Designing sgRNA using CRISPR Protocol

What is CRISPR/Cas9?

Until now, there are three generations of gene editing tools have been developed, Zinc Finger Nuclease (ZFN), Transcription Activator Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats associated systems (CRISPR/Cas9). As the great simplicity and high cutting efficiency, CRISPR/Cas9 systems has been applied most widely among them.

how to deisgn sgrna for your crispr ssl bdl ssl 01 (1) Adapted from Nature Protocols, ZhangFeng

As the above cartoon shown, CRISPR/Cas9 system is consisted of a Cas9 protein and a single guided RNA (sgRNA). The sgRNA could be divided into two parts: a 20 nucleotides targeting sequence and a scaffold sequence. The 20-nt sequence completely pairs to the genomic DNA and the scaffold RNA which is essential for Cas9 recognizing with sgRNA helps Cas9 bind to genome. For this reason, Cas9 could recognize target sequence and mediate a Double Stranded Breaks (DSB) that located nearly 3 base pairs upstream of Protospacer Adjacent Motif (PAM) sequence. Actually, PAM acts as the specific requirement for CRISPR/Cas9 systems that varies from different Cas9 orthologs, for example, PAM of S.Pyogenes Cas9 is 5’-NGG-3’ and Neisseria meningiditis Cas9 is 5’-NNNNGATT-3’. After DSBs are caused, Gene edition, no matter disruption or replacement, will be finished by gene repair mechanisms.

how to deisgn sgrna for your crispr ssl bdl ssl 01 (2) Adapted from Nature Protocols, ZhangFeng

As shown in the above picture, there are two main gene repair mechanisms: Non-Homologous End Joining (NHEJ) which leads to arbitrary mutations and disruptions (insertions and deletions, briefly indels) and Homologous Directed Repair (HDR) that requires the present of homologous templates and replace inherent genes with foreign genes. For gene knockout, we could utilize the NHEJ mechanism to cause indels in DSB to disrupt or silence the gene. For gene knockin, HDR mechanism may help exogenous gene integration.

Websites for sgRNA design

In the CRISPR/Cas9 system, it is crucial to choose a workable and effective sgRNA. Selection of sgRNAs for an experiment needs to balance two aspects: maximizing on-target activity while minimizing off-target activity, which sounds obviously but can often require thorough decisions. For example, would it be better to use a less-active sgRNA that targets a truly unique site in the genome, or a more-active sgRNA with one additional target site in a region of the genome with unknown function? Fortunately, we could use next websites to help us to design and score sgRNAs. The analysis function could help us to make final decision.

Example gene design (by using MIT website: http://crispr.mot.edu/)

First step: find your interested gene in NCBI and fulfill the basic information

how to deisgn sgrna for your crispr ssl bdl ssl 01 (3)

Tips: Do not forget to choose species, human, mouse or others. sgRNAs are scored by the likelihood of off target binding with the species genome.

Second step: Submit your sequence (250bp from the beginning of target gene)

how to deisgn sgrna for your crispr ssl bdl ssl 01 (4)

Tips: In this website, only 250bp could be submitted for each time. It is solvable to separate your sequence into several times. More important, for Gene knockout, we always use the upstream sequence close to 5’ terminal, because this part of sequence is much more important for protein translation than others.

Third step: select sgRNAs from the calculating results

how to deisgn sgrna for your crispr ssl bdl ssl 01 (5)

Tips: we normally choose sgRNAs whose score is higher than 90 or we choose 3-5 sgRNAs candidates with highest score.

* For research use only. Not intended for any clinical use.

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Designing sgRNA using CRISPR Protocol

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