Crispam is an allele-specific gRNA design tool that is based on the SNP-derived PAM targeting approach.
The SNP-derived PAM targeting approach allows highly specific targeting of an allele of interest.
Citation info:
“CrisPam: SNP-derived PAM Analysis Tool for Allele-Specific Targeting of Genetic Variants
Using CRISPR/Cas Systems”. Rabinowitz R., Almog S., Darnell R. and Offen D. (2020)
The CRISPR/Cas system can tolerate some mismatches between the crRNA and the target DNA. The bases at the 8th to 13th positions at the 3′ end of the spacer (regarding type II Cas proteins) are termed the seed sequence along with the first base at the 5′ end. Mismatches at the seed sequence are thought to be not tolerated and abolish DNA cleavage. Previous studies have shown that targeting an allele caused by a point mutation by incorporating the variant base within the gRNA is seemingly insufficient, resulting in a non-specific knockdown of both the target allele and the wildtype allele in some proportion [1,2].
An SNP-derived PAM approach overcomes this potential limitation of targeting the disease-causing allele while leaving the wildtype allele intact. This method dramatically increases the specificity of targeting the mutant allele alone by choosing a PAM sequence that is present exclusively within the mutant sequence. Meaning, the point mutation generates the PAM sequence (figure 1) [2,3].
Figure 1: SNP-derived PAM.
When targeting a gene without a particular DNA cleavage location preference, almost any of the Cas enzymes are
optional. However, when targeting a SNP in general, or if utilizing the SNP-derived PAM approach in particular,
the selection of Cas is limited mostly due to the condition of PAM presence in proximity to the SNP or having a
PAM generated by the SNP.
CrisPam is a pythonic code that obtains data of a given SNP, and tests whether it generates a unique PAM
sequence in the DNA of the mutation allele only. The candidate PAMs, 26 PAM sequences of 23 Cas variants
(Table 1), are tested for each given point mutation. The CrisPam Database is a list of SNPs that were found to
be generating at least one PAM.
The CrisPam web tool offers researchers to use their own SNP data (by manually typing DNA sequences,
automatically fetch sequence by rsID or genomic coordinates, or use the batch mode to analyse multiple
sequences) to find the most suitable Cas variant for their allele-specific targeting experiments using the
CRISPR system.
90% of the SNPs checked were found to be “PAM-generators”.
We have tested all human known
pathogenic and likely-pathogenic SNPs (obtained from NCBI dbSNP) using CrisPam. The resulted database
is available (See CrisPam DB page for more information).
Some SNPs are “multiple-PAM generators”; meaning
they generate more than one PAM (e.g. figure 1). For such SNPs, considerations such as delivery vector
capacity may also determine the most suitable Cas protein for the experiment. Some information regarding the
variety of Cas protein is available in the following table:
Table 1: The Cas Repository - 26 unique PAMs of 23 Cas enzymes
It is recommended to use CrisPam as the first step in experiment design. Further off-targets and efficiency predictions of the desired gRNA are required.
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