Our results provide a framework for designing high-fidelity CRISPR-based knock-in experiments, in both research and therapeutic settings.īreast cancer brain metastases (BCBM) have a 5-20 year latency and account for 30% of mortality however, mechanisms governing adaptation to the brain microenvironment remain poorly defined. Finally, by comparing ssDNA donors with equivalent dsDNA sequences (PCR products or plasmids), we demonstrate that ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration. We next define a set of rules to maximize the efficiency of ssDNA-mediated knock-in by optimizing donor design.

Here, we describe an optimized method for the synthesis of long ssDNA templates and demonstrate that ssDNA donors can drive efficient integration of gene-sized reporters in human cell lines. While both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) forms of donors have been used as repair templates, a systematic comparison of the performance and specificity of repair using ssDNA versus dsDNA donors is still lacking. In particular, homology-directed repair after genomic cleavage allows for precise modification of genes using exogenous donor sequences as templates. CRISPR/Cas technologies have transformed our ability to manipulate genomes for research and gene-based therapy. The conclusions of this pre-print are outdated. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin.

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype.

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