The original approach from the Jaenisch lab uses two ssoligos and 2 guide RNAs. We were lucky to have obtained one (out of 40 genotyped) mice, though a single base pair deletion outside the CRE binding site was evident; this however did not affect cleavage. I am polling the globe to see who has had success in mice with the original method and if not, which method are you following?
Thanks all
Joe
Hi! Check the following (if you seen already neglect).
CRISPR/CAS GENOME ENGINEERING SYSTEM GENERATES VALUABLE CONDITIONAL MOUSE MODELS
(http://wi.mit.edu/news/archive/2013/crisprcas-genome-engineering-system-generates-valuable-conditional-mouse-models)
Generating rats with conditional alleles using CRISPR/Cas9
(http://www.nature.com/cr/journal/v24/n1/pdf/cr2013157a.pdf)
Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice
(http://ac.els-cdn.com/S221154631400062X/1-s2.0-S221154631400062X-main.pdf?_tid=41b5f628-ea03-11e6-9905-00000aacb360&acdnat=1486121219_6b1ae49fdeb8082d82bdc89d976dbf9d)
One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering
(http://ac.els-cdn.com/S0092867413010167/1-s2.0-S0092867413010167-main.pdf?_tid=856f64da-ea03-11e6-93da-00000aab0f01&acdnat=1486121332_ddfad14a0f7f39866c0eb6303728afb9)
Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes
(http://download.springer.com/static/pdf/714/art%253A10.1186%252Fs12896-016-0234-4.pdf?originUrl=http%3A%2F%2Fbmcbiotechnol.biomedcentral.com%2Farticle%2F10.1186%2Fs12896-016-0234-4&token2=exp=1486121410~acl=%2Fstatic%2Fpdf%2F714%2Fart%25253A10.1186%25252Fs12896-016-0234-4.pdf*~hmac=8b725759f945ddf3ea55c9f3e7f141470ad757f5fdac1cb325b598dfe9fc2ad7)
A Mouse Geneticist’s Practical Guide to CRISPR Applications
(http://www.genetics.org/content/genetics/199/1/1.full.pdf)
Thank you Jetty. I am familiar with all of thee papers; I am looking for anecdotal experiences with making cKO using CRISPR that have not been published.
May be the following or not!!!
A facile one-step strategy for the generation of conditional knockout mice to explore the role of Notch1 in oroesophageal tumorigenesis
(http://www.sciencedirect.com/science/article/pii/S0006291X15310111)
CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678847/pdf/gkv802.pdf)
Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System
(http://link.springer.com/protocol/10.1007%2F978-1-4939-2917-7_16)
Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes
(http://www.biotechniques.com/multimedia/archive/00251/BTN_A_000114339_O_251010a.pdf)
Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes
(http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0136690&type=printable)
4 DEVELOPMENTAL OUTCOMES AND EFFICIENCY OF TWO CRISPR/Cas9 MICROINJECTION METHODS IN BOVINE ZYGOTES
(http://www.publish.csiro.au/rd/Fulltext/RDv27n1Ab4)
Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing
(file:///C:/Users/Jetty/Downloads/srep11315.pdf)
ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes (attached)