Increasing the accuracy of genome editing

The Chinese gene editing of human embyros does not appear to have been using the latest techniques for maximum accuracy.

CRISPR-Cas9 is a powerful new tool for editing the genome. For researchers around the world, the CRISPR-Cas9 technique is an exciting innovation because it is faster and cheaper than previous methods. Now, using a molecular trick, Dr. Van Trung Chu and Professor Klaus Rajewsky of the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch and Dr. Ralf Kühn, MDC and Berlin Institute of Health (BIH), have found a solution to considerably increase the efficiency of precise genetic modifications by up to eightfold.

Many researchers, including Van Trung Chu, Klaus Rajewsky and Ralf Kühn, are seeking to promote the HDR repair pathway to make gene modification in the laboratory more precise in order to avoid editing errors and to increase efficiency. The MDC researchers succeeded in increasing the efficiency of the more precisely working HDR repair system by temporarily inhibiting the most dominant repair protein of NHEJ, the enzyme DNA Ligase IV. In their approach they used various inhibitors such as proteins and small molecules.

“But we also used a trick of nature and blocked Ligase IV with the proteins of adeno viruses. Thus we were able to increase the efficiency of the CRISPR-Cas9 technology up to eightfold,” Ralf Kühn explained. For example, they succeeded in inserting a gene into a predefined position in the genome (knock-in) in more than 60 per cent of all manipulated mouse cells. Kühn has just recently joined the MDC and is head of the research group for “iPS cell based disease modeling”. Before coming to the MDC, he was on the research staff of Helmholtz Zentrum München. “The expertise of Ralf Kühn is very important for gene research at MDC and especially for my research group,” Klaus Rajewsky said.

Nature Biotechnology – Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a ‘traffic light’ and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4–5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells

Double Nicking to avoid off target changes

Cell – Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity

Highlights

•Cas9 nickase can facilitate targeted DNA double-strand break using two guide RNAs
•Double nicking of DNA reduces off-target mutagenesis by 50- to 1,000-fold
•Multiplex nicking stimulates homology directed repair, microdeletion, and insertion
•Double nicking provides efficient modification of mouse zygotes

Summary

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

Genome germline engineered mice have gotten to 100% accuracy for the pups that were selected to be implanted and born.

There is other work to use computer algorithms to optimize the accuracy and targeting of Crispr gene editing

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Increasing the accuracy of genome editing

The Chinese gene editing of human embyros does not appear to have been using the latest techniques for maximum accuracy.

CRISPR-Cas9 is a powerful new tool for editing the genome. For researchers around the world, the CRISPR-Cas9 technique is an exciting innovation because it is faster and cheaper than previous methods. Now, using a molecular trick, Dr. Van Trung Chu and Professor Klaus Rajewsky of the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch and Dr. Ralf Kühn, MDC and Berlin Institute of Health (BIH), have found a solution to considerably increase the efficiency of precise genetic modifications by up to eightfold.

Many researchers, including Van Trung Chu, Klaus Rajewsky and Ralf Kühn, are seeking to promote the HDR repair pathway to make gene modification in the laboratory more precise in order to avoid editing errors and to increase efficiency. The MDC researchers succeeded in increasing the efficiency of the more precisely working HDR repair system by temporarily inhibiting the most dominant repair protein of NHEJ, the enzyme DNA Ligase IV. In their approach they used various inhibitors such as proteins and small molecules.

“But we also used a trick of nature and blocked Ligase IV with the proteins of adeno viruses. Thus we were able to increase the efficiency of the CRISPR-Cas9 technology up to eightfold,” Ralf Kühn explained. For example, they succeeded in inserting a gene into a predefined position in the genome (knock-in) in more than 60 per cent of all manipulated mouse cells. Kühn has just recently joined the MDC and is head of the research group for “iPS cell based disease modeling”. Before coming to the MDC, he was on the research staff of Helmholtz Zentrum München. “The expertise of Ralf Kühn is very important for gene research at MDC and especially for my research group,” Klaus Rajewsky said.

Nature Biotechnology – Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a ‘traffic light’ and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4–5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells

Double Nicking to avoid off target changes

Cell – Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity

Highlights

•Cas9 nickase can facilitate targeted DNA double-strand break using two guide RNAs
•Double nicking of DNA reduces off-target mutagenesis by 50- to 1,000-fold
•Multiplex nicking stimulates homology directed repair, microdeletion, and insertion
•Double nicking provides efficient modification of mouse zygotes

Summary

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

Genome germline engineered mice have gotten to 100% accuracy for the pups that were selected to be implanted and born.

There is other work to use computer algorithms to optimize the accuracy and targeting of Crispr gene editing

Subscribe on Google News