Gene Targeting

To deliver gene targeting capabilities including the production of transgenic mouse models.

The gene targeting facility aims to enable Babraham Institute scientists to carry out the latest genome editing methodologies. CRISPR/Cas9 allows for precise cleavage of double stranded DNA, these breaks are then repaired by various pathways. For example, a double stranded break (DSB) within a protEIn coding region will be repaired via non-homologous end joining (NHEJ) and will introduce an indel which can lead to a gene knock-out (KO). Thus, manipulating the genome has never been so accessible.

Key services

• Generation of bespoke mouse models; ranging from single nucleotide polymorphisms (SNP), deletion of certain regions and various tags such mAID.
• Guidance in CRISPR/Cas9-based technologies for creating your gene edited cell-line from design and implementation such as using electroporation via NEPA21.
• Generation of transgenic mice carrying large knock-ins introduced via PiggyBAC
• High-throughput production of single-guide RNA with efficiency validated in blastocysts.
• Designing strategies for Gibson assembly and recombineering in bacterial artificial chromosomes (BACs) for complex constructs.
• High-throughput screening of gene edited cell lines via Next Generation Sequencing.

Expertise & advice

We offer expertise on the use of CRISPR/Cas9 to achieve your desired gene edit. We provide advice on reagents based on mechanisms of DNA repair such as homology directed repair (HDR) or homologous recombination (HR). Additionally, we also advise on the optimal and most cost effective strategy to genotype clones or pools, as genotyping is the most crucial step. When gene targeting projects have been unsuccessful, we can help with troubleshooting or suggest alternative strategies.

Facility achievements

• Engineered triple gene KO embryos
• Introduced mAID and mini-turbo tags onto the end of genes
• Validated PCV-Cas9 in embryos and showed improved HDR rates

Contact:

Asif Nakhuda - Gene Targeting Specialist
asif.nakhuda@babraham.ac.uk