The application of the CRISPR/Cas‐system paved the way for the era of genome engineering and quickly became the standardly applied tool for targeted non‐homologous end joining (NHEJ) based mutagenesis. Despite various efforts, homologous recombination (HR) based gene targeting (GT) still requires further improvement regarding efficiency for general applicability in plants (Huang and Puchta, 2019). We developed the in planta GT (ipGT) method aimed to establish a technology for crops with meagre transformation efficiencies (Fauser et al., 2012). Here, the targeting vector including the nuclease is integrated into the genome and excised at the same time as the target site is activated by double‐strand break (DSB) induction (Figure 1a). We successfully adopted Cas9 for this application (Schiml et al., 2014) and improved GT efficiencies by replacing Cas9 from Streptococcus pyogenes (SpCas9) with Cas9 from Staphylococcus aureus (SaCas9), which is more efficient in DSB induction in Arabidopsis (Steinert et al., 2015). Egg‐cell specific expression and screening for the most efficient transgenic lines were further key points for GT improvement (Miki et al., 2018; Wolter et al., 2018). Most recently, we tested the CRISPR/LbCas12a‐system for ipGT and could demonstrate a further increase in GT efficiencies despite lower InDel rates induced by LbCas12a compared with SaCas9 at the ALS target locus (Wolter and Puchta, 2019). We speculated that the higher GT efficiency is caused by Cas12a‐mediated cleavage on the PAM distal site, leaving the seed sequence unaffected by mutagenesis. Thus, further cleavage of NHEJ repaired junctions might be much more frequent with Cas12a than with Cas9, enhancing the probability for HR to take over DSB repair. This is also in line with very recently published results on enhancing GT efficiency using LbCas12a in rice and tomato (Li et al., 2019; van Vu et al., 2020). Recently, by introducing the single amino acid substitution D156R, we were able to obtain a LbCas12a nuclease variant with improved, temperature‐tolerant cutting efficiency (ttLbCas12a) for plant gene editing, outperforming LbCas12a at 22°C and 28°C in mutation induction (Schindele and Puchta, 2019). Based on those findings, we were interested to test whether replacing LbCas12a by ttLbCas12a could further improve GT.

Therefore, we performed ipGT experiments in Arabidopsis testing LbCas12a and ttLbCas12a at 22°C as well as at 28°C in parallel for a direct comparison. As described before, the acetolactate synthase gene (ALS) was selected as target and both nucleases were expressed by an egg‐cell specific promoter to assure efficient germline transmission (Wolter et al., 2018). For GT induction, a ribozyme‐flanked crRNA cassette was used with a spacer targeting ALS, a respective GT donor molecule and a Kanamycin expression cassette for selection. The donor molecule contains the modification (S653N) conferring Imazapyr resistance and one silent point mutation to avoid DSB induction within the donor. For donor excision, the donor molecule is flanked by the respective spacer sequence (Figure 1a). The GT constructs, either containing LbCas12a or ttLbCas12a, were transformed as T‐DNAs into Arabidopsis thaliana Col‐0 plants via the floral dip method. After transformation, the T0 plants were grown either at 22°C or at 28°C until maturity and the harvested seeds subsequently sown out on Kanamycin selection medium. The resistant T1 primary transformants were grown until maturity at either 22°C or 28°C, respectively. The harvested T2 seeds were sown out on Imazapyr medium to verify ipGT events. At least 90 lines were tested per approach (Figure 1b). As expected, it turned out that raising the temperature from 22°C to 28°C is indeed enhancing GT efficiency. If we simply take the number of lines that produced resistant seedlings into account, ttLbCas12a outperforms the standard enzyme at 22°C by almost twofold. At 28°C, still one third more lines with GT events were detected for ttLbCas12a. The effect is even more pronounced if we take the number of GT events per line into account. For all lines, the individual GT frequencies were calculated by setting the total number of seeds into relation with the number of Imazapyr resistant seedlings. Mean GT frequencies were determined over all lines per approach. At 22°C, ttLbCas12a outperforms the native enzyme by 2.4‐fold and at 28°C still by 1.7‐fold. This is also obvious when we compare the targeting frequencies for the single lines individually for 28°C and for 22°C (Figure 1c and d). The difference in numbers and frequencies are especially impressive for the lower temperature but still clearly visible for the higher. Additionally, molecular analysis was performed to check the nature of GT events. DSB induced GT depends on the synthesis‐dependent strand annealing (SDSA) mechanism of HR (Huang and Puchta, 2019). In principle, besides perfect GT events, using HR at both junctions at the target locus also with a combination of HR and NHEJ, can lead to the restoration of the marker gene. Previously, we were able to demonstrate that in a large number of cases the marker gene is first restored by a homologous interaction with the target locus but then the vector integrates elsewhere in the genome by NHEJ. These ectopic targeting events can be discriminated from perfect GT events by PCR analysis of both donor‐genome junctions. We applied the same kind of analysis that we established in previous studies using the ALS gene as target (Wolter et al., 2018; Wolter and Puchta, 2019). All in all, over one hundred Imazapyr‐resistant plants, all representing independent GT events, were analysed. The respective result is shown in Figure 1e: the GT events induced by ttLbCas12a are in about half of the cases true GT events indicating that the modified enzyme, ttLbCas12a, is at least as efficient as the native one, which showed perfect GT events in about one third of the cases. Furthermore, for ttLbCas12a at 28°C molecular analysis indicated that 3% of the analysed T2 lines already comprised a homozygous GT event, which did not occur in any other approach in this study.

Recent results already demonstrated the beneficial effect of increased temperature on LbCas12a‐mediated GT in plants (van Vu et al., 2020). Here, we showed that the application of the recently developed ttLbCas12a (Schindele and Puchta, 2019) is not only an alternative way to boost GT for plants that cannot cope with high temperatures. GT can be elevated even further by its use in combination with a high‐temperature treatment. Yet, the potential of ttLbCas12a for GT might be even more promising as our data indicate. We were recently able to show that ttLbCas12a provides access to target sites that could hardly be edited at all before by LbCas12a even with a high‐temperature treatment (Schindele and Puchta, 2019). As we used a target locus that was accessible for native LbCas12a (Wolter and Puchta, 2019), the use of ttLbCas12a might increase GT efficiencies at those loci that are more difficult to access even much stronger.

CRISPR / Cas系统的应用为基因组工程的时代铺平了道路，并迅速成为基于靶向非同源末端连接（NHEJ）的诱变的标准应用工具。尽管进行了各种努力，基于同源重组（HR）的基因靶向（GT）仍需要进一步提高植物普遍适用性的效率（Huang和Puchta，2019）。我们开发了植物内GT（ipGT）方法，旨在为转化效率不高的农作物建立一种技术（Fauser等，2012）。）。在这里，包括核酸酶的靶向载体被整合到基因组中，并在通过双链断裂（DSB）诱导激活靶位的同时被切除（图1a）。我们成功地将Cas9用于该应用（Schiml等人，2014），并通过用金黄色葡萄球菌（Sa Cas9）的Cas9替代化脓性链球菌（Sp Cas9）的Cas9来提高GT效率，这在拟南芥中的DSB诱导更有效（Steinert）等人，2015）。卵细胞特异性表达和最有效转基因品系的筛选是进一步改善GT的关键（Miki等。，2018 ; Wolter et al。，2018）。最近，我们测试了用于ipGT的CRISPR / Lb Cas12a系统，尽管在ALS目标基因座上与Sa Cas9相比，Lb Cas12a诱导的InDel率更低，但可以证明GT效率进一步提高（Wolter和Puchta，2019年）。我们推测，较高的GT效率是由PAM远端位点上的Cas12a介导的切割引起的，而种子序列不受诱变的影响。因此，用Cas12a进行的NHEJ修复连接的进一步切割可能比用Cas9进行的更频繁，从而增加了HR接管DSB修复的可能性。这也与最近发表的有关在水稻和番茄中使用Lb Cas12a提高GT效率的结果相吻合（Li等，2019 ; van Vu等，2020）。最近，通过引入单个氨基酸取代D156R，我们能够获得具有改进的耐温切割效率的Lb Cas12a核酸酶变体（tt LbCas12a）用于植物基因编辑，在诱变诱导方面在22°C和28°C方面优于Lb Cas12a（Schindele和Puchta，2019年）。基于这些发现，我们有兴趣测试用tt Lb Cas12a替代Lb Cas12a是否可以进一步改善GT。

因此，我们在拟南芥中进行了ipGT实验，分别在22°C和28°C下测试Lb Cas12a和tt Lb Cas12a，以进行直接比较。如前所述，选择乙酰乳酸合酶基因（ALS）作为靶标，两个核​​酸酶均通过卵细胞特异性启动子表达，以确保有效的种系传递（Wolter等人，2018）。对于GT诱导，使用了带有核酶的侧翼crRNA盒，并带有靶向ALS的间隔，相应的GT供体分子和卡那霉素表达盒供选择。供体分子包含赋予Imazapyr抗性的修饰（S653N）和一个沉默位点突变，以避免在供体中诱导DSB。对于供体切除，供体分子两侧分别是间隔区序列（图1a）。含有Lb Cas12a或tt Lb Cas12a的GT构建体以T-DNA的形式转化到拟南芥中Col-0植物通过浸花法种植。转化后，将T0植物在22℃或28℃下生长直至成熟，然后将收获的种子播种在卡那霉素选择培养基上。抗性T1初级转化体分别生长直到分别在22℃或28℃下成熟。将收获的T2种子播种在Imazapyr培养基上，以验证ipGT事件。每种方法至少测试了90条线（图1b）。不出所料，事实证明，将温度从22°C升高到28°C确实可以提高GT效率。如果仅考虑产生抗性幼苗的品系数量，则在22°C时tt Lb Cas12a的性能要比标准酶高出将近两倍。在28°C时，还发现了TT Lb还有三分之一的GT事件发生Cas12a。如果我们考虑每行GT事件的数量，效果会更加明显。对于所有品系，通过将种子总数与抗Imazapyr幼苗的数量相关来计算单个GT频率。确定每种方法在所有线路上的平均GT频率。在22°C时tt LbCas12a胜过天然酶2.4倍，在28°C时仍胜过1.7倍。当我们分别比较单线的目标频率在28°C和22°C时，这也很明显（图1c和d）。数量和频率的差异对于较低的温度尤为明显，但对于较高的温度仍清晰可见。另外，进行了分子分析以检查GT事件的性质。DSB诱导的GT取决于HR的合成依赖链退火（SDSA）机制（Huang和Puchta，2019年）。原则上，除了完美的GT事件外，在目标基因座的两个连接处同时使用HR和HR和NHEJ的组合，还可以导致标记基因的恢复。以前，我们能够证明在许多情况下，标记基因首先通过与靶基因座的同源相互作用而被恢复，但随后载体通过NHEJ整合到基因组的其他位置。这些异位靶向事件可以通过两个供体-基因组连接的PCR分析与完美的GT事件区分开。我们应用了以前的研究中建立的相同分析类型，使用ALS基因作为靶标（Wolter等人，2018 ; Wolter和Puchta，2019）。总共分析了一百多株抗Imazapyr的植物，它们均代表独立的GT事件。相应的结果如图1e所示：在大约一半的情况下，由tt Lb Cas12a诱导的GT事件是真正的GT事件，这表明修饰的酶tt Lb Cas12a至少与天然酶一样有效，表现出完美的大约三分之一的案例中发生了GT事件。此外，对于tt Lb Cas12a在28°C的分子分析表明，分析的T2系中有3％已经包含纯合GT事件，在本研究中的任何其他方法中均未发生。

最近的结果已经证明了温度升高对植物中Lb Cas12a介导的GT的有益作用（van Vu等，2020）。在这里，我们表明，最近开发的tt Lb Cas12a（Schindele和Puchta，2019）的应用不仅是无法应对高温的植物提高GT的替代方法。通过与高温处理结合使用，GT可以进一步提高。然而，正如我们的数据表明的那样，tt Lb Cas12a在GT中的潜力可能甚至更大。我们最近能够证明tt Lb Cas12a提供了对目标站点的访问，而以前Lb几乎无法对其进行编辑Cas12a甚至经过高温处理（Schindele和Puchta，2019年）。由于我们使用了天然Lb Cas12a可以访问的目标基因座（Wolter and Puchta，2019），因此tt Lb Cas12a的使用可能会提高那些更难获得甚至更强大的基因座的GT效率。