custom construction services to meet the different demands of our customers.

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