The research team in CSIR-IGIB, Delhi led by Dr Debojyoti Chakraborty has characterized a naturally occurring Cas9 from Francisella novicida (FnCas9), which has negligible off-target binding and can correct a sickle cell anaemia mutation in patient-derived induced pluripotent stem cells.
Aswini | 19 October 2019
Genome editing is a technique to make specific changes to a desired region of the genome by adding, deleting or altering the genetic material. It has helped researchers in multiple ways to understand various biological functions. Numerous studies have shown promising results in its ability to treat or prevent various diseases and genetic conditions like cystic fibrosis, sickle cell disease and complex diseases like cancer and HIV infections. Of the different approaches developed for genome editing, CRISPR-Cas9 is a rapidly emerging technique that has excited researchers because it is fast, cheap, accurate and efficient in performing genome editing. However, the safety and efficiency of using CRISPR-Cas9 system in humans is still debatable due to its off-targeting effects.
The research team in CSIR-IGIB, Delhi led by Dr Debojyoti Chakraborty and Souvik Maiti has characterized a naturally occurring Cas9 from Francisella novicida (FnCas9) with regards to its genome editing properties. According to their study, unlike the commonly used SpCas9, FnCas9 has negligible binding affinity to off-targets, making it highly specific in target recognition. They also show that FnCas9 can direct homology-directed repair (HDR) at a higher rate compared to the normal SpCas9. “These two are the most important aspects of our work”, says Sundaram Acharya, one of the first authors of the paper. To extend their observations in a therapeutic scenario, they have used FnCas9 to edit and correct a sickle cell anaemia mutation in patient-derived induced pluripotent stem cells (iPSCs). They found higher events of HDR in the FnCas9 system compared to the SpCas9, demonstrating its potential in genome editing of disease-causing mutations.
Like every success story, this study is an outcome of the endurance of multiple rounds of failures and disappointments. Dr Chakraborty said, “We were looking for a Cas9 that could target RNA. There was a report that showed the ability of FnCas9 to target viral RNA. However, it didn’t work out well for us.” As the saying goes, ‘Failure is simply the opportunity to begin again. This time more intelligently’, after heaps of negative results they decided to investigate the ability of FnCas9 to target DNA, since its precision in gene correction, was unknown at that time. But the dark swamp of despair was still covered in the project while they were struggling to detect genome editing in mammalian cells. Acharya says, “It was a eureka moment for us when we found that FnCas9 can work in mammalian cells quite efficiently contrary to its inefficient editing outcomes reported earlier and most importantly this is the first hint of HDR-meditated editing by FnCas9”.
When asked about the precision of FnCas9, Dr Chakraborty says, “FnCas9 has evolved separately from the other Cas9 systems. It has distinct structural attributes, particularly with respect to DNA recognition which might lead to its specificity in targeting.” FnCas9 belongs to type II-B Cas system. Exploring more Cas9 belonging to this family may give us better genome editing Cas9 proteins. He also says, “FnCas9 can be structurally engineered to make them competent for base editing, which would be an attractive therapeutic application for monogenic disorders where the current generation of base editors have so far shown variability in off-targeting”. However, FnCas9 has a drawback as well. Its large size may hinder its efficient delivery into cells.
The lab is currently proceeding into pre-clinical studies in establishing the efficiency of FnCas9 for genome-wide binding and targeting using patient-derived cells. “Our goal is to take FnCas9-based genome editing tool 'from bench to bedside'!” says Acharya. Dr Chakraborty from a background of lncRNA biology is now excelling in CRISPR-Cas9 biology. Their team has put a tremendous effort in this study which would probably open new avenue into the field of therapeutic genomic editing. This work contributes to some of the promising usages of CRISPR in humans for therapeutic genomics.
This work was published in PNAS in October 2019. The article is available online at https://doi.org/10.1073/pnas.1818461116
Aswini | 19 October 2019
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| Art by Manoj Kumar (CSIR-IGIB) |
The research team in CSIR-IGIB, Delhi led by Dr Debojyoti Chakraborty and Souvik Maiti has characterized a naturally occurring Cas9 from Francisella novicida (FnCas9) with regards to its genome editing properties. According to their study, unlike the commonly used SpCas9, FnCas9 has negligible binding affinity to off-targets, making it highly specific in target recognition. They also show that FnCas9 can direct homology-directed repair (HDR) at a higher rate compared to the normal SpCas9. “These two are the most important aspects of our work”, says Sundaram Acharya, one of the first authors of the paper. To extend their observations in a therapeutic scenario, they have used FnCas9 to edit and correct a sickle cell anaemia mutation in patient-derived induced pluripotent stem cells (iPSCs). They found higher events of HDR in the FnCas9 system compared to the SpCas9, demonstrating its potential in genome editing of disease-causing mutations.
Like every success story, this study is an outcome of the endurance of multiple rounds of failures and disappointments. Dr Chakraborty said, “We were looking for a Cas9 that could target RNA. There was a report that showed the ability of FnCas9 to target viral RNA. However, it didn’t work out well for us.” As the saying goes, ‘Failure is simply the opportunity to begin again. This time more intelligently’, after heaps of negative results they decided to investigate the ability of FnCas9 to target DNA, since its precision in gene correction, was unknown at that time. But the dark swamp of despair was still covered in the project while they were struggling to detect genome editing in mammalian cells. Acharya says, “It was a eureka moment for us when we found that FnCas9 can work in mammalian cells quite efficiently contrary to its inefficient editing outcomes reported earlier and most importantly this is the first hint of HDR-meditated editing by FnCas9”.
When asked about the precision of FnCas9, Dr Chakraborty says, “FnCas9 has evolved separately from the other Cas9 systems. It has distinct structural attributes, particularly with respect to DNA recognition which might lead to its specificity in targeting.” FnCas9 belongs to type II-B Cas system. Exploring more Cas9 belonging to this family may give us better genome editing Cas9 proteins. He also says, “FnCas9 can be structurally engineered to make them competent for base editing, which would be an attractive therapeutic application for monogenic disorders where the current generation of base editors have so far shown variability in off-targeting”. However, FnCas9 has a drawback as well. Its large size may hinder its efficient delivery into cells.
The lab is currently proceeding into pre-clinical studies in establishing the efficiency of FnCas9 for genome-wide binding and targeting using patient-derived cells. “Our goal is to take FnCas9-based genome editing tool 'from bench to bedside'!” says Acharya. Dr Chakraborty from a background of lncRNA biology is now excelling in CRISPR-Cas9 biology. Their team has put a tremendous effort in this study which would probably open new avenue into the field of therapeutic genomic editing. This work contributes to some of the promising usages of CRISPR in humans for therapeutic genomics.
This work was published in PNAS in October 2019. The article is available online at https://doi.org/10.1073/pnas.1818461116