• MeSH
• alely MeSH
• CRISPR-Cas systémy * MeSH
• editace genu MeSH
• myši MeSH
• protein Cas9 * metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• dopisy MeSH
• komentáře MeSH

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.

• MeSH
• biologické modely MeSH
• buněčná diferenciace * MeSH
• CRISPR-Cas systémy genetika   MeSH
• delece genu MeSH
• flagella enzymologie   MeSH
• Leishmania mexicana cytologie   enzymologie   MeSH
• leishmanióza parazitologie   patologie   MeSH
• mutace genetika   MeSH
• myši inbrední BALB C MeSH
• protein Cas9 metabolismus   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• proteom metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

• MeSH
• alely * MeSH
• blastocysta metabolismus   MeSH
• CRISPR-Cas systémy genetika   MeSH
• faktorová analýza statistická MeSH
• mikroinjekce MeSH
• myši knockoutované MeSH
• protein 2 vázající methyl-CpG genetika   metabolismus   MeSH
• protein Cas9 metabolismus   MeSH
• regresní analýza MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH