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Cas12a Activity in Detecting Sickle Cell Anemia
Figure 1: Successful Cas12a Activity & Sickle Cell gRNA design:
Fragments observed in lanes 2 and 3 display that EnGen® Lba Cas12a (Cpf1) was successfully activated as the expected binding of template Cas12a occurred with the aid of gRNA and hence initiated the indiscriminate cleavage activity of the Cas12a. Moreover, this shows that optimized gRNA for the rs334_C mutation and the HBB_WT is effective as it gets used by Cas12a to target the gene of interest. In addition, in the control samples, lanes 4,5,6, no fragmentation was observed confirming that the lack of gRNA prevents the activation of the indiscriminate cleavage activity of Cas12a since there will be no binding. Nevertheless, while fragmentation was expected in lane 1 due to the Cas12a targeting the s334_T mutation in the HBB gene, none was observed. Experimental techniques and PAM sequence editing were taken in consideration and altered
Figure 2: Electropherogram of Sickle Cell Anemia Samples
As shown in the graphs, the major peaks in samples 5 and 6 which represents the template DNA for HBB_WT and HBB_rs334_C mutation respectively are fragmented into smaller peaks upon the addition of gRNA. The smaller fragments (circled in sample 2 and 3) reflect the activity of Cas12a upon detecting the desired sequence and fragementing the template DNA.
Figure 3: Replicate for validation of Cas12a Activity and Sickle Cell Anemia gRNA Design
Fragmentation was observed for lanes 2 and 6 corresponding to a successful Cas12a Activity as it uses the respective gRNAs to target the DNA template. The control samples lacking the gRNA showed no fragmentation in lanes 1,3,and 5 indicating no Cas12a activity due to its inability to bind. As for lane 4 where fragmentation was expected, nothing appeared. From the replicate we confirmed that there seems to be a low affinity binding between the Cas12a and the rs334_T gRNA due to the PAM site specificity.
Tóth, E., Czene, B. C., Kulcsár, P. I., Krausz, S. L., Tálas, A., Nyeste, A., … Welker, E. (2018). Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants. Nucleic acids research, 46(19), 10272–10285. doi:10.1093/nar/gky815
Cas12a Activity in Detecting Cystic Fibrosis
Figure 1 : Successful Cas12a Activity & Cystic Fibrosis gRNA design:
EnGen® Lba Cas12a (Cpf1) was added to the reaction tube containing DNA template and a corresponding gRNA to analyze the indiscriminate cleavage activity of Cas12a. Fragments observed in lanes 2 and 4 display that Cas12a was successfully activated as the expected binding of template Cas12a occurred with the aid of gRNA. To test for off-target binding, we mixed Cystic fibrosis WT DNA template and the gRNA corresponding to the Cystic Fibrosis G mutation in lane 5, and as expected there was no fragmentation observed which highlights the accuracy of the detection of the s75389940_G SNP
Note: Agilent Bioanalyzer was used to determine activity instead of an agarose gel for higher sensitivity of fragment detection by capillary migration.
Figure 2: Electropherogram of Cystic Fibrosis Samples
Results show that that the DNA Template of (134 bp) of the WT Cystic Fibrosis CFTR gene and rs75389840_G SNP had an intact peak shown between 50 and 55 ss in the control samples (samples 1 and 3) where no gRNA was used. Then, in samples 2 and 4, the peak shown in the control samples is fragmented to several peaks (circled) to reflect fragmentation due to Cas12a activity. In Sample 5 where WT_CF template is paired with a wrong guide (gRNA_rs75389940_G), the peak remained intact as expected.
Note: Agilent Bioanalyzer was used to determine activity instead of an agarose gel for higher sensitivity of fragment detection by capillary migration.
Figure 3: Replicate for validation of Cas12a Activity and Cystic Fibrosis gRNA Design
Fragments observed in lanes 1 and 2 display that EnGen® Lba Cas12a (Cpf1) was successfully activated as the expected binding of template Cas12a occurred with the aid of gRNA and hence initiated the indiscriminate cleavage activity of the Cas12a. On the other hands, lanes 4 and 5 showed no fragmentation as there was no gRNA added to help the Cas12a bind to the DNA template and become active. The off-target binding was proven to be almost null again as there is no fragmentation observed, hence confirming that a mismatch of gRNA and DNA template does not cause binding, and hence false results.
Figure 4: Electropherogram of Cystic Fibrosis Samples Replicate
Successful Replication of the Cas12a activity is observed here where the results are similar to those from Figure 2. Lanes 1 and 2 display that Cas12a was successfully activated due to the presence of the corresponding gRNA . On the other hand the control samples 4 and 5 had an intact peak reflecting no fragmentation and thus no Cas12a activity. Similarly, the peak in the mismatch (off-target) testing remained intact.
pY016 Transformation & Cas12a Purification
Figure 1. Cas 12 produced by BL21 cells measured by western blot.
The BL21 cells were inoculated and grew to the mid-log phase (OD 30 Kletts) in LB+Amp+Chl medium when it was induced by IPTG for 18 hours. The proteins were harvested by being centrifuged by 5000 rpm for 5 minutes. The western blot shows Cas12 protein was successfully produced(circled in red).
Cas 12 proteins were purified using the Pierce® Anti-HA Agarose column(Thermo Fischer Scientific 26181). Cas 12 proteins were then eluted using TBS. Different fractions and elutions were collected.
Our spectroscopy shows that E1, E2 and P have the highest amount of protein(5.495mg/ml, 2.040mg/ml and 2.147mg/ml). Samples of the highest protein concentration were loaded on the 12.5% SDS-PAGE Gel and ran at 100V for 1h.
Results showed that one band was observed in the gel, which corresponds to the molar weight of 150kDal from elution 1, elution 2, indicating the presence of purified Cas 12 protein in our sample.
Figure 4: Electropherogram of Cystic Fibrosis Samples Replicate:
Successful Replication of the Cas12a activity is observed here where the results are similar to those from Figure 2. Sample 2 and 6 display that Cas12a was successfully activated due to the fragmentation (circled) of the major peak (as indicated by the arrow in samples 1 and 5) . The major peak in samples 1 and 5 remained intact reflecting no no Cas12a activity.
Figure 1-2:
After transforming and Purifying Cas12a in lab, we measured the activity of the purified product using Bioanalyzer Agilent Biotechnologies .
As shown, lanes 2,6, and 8 containing Cas12a, template DNA, and the corresponding gRNA displayed activation of the Cas12a. The appearance of these fragments not only serves as a third replicate for our mechanism but it also proves successful purification of the Cas12a. Although, lanes 4 and 10 containing the CF_G and HBB_rss34_T reactions mix displayed no fragments despite the expected results, the controls and the lanes with the fragments hold true in which controls’ peaks remained intact.
*Note : Both EnGen® Lba Cas12a (Cpf1) and the purified Cas12a failed to detect the HBB_rs334_T.
DNAse Alert Kit Fluorescent Testing
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