Precise Insertions Research Articles

Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations

Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.

Ultrasound-Assisted Technology Versus the Conventional Landmark Location Method in Spinal Anesthesia for Cesarean Delivery in Obese Parturients: A Randomized Controlled Trial

(Anesth Analg. 2019;129(1):155–161) For cesarean deliveries, spinal anesthesia is typically performed with the use of manual palpation. However, some patients present challenges to this method, particularly patients with imperceptible surface landmarks or those who are obese. Because spinal anesthesia placement may be more difficult in these patients, there is an increased likelihood of postdural puncture headache, paresthesia, or spinal hematoma. It has been proposed that preprocedure ultrasound scanning can assist care providers with spinal anesthesia placement, but not much research has been done on this subject. These investigators conducted their study to determine whether lateral-positioned ultrasound scanning would result in more precise needle insertions than a blind manual palpation technique for obese patients.

True Homoplasy of Retrotransposon Insertions in Primates.

How reliable are the presence/absence insertion patterns of the supposedly homoplasy-free retrotransposons, which were randomly inserted in the quasi infinite genomic space? To systematically examine this question in an up-to-date, multigenome comparison, we screened millions of primate transposed Alu SINE elements for incidences of homoplasious precise insertions and deletions. In genome-wide analyses, we identified and manually verified nine cases of precise parallel Alu insertions of apparently identical elements at orthologous positions in two ape lineages and twelve incidences of precise deletions of previously established SINEs. Correspondingly, eight precise parallel insertions and no exact deletions were detected in a comparison of lemuriform primate and human insertions spanning the range of primate diversity. With an overall frequency of homoplasious Alu insertions of only 0.01% (for human-chimpanzee-rhesus macaque) and 0.02-0.04% (for human-bushbaby-lemurs) and precise Alu deletions of 0.001-0.002% (for human-chimpanzee-rhesus macaque), real homoplasy is not considered to be a quantitatively relevant source of evolutionary noise. Thus, presence/absence patterns of Alu retrotransposons and, presumably, all LINE1-mobilized elements represent indeed the virtually homoplasy-free markers they are considered to be. Therefore, ancestral incomplete lineage sorting and hybridization remain the only serious sources of conflicting presence/absence patterns of retrotransposon insertions, and as such are detectable and quantifiable. [Homoplasy; precise deletions; precise parallel insertions; primates; retrotransposons.].

Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases

Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. Both nucleases, in the presence of a guide RNA and repairing DNA template flanked by homology DNA fragments to the target site, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertion for these Cpf1 nucleases, up to 8%, is higher than most other genome editing nucleases, indicative of its effective enzymatic chemistry. Further refinements and broad adoption of the Cpf1 genome editing technology have the potential to make a dramatic impact on plant biotechnology.

Abstract 71: Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair

Abstract The comprehensive and coordinated efforts of The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) identified the somatic genomic alterations occurring in a broad panel of human cancers. The challenge ahead is to understand the functions of these somatically altered genes.Genome editing tools like the CRISPR/Cas9 system represent a transformative technology that allows the precise engineering of cells harboring specific (point) mutations found in specific tumor subtypes, enabling the investigation of the role of a candidate cancer gene in a stringently defined genetic context. Once recruited to its DNA target sequence in cells, Cas9 induces a DNA double strand break (DSB), which is either repaired by the non-homologous end-joining pathway (NHEJ), or by homology-directed repair (HDR). The HDR pathway allows for a precise site-specific knock-in and thus functional analysis of cancer-associated sequence variants. However, HDR activity is restricted to the late S and G2 phases of the cell cycle, while the NHEJ pathway dominates DNA repair throughout the cell cycle. Thus, the error-prone NHEJ pathway out-competes the HDR pathway, which reduces the likelihood for precise insertions, deletions or base substitutions at the DSB. Here, we devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1 resulting in a cell cycle tailored expression with low levels in G1, but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the relative rate of HDR by up to 87% at a single-copy reporter locus, and up to 42% at the endogenous MALAT1 locus compared to wild-type Cas9. In summary, our regulatory concept takes advantage of cell-autonomous pathways to enable fluctuation of Cas9 protein levels coupled to cell cycle dynamics. This resulted in higher site-specific integration events without sophisticated manipulation of cells by small molecules or additional gene targeting reagents. Future methodological developments may enable high-resolution expression of Cas9 and alternative genome engineering proteins like Cpf1 by combining cell cycle phase-specific transcriptional and post-translational regulatory elements. This might enhance HDR efficiencies even further, and will help to make sense of cancer genomic data. Citation Format: Tony Gutschner, Monika Hämmerle, Giannicola Genovese, Giulio F. Draetta, Lynda Chin. Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 71.

Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome

ABSTRACTModifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1) to precisely generate genomic deletions; (2) to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3) to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the “ends-in” recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair); either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5 kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome.

A method for generating precise gene deletions and insertions in Escherichia coli

A simple and general method for disrupting chromosomal genes and introducing insertions is described. This procedure involves eliminating wild-type bacterial genes and introducing mutant alleles or other insertions at the original locus of the wild-type gene. To demonstrate the utility of this approach, the tig gene of Escherichia coli was replaced by homologous recombination with a cassette containing the chloramphenicol resistance gene and the sacB gene. The cassette was then removed and the tig mutant alleles were moved into the native tig location. Sequencing and Western blotting results demonstrated that insertions or deletions can be introduced precisely in E. coli using our approach. Our system does not require extra in vitro manipulations such as restriction digestion or ligation, and does not require use of specific plasmids or strains which are used to prevent false positive transformants caused by template plasmid transformation. This technique can be used widely in bacterial genome analysis.

Distinct roles for sequences upstream of and downstream from Physarum editing sites

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within approximately 9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.

Anatomy of the intrinsic hand muscles revisited: part II. Lumbricals.

The authors discuss the anatomic variations and the precise origins and insertions of the lumbrical muscles, after dissecting 14 fresh cadavers (56 lumbrical muscles) and reviewing the scant published literature. They compare the findings with those of other investigators, and they describe the lumbrical muscle insertions discovered during the dissections. The authors conclude that descriptions of the origins and insertions of the lumbrical muscles have been oversimplified or not investigated at all, that lumbricals originate variably from the flexor digitorum profundus tendon and may even be bipinnate, and that three different major destinations exist for distal insertion of these intrinsic muscles: bone, volar plate, and extensor apparatus. Previous descriptions have focused only on the lumbrical continuation to the lateral band of the extensor mechanism.

Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.

Impact ofBacillus thuringiensis on pest control with emphasis on genetic manipulation

This paper will bringBacillus thuringiensis (B.t.) into scientific and industrial perspective, then look at the future, particularly at genetics. Since its discovery in 1911,B.t. has had fluctuating impacts on science and on the pest control industry. In the late 1950s, impact on industry was expected because of the great advantages possessed byB.t.. These include its safety for man and wild life, specificity to important groups of insect pests and rapid action due to the unique crystals of toxic protein formed at sporulation. Toxins in these crystals were new to science. However, many constraints—largely unrealised—resulted in its usage increasing unevently, although exponentially, to its present level of several thousand tons per year. Among the most important constraints are its failure to spread in insect infestations and the action of the crystal toxins only as larvicidal stomach poisons, so that application has to be even and frequent. The moderate and variable potency of early products made their use expensive. By 1972, wide ranging research into constraints increased efficacy ofB.t. byca. 100-fold against the commonest assay species,Trichoplusia ni. Industry adopted one very good strain, HD-1. Later, an organised International Selection Programme revealed strains up to 60-fold more potent than HD-1 against certain pests. Studies on the chemistry and modes of action of the crystal toxins explained many differences, indicating how genetic manipulation might lead to dramatic progress. Up to 1980, manipulation ofB.t. was laborious. In two years, single genes encoding toxins were found on plasmids and a conjugation-like process was discovered that effected plasmid exchange at high frequencies, so recombinants could be screened directly by insect bioassay. Strains were tailored with greater potency and/or a better host range than HD1: some were patented. Progress inB.t. has been almost comparable to that in the production of antibiotics. Genetic engineering withB.t. began with the cloning and expression of toxin gene in other bacteria. From these, it can be inserted directly into bacteria inhabiting plant rhizospheres, with the aim of killing root feeding caterpillars. Using further vectors, a gene has been transferred to and expressed in the tobacco plant, making the plant toxic to caterpillars. In future, sequencing ofB.t. DNA should permit more precise insertions into more microorganisms and plants. The very powerful promoter sequence of the toxin gene might be used to improve yields of products of other bacteria beyond the realm of insect control. Recent phases ofB.t. research have had strong impact on science, and industry should increasingly reap benefits from it. However, there are many problems in the developent of industrial products and some ideas may fail at various stages.