Easi-CRISPR: Efficient germline modification with long ssDNA donors

Abstract

CRISPR/Cas9 technology efficiently produces short insertions or deletions (indels) and can insert short exogenous sequences at Cas9 cut sites. However, targeting long inserts is still a major technical challenge. To overcome this challenge, we developed Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a method that uses long, in vitro-synthesized, single-stranded DNAs with 50-100 base homology arms as repair templates. We demonstrate that Easi-CRISPR can generate knock-in and floxed alleles in mice with an efficiency at many loci as high as 100%. The simple design requirements for donor DNAs and the reproducibly high-efficiency of Easi-CRISPR enables rapid development of many types of commonly used animal and cell models.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

In situgenome editing method suitable for routine generation of germline modified animal models

“…We also successfully inserted a long donor fragment into the targeted locus using the i-GONAD method. Long ssDNA donors were prepared using the ivTRT method described in our highly efficient knock-in method, "Easi-CRISPR" [18][19][20] . Since a large amount of ssDNA is required for the i-GONAD experiment, we used the spin column-based nucleic acid purification instead of gel purification where loss of sample recovery is very high.…”

“…We previously demonstrated that knocking-in of longer sequences can be efficiently achieved using ssDNA donors [18][19][20] . We tested whether long ssDNA donors can be used with i-GONAD to create knock-in alleles.…”

The amphipod crustacean Parhyale hawaiensis: An emerging comparative model of arthropod development, evolution, and regeneration

“…Within the arthropods, crustaceans offer an interesting opportunity for Hox gene analysis, as they exhibit immense diversity, particularly in regards to limb morphology, yet have body regions that can be readily homologized (VanHook & Patel, ), at least within some clades. Crustacean Hox gene expression domains correlate with the presence of particular limb types, suggesting that AP body regionalization is controlled by Hox expression.…”

AIDing‐targeted protein degradation in Drosophila

“…This is likely to be tolerated more easily within a complex protein compared with GFP and it might also be simpler to insert it into the genome using CRISPR. In the future, it will be interesting to determine if providing a long single‐strand DNA oligo (coding for the AID degron) as a simple donor for CRISPR will indeed result in very high efficiencies as reported for small genome manipulations . This may pave the way to AID‐targeted protein degradation in Drosophila going large scale.…”