Length Of Homology Research Articles

Insight into RecA-mediated repair of double strand breaks is provided by probing how contiguous heterology affects recombination

Homologous recombination can promote correct repair of double strand breaks (DSB) in DNA by aligning a sequence region in the broken chromosome with the corresponding sequence region in an unbroken chromosome. D-loops join the broken and unbroken chromosomes during homology testing. Previous work studied how some mismatches affect the stability of D-loops, but they did not probe whether the D-loops disrupt regions of contiguous mismatches or simply bypass them. Furthermore, previous work has not considered how the length of flanking homology affects D-loop disruption of regions of contiguous mismatches. Finally, there are conflicts about the polarity of D-loop extension. We demonstrate that with or without ATP hydrolysis invading strands with 6 contiguous mismatches and sufficient flanking homology readily form D-loops that disrupt the structure of the mismatched region and incorporate both flanking homologous regions. Unsurprisingly, the probability that D-loops will incorporate both flanking homologous regions decreases as the number of mismatched bases increases. Furthermore, though D-loops may progress through homologous regions initially and dominantly in the 5′ to 3′ direction with respect to the single strand in the broken chromosome, our results suggest that progress through contiguous mismatches proceeds dominantly in the 3′ to 5′ direction. These results may reconcile previous conflicts about the polarity of D-loop extension. Additionally, the results suggest that homology recognition is not characterized by any simple iterative decision tree model that considers each homology testing step separately. Instead, homology recognition involves collective interactions. Finally, we consider implications for DSB repair.

Peptide Nucleic Acid-Mediated Regulation of CRISPR-Cas9 Specificity.

Although CRISPR-Cas9 gene therapies have proven to be a powerful tool across many applications, improvements are necessary to increase the specificity of this technology. Cas9 cutting in off-target sites remains an issue that limits CRISPR's application in human-based therapies. Treatment of autosomal dominant diseases also remains a challenge when mutant alleles differ from the wild-type sequence by only one base pair. Here, we utilize synthetic peptide nucleic acids (PNAs) that bind selected spacer sequences in the guide RNA (gRNA) to increase Cas9 specificity up to 10-fold. We interrogate variations in PNA length, binding position, and degree of homology with the gRNA. Our findings reveal that PNAs bound in the region distal to the protospacer adjacent motif (PAM) site effectively enhance specificity in both on-target/off-target and allele-specific scenarios. In addition, we demonstrate that introducing deliberate mismatches between PNAs bound in the PAM-proximal region of the gRNA can modulate Cas9 activity in an allele-specific manner. These advancements hold promise for addressing current limitations and expanding the therapeutic potential of CRISPR technology.

The Influence of Homologous Arm Length on Homologous Recombination Gene Editing Efficiency Mediated by SSB/CRISPR-Cas9 in Escherichia coli.

The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10-100 bp homologous arms, while the other set included donors with homologous arms ranging from 10-100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10-100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10-60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60-100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10-90 bp, plateauing at 90-100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90-100 bp non-selectable homologous arms or 60-100 bp selectable homologous arms.

A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification.

The point-of-care testing (POCT) of miRNA has significant application in medical diagnosis, yet presents challenges due to their characteristics of high homology, low abundance, and short length, which hinders the achievement of quick detection with high specificity and sensitivity. In this study, a lateral flow assay based on the CRISPR/Cas13a system and MnO2 nanozyme was developed for highlysensitive detection of microRNA-21 (miR-21). The CRISPR/Cas13a cleavage system exhibits the ability to recognize the specific oligonucleotide sequence, where two-base mismatches significantly impact the cleavage activity of the Cas13a. Upon binding of the target to crRNA, the cleavage activity of Cas13a is activated, resulting in the unlocking of the sequence and initiating strand displacement, thereby enabling signal amplification to produce a new sequence P1. When applying the reaction solution to the lateral flow test strip, P1 mediates the capture of MnO2 nanosheets (MnO2 NSs) on the T zone, which catalyzes the oxidation of the pre-immobilized colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) on the T zone and generates the blue-green product (ox-TMB). The change in gray value is directly proportional to the concentration of miR-21, allowing for qualitative detection through visual inspection and quantitative measurement using ImageJ software. This method achieves the detection of miR-21 within a rapid 10-min timeframe, and the limit of detection (LOD) is 0.33pM. With the advantages of high specificity, simplicity, and sensitivity, the lateral flow test strip and the design strategy hold great potential for the early diagnosis of related diseases.

Expression of human BRCA2 in Saccharomyces cerevisiae complements the loss of RAD52 in double-strand break repair.

BRCA2 is a tumor-suppressor gene that is normally expressed in the breast and ovarian tissue of mammals. The BRCA2 protein mediates the repair of double-strand breaks (DSBs) using homologous recombination, which is a conserved pathway in eukaryotes. Women who express missense mutations in the BRCA2 gene are predisposed to an elevated lifetime risk for both breast cancer and ovarian cancer. In the present study, the efficiency of human BRCA2 (hBRCA2) in DSB repair was investigated in the budding yeast Saccharomyces cerevisiae. While budding yeast does not possess a true BRCA2 homolog, they have a potential functional homolog known as Rad52, which is an essential repair protein involved in mediating homologous recombination using the same mechanism as BRCA2 in humans. Therefore, to examine the functional overlap between Rad52 in yeast and hBRCA2, we expressed the wild-type hBRCA2 gene in budding yeast with or without Rad52 and monitored ionizing radiation resistance and DSB repair efficiency. We found that the expression of hBRCA2 in rad52 mutants increases both radiation resistance and DSB repair frequency compared to cells not expressing BRCA2. Specifically, BRCA2 improved the protection against ionizing radiation byat least 1.93-fold and the repair frequency by 6.1-fold. In addition, our results show that homology length influences repair efficiency in rad52 mutant cells, which impacts BRCA2 mediated repair of DSBs. This study provides evidence that S. cerevisiae could be used to monitor BRCA2 function, which can help in understanding the genetic consequences of BRCA2 variants and how they may contribute to cancer progression.

Highly mismatch-tolerant homology testing by RecA could explain how homology length affects recombination.

In E. coli, double strand breaks (DSBs) are resected and loaded with RecA protein. The genome is then rapidly searched for a sequence that is homologous to the DNA flanking the DSB. Mismatches in homologous partners are rare, suggesting that RecA should rapidly reject mismatched recombination products; however, this is not the case. Decades of work have shown that long lasting recombination products can include many mismatches. In this work, we show that in vitro RecA forms readily observable recombination products when 16% of the bases in the product are mismatched. We also consider various theoretical models of mismatch-tolerant homology testing. The models test homology by comparing the sequences of Ltest bases in two single-stranded DNAs (ssDNA) from the same genome. If the two sequences pass the homology test, the pairing between the two ssDNA becomes permanent. Stringency is the fraction of permanent pairings that join ssDNA from the same positions in the genome. We applied the models to both randomly generated genomes and bacterial genomes. For both randomly generated genomes and bacterial genomes, the models show that if no mismatches are accepted stringency is ∼ 99% when Ltest = 14 bp. For randomly generated genomes, stringency decreases with increasing mismatch tolerance, and stringency improves with increasing Ltest. In contrast, in bacterial genomes when Ltest ∼ 75 bp, stringency is ∼ 99% for both mismatch-intolerant and mismatch-tolerant homology testing. Furthermore, increasing Ltest does not improve stringency because most incorrect pairings join different copies of repeats. In sum, for bacterial genomes highly mismatch tolerant homology testing of 75 bp provides the same stringency as homology testing that rejects all mismatches and testing more than ∼75 base pairs is not useful. Interestingly, in vivo commitment to recombination typically requires homology testing of ∼ 75 bp, consistent with highly mismatch intolerant testing.

CRISPR-mediated genome editing in poplar issued by efficient transformation.

CRISPR has been increasingly used for plant genetic improvements because of its high efficiency and precision. Recently, the authors have reported the possibility of homology-directed repair (HDR) using CRISPR/Cas9 through woody plants such as poplar. HDR often replaces nucleotides with one donor DNA template (DDT), including homologous sequences. CRISPR-Cas9 was recruited, and three variables, Agrobacteria inoculator concentration, pDDT/pgRNA ratio, and homologous arm length, were designed to integrate nptII and 2XCamV 35S into the MKK2 promoter zone. Here, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by the precise integration of 2XcamV 35S and nptII, improving biochemical and phenotypic properties. Our findings confirmed that Agrobacterium inoculator OD600 = 2.5, increased DDT numbers during cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused efficient HDR and increased MKK2 expression. Efficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar.

Specific targeting of plasmids with Argonaute enables genome editing.

Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. In vitro, pAgos can bind small single-stranded guide DNAs to recognize and cleave complementary DNA. In vivo, pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we show that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA engineering in bacteria. We demonstrate that CbAgo loaded with plasmid-derived guide DNAs can recognize and cleave homologous chromosomal loci, and define the minimal length of homology required for this targeting. Cleavage of plasmid DNA at an engineered site of the I-SceI meganuclease increases guide DNA loading into CbAgo and enhances processing of homologous chromosomal loci. Analysis of guide DNA loading into CbAgo also reveals off-target sites of I-SceI in the Escherichia coli genome, demonstrating that pAgos can be used for highly sensitive detection of double-stranded breaks in genomic DNA. Finally, we show that CbAgo-dependent targeting of genomic loci with plasmid-derived guide DNAs promotes homologous recombination between plasmid and chromosomal DNA, depending on the catalytic activity of CbAgo. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing.

The effect of repeat length on Marcal1-dependent single-strand annealing in Drosophila.

Proper repair of DNA double-strand breaks is essential to the maintenance of genomic stability and avoidance of genetic disease. Organisms have many ways of repairing double-strand breaks, including the use of homologous sequences through homology-directed repair. While homology-directed repair is often error free, in single-strand annealing homologous repeats flanking a double-strand break are annealed to one another, leading to the deletion of one repeat and the intervening sequences. Studies in yeast have shown a relationship between the length of the repeat and single-strand annealing efficacy. We sought to determine the effects of homology length on single-strand annealing in Drosophila, as Drosophila uses a different annealing enzyme (Marcal1) than yeast. Using an in vivo single-strand annealing assay, we show that 50 base pairs are insufficient to promote single-strand annealing and that 500-2,000 base pairs are required for maximum efficiency. Loss of Marcal1 generally followed the same homology length trend as wild-type flies, with single-strand annealing frequencies reduced to about a third of wild-type frequencies regardless of homology length. Interestingly, we find a difference in single-strand annealing rates between 500-base pair homologies that align to the annealing target either nearer or further from the double-strand break, a phenomenon that may be explained by Marcal1 dynamics. This study gives insights into Marcal1 function and provides important information to guide the design of genome engineering strategies that use single-strand annealing to integrate linear DNA constructs into a chromosomal double-strand break.

Homology length dictates the requirement for Rad51 and Rad52 in gene targeting in the Basidiomycota yeast Naganishia liquefaciens.

Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast, Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80bp or 1kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 in N. liquefaciens conferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.

Optimizing recombineering in Corynebacterium glutamicum.

Owing to theincreasing demand for amino acids and valuable commodities that can be produced by Corynebacterium glutamicum, there is a pressing need for new rapid genome engineering tools that improve the speed and efficiency of genomic insertions, deletions, and mutations. Recombineering using the λ Red system in Escherichia coli has proven very successful at genetically modifying this organism in a quick and efficient manner, suggesting that optimizing a recombineering system for C. glutamicum will also improve the speed for genomic modifications. Here, we maximized the recombineering efficiency in C. glutamicum by testing the efficacy of seven different recombinase/exonuclease pairs for integrating single-stranded DNAand double-stranded DNA (dsDNA) into the genome. By optimizing the homologous arm length and the amount of dsDNA transformed, as well as eliminating codon bias, a dsDNA recombineering efficiency of 13,250 transformed colonies/109 viable cells was achieved, the highest efficiency currently reported in the literature. Using this optimized system, over 40,000 bp could be deleted in one transformation step. This recombineering strategy will greatly improve the speed of genetic modifications in C. glutamicum and assist other systems, such as clustered regularly interspaced short palindromic repeats and multiplexed automated genome engineering, in improving targeted genome editing.

Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system.

The ability to genetically manipulate microorganisms has been essential for understanding their biology and metabolism. Targeted genome editing relies on highly efficient homologous recombination, and while this is readily observed in the yeast Saccharomyces cerevisiae, most non-conventional yeast species do not display this trait and remain recalcitrant to targeted editing methods. CRISPR-based editing can bypass the requirement for high levels of native homologous recombination, enabling targeted modification to be more broadly implemented. While genetic transformation has been reported previously in Brettanomyces bruxellensis, a yeast with broad biotechnological potential and responsible for significant economic losses during the production of fermented beverages, targeted editing approaches have not been reported. Here, we describe the use of an expression-free CRISPR-Cas9 system, in combination with gene transformation cassettes tailored for B. bruxellensis, to provide the means for targeted gene deletion in this species. Deletion efficiency was shown to be dependent on homologous flanking DNA length, with higher targeting efficiencies observed with cassettes containing longer flanking regions. In a diploid strain, it was not possible to delete multiple alleles in one step, with heterozygous deletants only obtained when using DNA cassettes with long flanking regions. However, stepwise transformations (using two different marker genes) were successfully used to delete both wild-type alleles. Thus, the approach reported here will be crucial to understand the complex physiology of B. bruxellensis. Key points • The use of CRISPR-Cas9 enables targeted gene deletion in Brettanomyces bruxellensis. • Homozygous diploid deletions are possible with step-wise transformations. • Deletion of SSU1 confirmed the role of this gene in sulphite tolerance.

Structural transitions and mechanochemical coupling in the nucleoprotein filament explain homology selectivity and Rad51 protein cooperativity in cellular DNA repair.

The nucleoprotein filament (NPF) is the fundamental element of homologous recombination (HR), a major mechanism for the repair of double-strand DNA breaks in the cell. The NPF is made of the damaged DNA strand surrounded by recombinase proteins, and its sensitivity to base-pairing mismatches is a crucial feature that guarantees the fidelity of the repair. The concurrent recombinases are also essential for several steps of HR. In this work, we used torque-sensitive magnetic tweezers to probe and apply mechanical constraints to single nucleoprotein filaments (NPFs). We demonstrated that the NPF undergoes structural transitions from a stretched to a compact state, and we measured the corresponding mechanochemical signatures. Using an active two-state model, we proposed a free-energy landscape for the NPF transition. Using this quantitative model, we explained both how the sensitivity of the NPF to the homology length is regulated by its structural transition and how the cooperativity of Rad51 favors selectivity to relatively long homologous sequences.

Sex Chromosome Pairing Mediated by Euchromatic Homology in Drosophila Male Meiosis.

Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the ribosomal DNA (rDNA). Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y) Using fluorescence in situ hybridization to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis, while the autosomal-specific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.

Identification and functional analysis of potential prophage-derived recombinases for genome editing in Lactobacillus casei.

Numerous lactic acid bacteria (LAB) bacteriophage genomes have been sequenced, while the functional genes are yet to be exploited. In this study, a λ Red-like recombinase operon LCABL_13040-50-60 was identified from a prophage PLE3 in Lactobacillus casei BL23 genome, and its recombination function was confirmed by the replacement of a 167-bp galK fragment with chloramphenicol-resistant gene (cat) in the L. casei BL23 genome. Further functional analysis showed that LCABL_13040 and LCABL_13060 were analogs to the host nuclease inhibitor (Redγ) and 5΄-3΄ exonuclease (Redα/RecE), respectively. After optimization of recombineering conditions, including induction, homology length, recovery time and double-strand DNA substrates quantity, the recombineering efficiency reached ∼2.2×10-7. Subsequently, combining cre-lox technology, the optimal LCABL_13040-50-60 proteins could catalyze markerless deletion of a 167-bp galK fragment and insertion of the gfp gene as well as precision point mutation of rpoB gene in the L. casei BL23 genome, suggesting the LCABL_13040-50-60 operon encoded for three recombineering proteins. Moreover, with the assistance of Redγ, the LCABL_13040-50-60 proteins also showed recombinase activity in six other L. casei strains, L. paracasei OY and L. plantarum WCSF1. All the results demonstrated that the prophage-associated recombinases LCABL_13040-50-60 have great potential to be used for genome editing in LAB.

On systolic zeta functions

We define Dirichlet type series associated with homology length spectra of Riemannian, or Finsler, manifolds, or polyhedra, and investigate some of their analytical properties. As a consequence we obtain an inequality analogous to Gromov’s classical intersystolic inequality, but taking the whole homology length spectrum into account rather than just the systole.

PARP2 controls double-strand break repair pathway choice by limiting 53BP1 accumulation at DNA damage sites and promoting end-resection.

Double strand breaks (DSBs) are one of the most toxic lesions to cells. DSB repair by the canonical non-homologous end-joining (C-EJ) pathway involves minor, if any, processing of the broken DNA-ends, whereas the initiation of DNA resection channels the broken-ends toward DNA repair pathways using various lengths of homology. Mechanisms that control the resection initiation are thus central to the regulation to the choice of DSB repair pathway. Therefore, understanding the mechanisms which regulate the initiation of DNA end-resection is of prime importance. Our findings reveal that poly(ADP-ribose) polymerase 2 (PARP2) is involved in DSBR pathway choice independently of its PAR synthesis activity. We show that PARP2 favors repair by homologous recombination (HR), single strand annealing (SSA) and alternative-end joining (A-EJ) rather than the C-EJ pathway and increases the deletion sizes at A-EJ junctions. We demonstrate that PARP2 specifically limits the accumulation of the resection barrier factor 53BP1 at DNA damage sites, allowing efficient CtIP-dependent DNA end-resection. Collectively, we have identified a new PARP2 function, independent of its PAR synthesis activity, which directs DSBs toward resection-dependent repair pathways.

Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning.

Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s-2.5 min) over a wide range of temperatures (25-75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments.

Multi-invasions Are Recombination Byproducts that Induce Chromosomal Rearrangements

Inaccurate repair of broken chromosomes generates structural variants that can fuel evolution and inflict pathology. We describe a novel rearrangement mechanism in which translocation between intact chromosomes is induced by a lesion on a third chromosome. This multi-invasion-induced rearrangement (MIR) stems from a homologous recombination byproduct, where a broken DNA end simultaneously invades two intact donors. No homology is required between the donors, and the intervening sequence from the invading molecule is inserted at the translocation site. MIR is stimulated by increasing homology length and spatial proximity of the donors and depends on the overlapping activities of the structure-selective endonucleases Mus81-Mms4, Slx1-Slx4, and Yen1. Conversely, the 3'-flap nuclease Rad1-Rad10 and enzymes known to disrupt recombination intermediates (Sgs1-Top3-Rmi1, Srs2, and Mph1) inhibit MIR. Resolution of MIR intermediates propagates secondary chromosome breaks that frequently cause additional rearrangements. MIR features have implications for the formation of simple and complex rearrangements underlying human pathologies.

The contribution of homology arms to nuclease-assisted genome engineering.

Designer nucleases like CRISPR/Cas9 enable fluent site-directed damage or small mutations in many genomes. Strategies for their use to achieve more complex tasks like regional exchanges for gene humanization or the establishment of conditional alleles are still emerging. To optimize Cas9-assisted targeting, we measured the relationship between targeting frequency and homology length in targeting constructs using a hypoxanthine-guanine phosphoribosyl-transferase assay in mouse embryonic stem cells. Targeting frequency with supercoiled plasmids improved steeply up to 2 kb total homology and continued to increase with even longer homology arms, thereby implying that Cas9-assisted targeting efficiencies can be improved using homology arms of 1 kb or greater. To humanize the Kmt2d gene, we built a hybrid mouse/human targeting construct in a bacterial artificial chromosome by recombineering. To simplify the possible outcomes, we employed a single Cas9 cleavage strategy and best achieved the intended 42 kb regional exchange with a targeting construct including a very long homology arm to recombine ∼42 kb away from the cleavage site. We recommend the use of long homology arm targeting constructs for accurate and efficient complex genome engineering, particularly when combined with the simplifying advantages of using just one Cas9 cleavage at the genome target site.